Dual function fusion proteins and their preparation and use

Abstract

The present invention relates to the field of biotechnology and discloses a dual-function fusion protein and its preparation and use. The dual-function fusion protein comprises a VEGF antagonist fragment and a TGF-β antagonist fragment, the VEGF antagonist fragment comprises SEQ ID NO: 1 and 2 or a polypeptide fragment having 90% or more sequence identity thereto, and the TGF-β antagonist fragment comprises SEQ ID NO: 3 or a polypeptide fragment having 90% or more sequence identity thereto. The present invention also provides a method for preparing the dual-function protein and its use in the preparation of a drug for treating diseases associated with the expression of VEGF and / or TGF-β. The dual-function protein provided by the present invention is not easily cleaved during the purification process, which can greatly improve the purification efficiency and reduce the production cost, and the finished drug has good stability and has a wide range of future applications.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, in particular to dual function fusion proteins and their production and use. [Background technology]

[0002] In the tumor microenvironment, transforming growth factor-β (TGF-β) Transforming growth factor-β (TGF-β) plays an important role in immunosuppression. TGF-β regulates the generation and function of many types of immune cells. It directly promotes the proliferation of Treg cells, suppresses the generation and function of effector cells (Teff) and antigen-presenting dendritic cells (DC), and controls adaptive immunity. Similarly, TGF-β controls the innate immune system by suppressing natural killer cells (NK cells) and regulating the complex behavior of macrophages and neutrophils, forming an input network of negative immune regulation. Research has shown that angiogenesis is one of the factors in the growth of malignant tumors, and that vascular endothelial growth factor (VEGF) is a highly specific vascular endothelial cell growth-promoting factor, and its inhibition can effectively suppress tumor growth. Antiangiogenesis drugs, represented by VEGF antibodies, have good specificity, good therapeutic effect, and are less likely to cause drug resistance, making them a hot topic in the research and development of antitumor targeted drugs.

[0003] Bevacizumab (Avastin) is a recombinant humanized immunoglobulin G1 (IgG1) monoclonal antibody developed by Genetech, a subsidiary of Roche. It binds to VEGF-A and inhibits its binding to VEGF receptor-2 (VEGFR-2), and inhibits the biological effects of VEGF, including the effects on vascular permeability, proliferation, and endothelial cell migration and survival, thereby achieving the effect of inhibiting tumor angiogenesis, growth, and metastasis. Currently, the combination therapy of many small and large molecule VEGF antagonists with PD-1 or PD-L1 has already shown excellent therapeutic effects in many cancers, including end-stage liver cancer, and has become one of the important combination therapies for cancer.

[0004] The negative inhibitory tumor microenvironment is a factor that inhibits and limits the effective clinical therapeutic effects of immune checkpoint drugs, including industry-recognized programmed cell death receptor 1 (PD-1) and programmed cell death ligand (PD-L1). The negative impact of the tumor microenvironment is particularly prominent in solid tumors, and is a key point in drug discovery in terms of both response rate and drug resistance. For example, M7824 is a dual-function fusion protein developed by Merck Serono, a subsidiary of Merck, which consists of an anti-PD-L1 monoclonal antibody and the extracellular domain of human TGF-β receptor II (TGF-βRII), and can antagonize PD-L1 and TGF-β simultaneously. M7824 showed good activity in early clinical studies, but did not show superiority in clinical phase III studies. It can be seen that the inhibition of TGF-β alone is still insufficient to overcome the negative regulation of the tumor microenvironment.

[0005] In addition, existing dual-function fusion proteins containing TGF-β receptors, such as Merck's M7824, will be fragmented during the purification process of such dual-function fusion proteins due to the inherent physiological properties of TGF-β receptors, and these fragments will significantly reduce the purification efficiency, increase the production cost, and ultimately affect the efficacy of the finished drug. In addition, the clinical dosage of PD-L1 antibodies is much higher than that of PD-1 antibodies, further increasing the difficulty of clinical development of M7824 and limiting its future clinical application. Summary of the Invention

[0006] In view of the shortcomings of the existing theories and techniques described above, the objective of the present invention is to provide a dual-function fusion protein and its preparation and use, so as to solve the problems in the existing techniques, and at the same time provide a new technological approach in terms of efficiently overcoming the tumor microenvironment, and enhance the therapeutic effect of combination treatment with PD-1 or PD-L1.

[0007] In one aspect of the invention, the antibody comprises a VEGF antagonistic fragment and a TGF-β antagonistic fragment, The VEGF antagonist fragment provides a dual-function fusion protein comprising a heavy chain that includes at least an amino acid mutation at position 453 compared to the original heavy chain, and / or the TGF-β antagonist fragment includes at least an amino acid mutation at position 8 compared to the original TGF-β antagonist fragment.

[0008] In another aspect of the invention, there is provided an isolated polynucleotide encoding the aforementioned dual function fusion protein.

[0009] In another aspect of the invention, there is provided a construct containing the aforementioned isolated polynucleotide.

[0010] In another aspect of the invention, there is provided an expression system which contains the above-described construct or has integrated into its genome an exogenous above-described polynucleotide.

[0011] In another aspect of the present invention, there is provided a method for producing the dual function fusion protein, comprising culturing the expression system under suitable conditions to express the dual function fusion protein, and isolating and purifying the dual function fusion protein.

[0012] Another aspect of the present invention provides the use of the above-mentioned dual function fusion protein in the manufacture of a medicament for treating a disease associated with the expression of VEGF and / or TGF-β, preferably for treating a tumor, including inhibiting tumor angiogenesis, growth and metastasis, inhibiting tumor cell formation, proliferation, metastasis and invasion, promoting apoptosis of tumor cells, etc.

[0013] In another aspect of the invention there is provided a pharmaceutical composition comprising said dual function fusion protein or said expression system or a culture of said expression system.

[0014] In the present invention, the dual-function fusion protein and the biological material related to the dual-function fusion protein can be artificially synthesized, or the coding gene can be synthesized first and then obtained by biological expression. In one embodiment, the dual-function fusion protein and the biological material related to the dual-function fusion protein described in the present invention are produced by introducing a gene into E. coli, yeast, or mammalian cells, such as CHO cells, HEK293 cells, and expressing the gene using E. coli, yeast, or mammalian cells.

[0015] Beneficial effects of the present invention include the following:

[0016] 1) In the present invention, the VEGF antagonistic fragment is prepared by deleting the 453rd amino acid residue K in the heavy chain, and the sequence of the TGF-β antagonistic fragment is adjusted by mutating the 8th amino acid residue K in the amino acid sequence to A (K→A mutation). The dual-function fusion protein thus prepared has good stability and is not easily broken during purification, and can improve purification efficiency during production and reduce production costs. The purified dual-function fusion protein of the present invention has a molecular weight of about 179 kD and a purity of more than 98%.

[0017] 2) The dual-function fusion protein described in the present invention has very good affinity and can be expressed in recombinant human The dual function fusion protein can bind to TGF-β1 / 2 / 3 with an affinity approximately equivalent to that of M7824. The dual function fusion protein can bind to recombinant human VEGF with an affinity approximately equivalent to that of Avastin. The dual function fusion protein of the present invention can also bind to recombinant human TGF-β1 / 2 / 3 and Avastin at the same time. Therefore, the dual function fusion protein of the present invention can be applied to the treatment of diseases associated with the expression of VEGF and / or TGF-β.

[0018] 3) In vivo antitumor efficacy experiments demonstrated that the dual-function fusion protein described in the present invention can effectively inhibit tumor growth in mice, and is useful in tumor treatment. Description of the drawings [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows a schematic diagram of the results of a reduction electrophoresis identification experiment of a dual-function fusion protein in Example 4 of the present invention. [Diagram 2] FIG. 1 shows a schematic diagram of the results of a non-reducing electrophoresis identification experiment of a dual-function fusion protein in Example 4 of the present invention. [Diagram 3] 1 shows the results of identification of the stability of a dual-function fusion protein by reduced electrophoresis in Example 4 of the present invention. [Figure 4]1 shows a schematic diagram of the results of an ELISA experiment for TGF-β1 in Example 5 of the present invention. [Diagram 5] 1 shows a schematic diagram of the results of an ELISA experiment for TGF-β2 in Example 5 of the present invention. [Figure 6] 1 shows a schematic diagram of the results of an ELISA experiment for TGF-β3 in Example 5 of the present invention. [Figure 7] FIG. 1 shows a schematic diagram of the results of an ELISA experiment for VEGF in Example 6 of the present invention. [Figure 8] 1 shows a schematic diagram of the results of an ELISA experiment to simultaneously detect the binding of TGF-β and VEGF in Example 7 of the present invention. [Figure 9] FIG. 1 shows a schematic diagram of the results of an experiment in Example 8 of the present invention on suppressing VEGF-induced HUVEC proliferation. [Figure 10] FIG. 1 shows a schematic diagram of the results of an experiment in Example 9 of the present invention to suppress the transition of human breast cancer MDA-MB-231 cells induced by TGF-β. [Figure 11] FIG. 1 shows a schematic diagram of the statistical results of an experiment in Example 9 of the present invention for suppressing the transition of human breast cancer MDA-MB-231 cells induced by TGF-β. [Figure 12] 1 shows a schematic diagram of the experimental results of the experiment on the neutralizing effect of dual-function fusion protein on TGF-β in Example 10 of the present invention, in which control is PBS, control 2 is PBS+TGF-β1, control 3 is 10 nM HSP088-01+TGF-β1, test 1 is 1 nM dual-function fusion protein+TGF-β1, test 2 is 10 nM dual-function fusion protein+TGF-β1, and test 3 is 100 nM dual-function fusion protein+TGF-β1. [Figure 13-1] , [Figure 13-2] , [Figure 13-3] , [Figure 13-4] , [Figure 13-5] , [Figure 13-6] , [Figure 13-7]1 shows schematic diagrams of the results of in vivo tumor suppression experiments of dual-function fusion proteins in mice in Example 11 of the present invention, where each curve in each diagram represents the tumor growth curve of each animal in the corresponding experimental group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The following describes the present invention in order to make the purpose, technical solution and beneficial technical effects of the present invention clearer. The present invention will be described in more detail with reference to examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0021] After a great deal of practice and research, the inventors of the present invention have used gene site-directed mutagenesis technology to carry out gene mutations on the VEGF and TGF-β receptors in the fusion protein, thereby significantly improving the problem of peptide bond cleavage during the protein purification process and reducing the difficulty of purification, thereby producing a highly stable dual-function fusion protein that simultaneously inhibits VEGF and TGF-β, and have completed the present invention based on this.

[0022] In a first aspect of the present invention, a dual-function fusion protein is provided, comprising a VEGF antagonistic fragment and a TGF-β antagonistic fragment. The VEGF antagonistic fragment in the dual-function fusion protein is usually a fragment that has undergone genetic mutation, for example, any known VEGF antagonistic fragment. The VEGF antagonistic fragment may be a VEGF receptor fragment. The TGF-β antagonistic fragment is a fragment that has undergone genetic mutation of a TGF-β receptor fragment, for example, any known TGF-β antagonistic fragment.

[0023] The VEGF antagonistic fragment comprises a heavy chain that includes at least an amino acid mutation at position 453 compared to the original heavy chain.

[0024] The TGF-β antagonist fragment comprises at least an eighth amino acid mutation compared to the original TGF-β antagonist fragment.

[0025] Preferably, the mutation at amino acid 453 of the heavy chain of the VEGF antagonist fragment comprises an amino acid deletion or substitution, such as replacing the amino acid residue K at 453 with another amino acid other than K or its derivative, such as G, A, V, L, I, P, F, W, M, Y, S, T, C, N, Q, D, E, R, or H amino acid residue.

[0026] Preferably, the mutation at the 8th amino acid residue of the TGF-β antagonistic fragment comprises an amino acid deletion or substitution, i.e., the substitution of the 8th amino acid residue K with another amino acid other than K or its derivative, such as G, A, V, L, I, P, F, W, M, Y, S, T, C, N, Q, D, E, R, or H amino acid residue. Preferably, the mutation at the 8th amino acid residue of the TGF-β antagonistic fragment is a mutation of K to A (K→A).

[0027] Preferably, a dual function fusion protein according to the invention comprises at least one of the mutations described above.

[0028] Preferably, the amino acid sequence of the original heavy chain is shown in SEQ ID NO:6.

[0029] Preferably, the amino acid sequence of said original TGF-β antagonistic fragment is shown in SEQ ID NO:7.

[0030] In some preferred embodiments, the dual function fusion protein comprises a deletion mutation at amino acid 453 and an 8th amino acid mutation that is a K→A mutation.

[0031] In some preferred embodiments, the heavy chain of the VEGF antagonistic fragment may specifically comprise:

[0032] a-2) a polypeptide fragment having an amino acid sequence as set forth in SEQ ID NO: 2, or b-2) A polypeptide fragment having an amino acid sequence with 90% or more sequence identity to SEQ ID NO: 2, containing a deletion mutation at amino acid residue 453, and having the function of the polypeptide fragment defined in a-2). Specifically, the polypeptide fragment in b-2) is a polypeptide fragment obtained by substituting, deleting or adding one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) in the amino acid sequence shown in SEQ ID NO: 2, or by adding one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) to the N-terminus and / or C-terminus, and having the function of the polypeptide fragment having an amino acid sequence shown in SEQ ID NO: 2, which may be, for example, a function of antagonizing VEGF. The amino acid sequence in b-2) may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more to SEQ ID NO:2.

[0033] In some preferred embodiments, the TGF-β antagonistic fragment may specifically include:

[0034] c) a polypeptide fragment having the amino acid sequence set forth in SEQ ID NO:3; or d) A polypeptide fragment having an amino acid sequence with 90% or more sequence identity to SEQ ID NO: 3, containing a K8A mutation (mutation from K to A) at the 8th amino acid residue, and having the function of the polypeptide fragment defined in c). Specifically, the polypeptide fragment in d) is a polypeptide fragment obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) amino acids in the amino acid sequence shown in SEQ ID NO: 3, or by adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) amino acids to the N-terminus and / or C-terminus, and having the function of the polypeptide fragment having an amino acid sequence shown in SEQ ID NO: 3, which may be, for example, a function of antagonizing TGF-β. The amino acid sequence in d) may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:3.

[0035] In some preferred embodiments, the VEGF antagonistic fragment further comprises a light chain. The light chain may specifically comprise:

[0036] a-1) a polypeptide fragment having an amino acid sequence as set forth in SEQ ID NO: 1, or b-1) A polypeptide fragment having an amino acid sequence with 90% or more sequence identity to SEQ ID NO: 1 and having the function of the polypeptide fragment defined in a-1). Specifically, the polypeptide fragment in b-1) is a polypeptide fragment obtained by substituting, deleting or adding one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) in the amino acid sequence shown in SEQ ID NO: 1, or by adding one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) to the N-terminus and / or C-terminus, and having the function of the polypeptide fragment having an amino acid sequence shown in SEQ ID NO: 1, which may be, for example, a function of antagonizing VEGF. The amino acid sequence in b) may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:1.

[0037] In the dual-function fusion protein provided by the present invention, the dual-function fusion protein may further comprise a linked peptide fragment. Preferably, the linked peptide fragment is rich in G, S and / or A, where "rich in G, S and / or A" means rich in G, S and A, or G and S, or G and A. More preferably, the linked peptide fragment comprises a polypeptide fragment whose amino acid sequence may be as set forth in SEQ ID NO:5.

[0038] In the dual-function fusion protein provided by the present invention, the dual-function fusion protein may comprise, from the N-terminus to the C-terminus, a VEGF antagonistic fragment and a TGF-β antagonistic fragment.

[0039] In one specific embodiment of the present invention, the amino acid sequence of the dual function fusion protein may comprise the heavy chain sequence shown in SEQ ID NO:4 and the light chain sequence shown in SEQ ID NO:1.

[0040] In a second aspect of the invention, there is provided an isolated polynucleotide encoding the dual function fusion protein provided by the first aspect of the invention.

[0041] In a third aspect of the present invention, a construct is provided that contains the isolated polynucleotide provided by the second aspect of the present invention. Methods for providing a suitable construct are known to those skilled in the art. For example, the construct can usually be obtained by inserting the isolated polynucleotide into a suitable vector.

[0042] In a fourth aspect of the invention, there is provided an expression system which contains a construct as provided by the third aspect of the invention or has integrated into its genome an exogenous polynucleotide as provided by the second aspect of the invention, thereby enabling expression of the fusion protein as described above. The expression system may typically be a host cell, which may be one or more of a bacterial, fungal or mammalian cell, etc.

[0043] Wherein, the bacterium is selected from the group consisting of Escherichia coli, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Bacteroides ovatus, Campylobacter jejuni, and the like. The bacterium is one or more selected from the group consisting of Bacteroides jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Parabacteroides merdae, Fusobacterium mortiferum, and Bifidobacterium breve.

[0044] The fungus is selected from yeast and / or mold, and examples of the yeast include Saccharomyces cerevisiae, Candida dubliniensis, Candida glabrata, Candida guiliermondii, Candida kefir, Candida krusei, Hansenula polymorpha, Pichia pastoris, and the like. is, Kluyveromyces fragilis, Kluyveromyces lactis, Schizosaccharomyces pombe, Candida albicans, Candida lusitaniae, Candida melinii, Candida oleophila, Candida parapsilosis, Candida tropicalis and Candida utilis, and the mold fungus is one or more selected from Aspergillus clavatus, Aspergillus glaucus and Aspergillus glaucus group, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae oryzae, Aspergillus terreus, Aspergillus ustus and Aspergillus versicolor.

[0045] Wherein, the mammalian cell is one or more selected from CHO cell, HEK 293 human embryonic kidney cell, Bowes melanoma cell, COS-7 cell, C127 cell, HeLa cell, BHK cell, SP2 / 0 mouse plasma cell, NS0 mouse plasma cell, COS monkey kidney cell, CHO-S, R1 mouse embryonic cell, 3T3 mouse fibroblast cell, BHK21 golden hamster fibroblast cell, MDCK canine epithelial cell, PtK1 mouse kangaroo epithelial cell, E14.1 mouse embryonic cell, H1 human embryonic cell, H9 human embryonic cell and PER C.6 human embryonic cell. In some preferred embodiments, the mammalian cell is selected from CHO cell and / or HEK293 human embryonic kidney cell. In another preferred embodiment, the host cell is CHO-K1 cell.

[0046] In the fifth aspect of the present invention, there is provided a method for producing the dual-function fusion protein provided by the first aspect of the present invention. Those skilled in the art can select an appropriate method to produce the fusion protein. For example, the method for producing the multi-function fusion protein may include culturing an expression system provided by the fourth aspect of the present invention under appropriate conditions to express the fusion protein, collecting a culture containing the fusion protein, and further isolating and purifying the fusion protein to provide the fusion protein.

[0047] In a sixth aspect of the present invention, there is provided a use of the dual function fusion protein provided by the first aspect of the present invention in the manufacture of a medicament, the medicament being for treating a disease associated with the expression of VEGF and / or TGF-β, preferably the disease being one or more selected from tumors, autoimmune diseases and ophthalmological diseases.The dual function fusion protein provided by the present invention has good stability and at the same time has very good affinity with TGF-β and VEGF, and has similar antigen binding ability compared with the original drug Avastin and the control drug M7824, so that the dual function fusion protein can be applied to the treatment of diseases associated with the expression of VEGF and / or TGF-β.

[0048] The diseases associated with the expression of VEGF and / or TGF-β include tumors, specifically solid tumors or hematological tumors, more specifically colorectal cancer (CRC), hepatocellular carcinoma (HCC), ovarian cancer (OC), cervical cancer (CCA), thyroid cancer (TC), melanoma (MM), lung cancer (LC), head and neck cancer (HNC), nasopharyngeal carcinoma (NPC), glioblastoma tumor (GBM), prostate cancer (PCA). , pheochromocytoma-paraganglioma (PPGL), gastric cancer (GC), esophageal cancer (EC), renal cell carcinoma (RCC), bladder urothelial carcinoma (BLCA), pancreatic cancer (PAC), breast cancer (BRCA), lymphoma, sarcoma (SAR), acute or chronic leukemia, etc.

[0049] The disease associated with the expression of VEGF and / or TGF-β may be an autoimmune disease, and the autoimmune disease is one or more selected from systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, and polymyositis.

[0050] The disease associated with the expression of VEGF and / or TGF-β may be an ophthalmic disease, including, but not limited to, dry AMD, wet AMD, or choroidal neovascularization (CNV), such as age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), cystoid macular edema (CME), preretinal membrane (ERM) and macular hole, CNVM associated with myopia, pigmented streaks, retinal shedding, diabetic retinopathy, diabetic macular edema (DME), and the like. The present invention may be one or more selected from the group consisting of DME, atrophic or hypertrophic lesions of retinal pigment epithelium (RPE), retinal vein occlusion, choroidal retinal vein occlusion, macular edema, corneal neovascularization due to hypoxia, pterygium conjunctiva, subretinal edema and intraretinal edema, macular edema due to retinal vein occlusion, retinitis pigmentosa, Stargardt's disease, glaucoma, inflammatory diseases, cataracts, intractable abnormal phenomena, keratoconus, retinopathy of prematurity, anterior segment neovascularization, post-keratitis corneal neovascularization, corneal transplantation, and corneal molding.

[0051] In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising a dual function fusion protein as provided by the first aspect of the present invention or an expression system or a culture of an expression system as provided by the fourth aspect of the present invention. The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier. The carrier may include various excipients and diluents, but these carriers themselves are not essential active ingredients and are not excessively toxic after application. Suitable carriers will be familiar to those skilled in the art and can be found, for example, in Remington's Pharmaceutical A thorough discussion of pharma- ceutically acceptable carriers can be found in Sciences (Mack Pub. Co., NJ, 1991).

[0052] In the present invention, the dual-function fusion protein provided by the first aspect of the present invention may be used as the sole active ingredient in the above-mentioned drug or composition, or may be used in combination with other active ingredients.

[0053] In an eighth aspect of the invention, there is provided a method of treatment comprising administering to an individual a therapeutically effective dose of a dual function fusion protein provided by the first aspect of the invention, or a composition provided by the seventh aspect of the invention. The method of treatment provided by the invention may be used to treat diseases associated with expression of VEGF and / or TGF-β, such as in particular tumors.

[0054] In the antibody, use or pharmaceutical composition according to the present invention, the dual-function fusion protein may be used in combination with other means (standard tumor treatment methods / means) or drugs for tumor treatment (including standard tumor treatment methods / means), including but not limited to surgery, radiation therapy, chemotherapy, tumor immunotherapy, etc. When the dual-function fusion protein is used in combination with other drugs for tumor treatment, it may be formulated into a pharmaceutical composition or may be administered separately from other drugs for tumor treatment. When prepared into a pharmaceutical composition, the active ingredient of the pharmaceutical composition may contain other drugs for tumor treatment in addition to the dual-function fusion protein of the present invention, and the mass ratio of the dual-function fusion protein to other drugs for tumor treatment may be any suitable ratio, for example, 1: (1 to 99), specifically, for example, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85, 1:84, 1:83, 1:82, 1:81, 1:80, 1:79, 1:78, 1:77, 1:76, 1:75, 1:74, 1:73, 1:72, 1:71, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1 are also acceptable. The mass ratio of the other drug for tumor treatment and the dual-function fusion protein may be any suitable ratio, for example, 1: (1 to 99), specifically, for example, 1:99, 1:98, 1:97, 1:96, 1:95, 1:94, 1:93, 1:92, 1:91, 1:90, 1:89, 1:88, 1:87, 1:86, 1:85 ...8, :84, 1:83, 1:82, 1:81, 1:80, 1:79, 1:78, 1:77, 1:76, 1:75, 1:74, 1:73, 1:72, 1:71, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57 ,1:56,1:55,1:54,1:53,1:52,1:51,1:50,1:49,1:48,1:47,1:46,1:45,1:44,1:43,1:42,1:41,1:40,1:39,1:38,1:37,1:36,1:35,1:34,1:33,1:32,1:31,1:30,1: 29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1. Said separate administration may be before, simultaneously with or after administration of the bifunctional fusion antibody according to the present invention, other drugs for tumor treatment.

[0055] For tumor radiation therapy, common types of radiation include, but are not limited to, X-rays, gamma rays, and charged particles such as electrons, protons, and heavy ions.

[0056] Regarding chemotherapy of tumors, the common chemotherapy drugs are: (1) cytotoxic drugs: alkylating agents such as nitrogen mustard, carmustine (Gliadel), cyclophosphamide, busulfan (Myleran), lomustine (CCNU), etc.; (2) antimetabolites: fluorouracil, methotrexate, azacitidine, mercaptopurine, tegafur (Ftraful), etc.; (3) antibiotics: actinomycin D (dactinomycin), mitomycin, bleomycin, doxorubicin, pingyangmycin, daunorubicin, mitomycin, cytomegalovirus ... (4) Alkaloids: for example, vincristine, vinblastine, hydroxycamptothecin and podophyllotoxins, etoposide (VP-16), teniposide (VM-26); (5) Hormones: for example, tamoxifen, diethylstilbestrol, progesterone, testosterone propionate, thyroxine, prednisone and dexamethasone; (6) Others: for example, procarbazine, hydroxyurea, L-asparaginase, cisplatin, carboplatin, antimony aminotriacetate, dacarbazine, etc.

[0057] Tumor immunotherapy mainly includes immunomodulators, adoptive cell transfer therapy (ACT), tumor-specific vaccines, small molecule immuno-drugs, etc.

[0058] Here, immunomodulatory agents include costimulatory molecule agonists, immune checkpoint inhibitors (ICBs), immune checkpoint agonists, etc.

[0059] In some embodiments, the immunomodulatory agent is an agonist of a costimulatory molecule. In one embodiment, the costimulatory molecule agonist is selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or a CD83 ligand agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion protein).

[0060] In some embodiments, the immunomodulatory agent is an inhibitor (ICB) or agonist of an immune checkpoint molecule, and the immune checkpoint molecule is a molecule of the immune checkpoint stimulatory or inhibitory pathway. Immune checkpoint inhibitors are monoclonal antibody-based drugs against the corresponding immune checkpoint, and their main action is to block the interaction between tumor cells expressing immune checkpoints and immune cells, thereby blocking the inhibitory action of tumor cells on immune cells, thereby affecting the immune response. In one embodiment, the monoclonal antibody molecule is a complete antibody or a fragment thereof (e.g., Fab, F(ab')2, Fv, or single chain Fv fragment (scFv)). In another embodiment, the monoclonal antibody molecule has a heavy chain constant region (Fc), for example, selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In one embodiment, the constant region is altered (e.g., mutated) to modify the properties of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0061] In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, PD-L3, CTLA4, TIM-3, LAG3, CEACAM (such as CEACAM-1, CEACAM-3 and / or CEACAM-5), VISTA, VSIR, BTLA, TIGIT, LAIR1, LMTK3, TIGHT, IDO, CD27L, CD40, CD47, CD137, CD160, CD244, CD270, GITR, B7-H1, B7-1, 2B4 and / or TGF-βR. In one embodiment, the immune checkpoint inhibitor can inhibit PD-1, PD-L1, PD-L2, PD-L3, CTLA4, TIM-3, LAG3, CEACAM (such as CEACAM-1, CEACAM-3 and / or CEACAM-5), VISTA, VSIR, BTLA, TIGIT, LAIR1, LMTK3, TIGHT, IDO, CD27L, CD40, CD47, CD137, CD160, CD244, CD270, GITR, B7-H1, B7-1, 2B4 and / or TGFRβ, or any combination thereof. In another embodiment, the immune checkpoint inhibitor can inhibit PD-1, PD-L1, LAG-3, TIM-3, CEACAM (such as CEACAM-1, -3 and / or -5) or CTLA4, or any combination thereof. In another embodiment, the dual function fusion protein is used in combination with an immune checkpoint inhibitor capable of inhibiting PD-1 and / or PD-L1. The term "inhibition" or "inhibitor" includes inhibiting the activity of a given molecule, e.g., PD-1 or PD-L1 activity, by at least 5%, 10%, 20%, 30%, 40% or more. Thus, inhibition does not necessarily have to be 100%.

[0062] Here, adoptive cell transfer therapy (ACT) includes tumor-infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) therapy, chimeric antigen receptor T cell (CAR-T) therapy, and NK cell therapy.

[0063] Here, the tumor-specific vaccine includes a tumor whole cell vaccine, a dendritic cell vaccine, a gene vaccine, etc. These include vaccines that target specific antigens, RNA vaccines and protein polypeptide vaccines.

[0064] Tumor whole cell vaccines are made by treating autologous or allogeneic tumor cells with physical, chemical, biological or other methods to turn them into tumor vaccines that have lost their carcinogenicity but retain their antigenicity, and then combining them with non-specific stimulatory factors (e.g., BCG vaccine) to provide active immunotherapy to tumor patients.

[0065] Dendritic cell vaccine: Dendritic cells are heterogeneous immune cells that have the strongest antigen-presenting function and are the only specialized antigen-presenting cells that can activate naive T cells.

[0066] Gene vaccines are the products of cloning gene fragments encoding conserved antigens of pathogens into expression vectors and transfecting them into cells or eukaryotic and prokaryotic microorganisms, or of deleting virulence-related genes of pathogens to produce gene-deleted vaccines that do not have virulence-related genes. They mainly include viral vector vaccines and bacterial vector vaccines. Many viruses, such as adenoviruses and poxviruses, are used as carriers for recombinant vaccines, and bacterial vectors are mainly Listeria and Salmonella.

[0067] Protein polypeptide vaccine: A vaccine produced by chemical synthesis technology based on the amino acid sequence of a known or predicted antigen epitope in a pathogen antigen gene. If a specific antigen in a tumor cell is found and a cancer vaccine can be developed based on the amino acid sequence, it can activate the relevant immune cells to kill tumor cells with the same antigen, for example, EGF vaccine (Cimavax), MAGE-A3, TG4010, etc.

[0068] As used herein, sequence identity refers to the percentage of similar residues in the sequences involved in the alignment. The sequence identity of two or more sequences can be calculated using software well known in the art, and such software can be obtained from sources such as NCBI.

[0069] In the present invention, an "individual" generally includes humans, non-human primates, and other mammals, such as murines, canines, felines, equines, ovines, porcines, bovines, etc., that may benefit from treatment with the above-described drugs, compositions, formulations, kits, or combination formulations.

[0070] In the present invention, the term "therapeutically effective dose" generally means a dose that can achieve the effect of treating the above-listed diseases after an appropriate administration period.

[0071] The dual-function fusion protein provided by the present invention can simultaneously and effectively antagonize VEGF and TGF-β, and the stability of the modified protein is significantly improved, and it is not prone to spontaneous cleavage during purification, which significantly reduces the difficulty of protein purification under the premise of guaranteed efficacy, improves purification efficiency, and reduces production costs.In addition, the VEGF / TGF-β dual-function fusion protein provided by the present invention also has the advantages of high efficacy and low side effects, and has good prospects for industrialization.

[0072] The present invention will be further described below with reference to examples, but the scope of the present invention is not limited thereto.

[0073] Unless otherwise stated, all of the experimental, detection and manufacturing methods disclosed in the present invention employ conventional techniques in molecular biology, biochemistry, chromatin construction and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields, which are fully described in the existing literature, in particular Sambrook et al., MOLECULAR CLON ING:A LABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989 and Third edition,2001;Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;the series METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATIN STRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS IN ENZYMOLOGY,Vol.304,Chromatin(PMWassarman and See, for example, AP Wolfe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999.

[0074] Example 1. Preparation of a dual-function fusion protein A polynucleotide encoding the heavy chain (SEQ ID NO: 4) and a polynucleotide encoding the light chain (SEQ ID NO: 1) of the dual-function fusion protein were co-transduced into CHO-K1 expression host cells using the same expression vector, and high-expressing clones were pressure screened using methionine sulfoximine (MSX). The high-expressing clones obtained by screening were mass-cultured in EmCD CHO 104 medium (Eminence) at 37°C and 5% CO2 conditions for inducible expression. After culturing for 12 to 14 days, the cell culture medium was collected.

[0075] Example 2 Purification of dual function fusion proteins The cell culture fluid collected in Example 1 was centrifuged and centrifuged at high speed, and then purified by affinity chromatography column. Unimab 50HC purification filler from Suzhou NanoMicro Technology Co., Ltd. and AKTA (GE healthcare) protein purification system were used, and the dosage was 30 mg protein / ml medium. The equilibration buffer was 50 mM Tris-HCl + 150 mM NaCl solution at pH 7.5, and the elution buffer was 20 mM Na-Citrater solution at pH 3.0. The flow rate was 5 ml / min. After collecting the eluate containing the target protein, the pH was adjusted to 7.0, and the physical and chemical properties and biological activity were detected and identified.

[0076] Example 3 Production and purification of control fusion proteins The amino acid sequence of the control fusion protein comprises the heavy chain sequence shown in SEQ ID NO:8 and the light chain sequence shown in SEQ ID NO:7.

[0077] The manufacturing process is the same as in Example 1, and the purification process is the same as in Example 2.

[0078] Example 4 Identification of dual-function fusion proteins by SDS-PAGE electrophoresis 4.1 Determination of molecular weight and purity of dual-function fusion proteins by SDS-PAGE electrophoresis

[0079] The two compounds obtained in Example 2 were synthesized under reducing (β-mercaptoethanol) and non-reducing conditions. The dual-function fusion proteins were identified by SDS-PAGE electrophoresis, and electrophoresis was performed on polyacrylamide gels with separation gel concentrations of 12% and 8%, respectively, and the loading amount of each well was 3 μg of protein. The electrophoresis results are shown in Figure 1 (reduced SDS-PAGE) and Figure 2 (non-reduced SDS-PAGE), and lanes 1, 2, and 3 are the duplicate samples of the three dual-function fusion proteins targeting VEGF and TGF-β, respectively. The electrophoresis results show that the molecular weight of the target protein is about 179 kD and the purity is more than 98%.

[0080] 4.2 Determining the stability of dual-function fusion proteins by SDS-PAGE electrophoresis

[0081] Under the condition of reduction (β-mercaptoethanol), the dual-function fusion protein obtained in Example 2 and the control fusion protein obtained in Example 3 were identified by SDS-PAGE electrophoresis, and electrophoresis was performed on polyacrylamide gels with separation gel concentrations of 12% and 8%, respectively, and the loading amount of each well was 3 μg of protein. The electrophoresis results are shown in FIG. 3 (reduced SDS-PAGE), where lanes 1 and 2 are the control fusion protein obtained in Example 3 and the dual-function fusion protein obtained in Example 2, respectively. From FIG. 3, it can be seen that the control fusion protein, i.e., the protein without K→A mutation, is easily cleaved (there is obvious cleaved protein at the position marked by the arrow), while the dual-function fusion protein containing K→A mutation did not have obvious cleaved protein at the position corresponding to the arrow in lane 1. It was shown that the dual-function fusion protein obtained in Example 2 of the present invention has very high stability.

[0082] Example 5 ELISA experiment of recombinant human TGF-β1 / 2 / 3 ELISA experiment according to standard process: 5.1 96-well plates were coated with recombinant human TGF-β1, TGF-β2 or TGF-β3 dissolved in a solution containing 15 mmol / L Na2CO3, 35 mmol / L NaHCO3 and 7.7 mmol / L NaN3 at pH 9.6, and allowed to coat overnight at room temperature, with the coating amount in each well being 0.5 μg.

[0083] 5.2 The next day, the 96-well plate was washed four times with TBS solution containing 0.05% Tween-20 at pH 7.4, and then blocked with TBS solution containing 0.05% Tween-20 and 5% skim milk powder at pH 7.4 at room temperature for 1 hour. After removing the blocking solution, the well plate was loaded with different concentrations of dual-function fusion protein or control drug M7824, and incubated in duplicate wells at room temperature for 2 hours.

[0084] 5.3 Then, the well plate was washed four times with washing solution, and anti-human IgG (Fc specific)-peroxidase goat antibody was added and incubated at room temperature for 30 minutes in the dark. The color reaction was then stopped with 1M H2SO4.

[0085] 5.4 The absorbance was read at 450 nm by Envision reader and calibrated at 570 nm, detailed results are shown in Figures 4, 5 and 6.

[0086] 4, 5 and 6 show that the dual function fusion protein prepared in Example 1 has an affinity for TGF-β almost equivalent to that of M7824, and both have weak affinities for TGF-β2. The dual function fusion protein prepared in Example 1 and M7824 have affinity EC50s for TGF-β1 of 2.843 nM and 2.076 nM, respectively, and affinity EC50s for TGF-β3 of 10.309 nM and 3.323 nM, respectively.

[0087] Example 6 ELISA Experiments of Recombinant Human VEGF To verify whether the dual-function fusion protein prepared in Example 1 binds to human VEGF, recombinant human VEGF (Novoprotein) was coated on a 96-well plate and left to coat overnight at room temperature, with each well coated with 0.5 μg of recombinant human VEGF. The dual-function fusion protein prepared in Example 1 or the control drug Avastin was used in the experiment, and the other implementation methods and procedures were the same as those in Example 3. The specific results are shown in Figure 7.

[0088] From FIG. 7, it can be seen that the affinity of the dual function fusion protein prepared in Example 1 for VEGF was almost equivalent to that of Avastin, and the EC50 of the affinity for VEGF was 0.0659 nM and 0.0168 nM, respectively.

[0089] Example 7 ELISA experiment to simultaneously detect binding of TGF-β and VEGF In order to verify whether the dual-function fusion protein prepared in Example 1 can bind with recombinant human TGF-β1 / 2 / 3 and VEGF at the same time, recombinant human TGF-β1 / 2 / 3 and VEGF were coated on a 96-well plate and coated overnight at room temperature, and the coating amount of TGF-β1 / 2 / 3 and VEGF in each well was 0.5μg respectively. The dual-function fusion protein prepared in Example 1 or the control drug M7824, Avastin were used for the experiment, and other implementation methods and procedures were the same as those in Example 3, and the specific results are shown in Figure 8.

[0090] From Figure 8, it can be seen that the dual function fusion protein prepared in Example 1 simultaneously binds to recombinant human TGF-β and VEGF, with an EC50 of 0.0365nM. The dual function fusion protein of the present invention simultaneously binds to recombinant human TGF-β and VEGF, and simultaneously achieves the binding of VEGF and VEGF receptor and the inhibition of the regulatory effect of TGF-β on the immune system, thereby effectively inhibiting the biological effects of VEGF and TGF-β, including the effects on vascular permeability, proliferation, and migration and production of endothelial cells, improving the production and function of effector T cells and DC cells, and further inhibiting the growth and proliferation of tumors, thereby achieving anti-tumor effects.

[0091] Example 8: Experiment on the inhibition of VEGF-induced proliferation of human umbilical vein endothelial cells (HUVEC) by the dual-function fusion protein HUVEC cells were resuspended in complete medium, diluted, and then seeded at 5000 cells / well in a 96-well plate, and the culture plate was further incubated overnight in a 37°C, 5% CO2, humid incubator. The next day, the original medium was discarded, and ECM+1% FBS medium was added and starved for 24 h. Avastin, the dual-function fusion protein prepared in Example 1, and HSP088-01 (an isotype control protein that only retains the TGF-β fragment activity of the dual-function fusion protein prepared in Example 1) were diluted in a 3-fold gradient from an initial concentration of 10 nM, and then added to the plate wells containing the cells, and further incubated for 72 hours in a 37°C, 5% CO2, humid incubator. After removing the 96-well plate, add freshly prepared CCK-8 (10%) detection solution, incubate in a 37°C incubator for 2-4 hours, shake gently, and then measure the absorbance at a wavelength of 450 nm using a SpectraMax M5 instrument. The absorbance at 650 nm was used as a reference to calculate the inhibition rate (inhibition rate% = [1-(OD 薬物+VEGF -OD VEGF無し ) / (OD VEGFのみ -OD VEGF無し )] × 100) and the inhibition curve was fitted using Graphpad Prism 6 software to obtain the IC50 The values ​​were calculated and the results are shown in FIG.

[0092] The results showed that Avastin and the dual-function fusion protein prepared in Example 1 could effectively inhibit the proliferation of HUVEC cells induced by VEGF, and the IC 50 were 0.3869 nM and 0.1964 nM, respectively.

[0093] Example 9: Experiment of the dual-function fusion protein suppressing the migration of human breast cancer MDA-MB-231 cells induced by TGF-β MDA-MB-231 cells were cultured and subcultured in complete medium, and cells in the logarithmic growth phase were harvested. When the cells attached, they were starved in L15+1%FBS medium for 16 hours. Matrigel was diluted with serum-free L15 medium at a ratio of 1:2, then placed in a Transwell chamber and incubated at 37°C for 30 minutes. The starved MDA-MB-231 cells were resuspended in serum-free L15 medium and placed in each Matrigel-containing Transwell chamber, and then respectively placed in blank medium, the dual-function fusion protein prepared in Example 1, and HSP088-01 (an isotype control protein that only retains the TGF-β fragment activity of the dual-function fusion protein prepared in Example 1). After 1 h of incubation, the upper chamber was further filled with TGF-β stock solution to a final concentration of 0.23 nM (the blank medium group was filled with the same volume of medium alone), the total volume of the chamber was 400 μL (containing 200 μL of Matrigel), and the lower chamber contained 600 μL of fresh L-15 medium, and incubated at 37°C for 24 h. After incubation, the cells were fixed with paraformaldehyde for 20 min and stained with crystal violet for 20 min. After washing with PBS, the cells were photographed under an inverted microscope, and statistical analysis was performed on the cell counts using the software Image J. The results are shown in Figure 10 and Figure 11, respectively.

[0094] The results showed that TGF-β could induce the migration of MDA-MB-231 cells, and after the addition of the dual-function fusion protein prepared in Example 1 and HSP088-01, the number of MDA-MB-231 cells was obviously reduced, and with the increase in the concentration of the dual-function fusion protein prepared in Example 1, the inhibitory effect on MDA-MB-231 cell migration became stronger and stronger. The inhibitory effect of 100nM FS8002 on MDA-MB-231 cell migration was significantly different from the negative control containing only TGF-β (p<0.05 ** ).

[0095] Example 10: Experiment on the neutralizing effect of the dual-function fusion protein against TGF-β MDA-MB-231 cells were seeded in 6-well plates (1 × 10 6 After culturing at 37°C for 24 hours, the culture medium was replaced with PBS, PBS+TGF-β1 or TGF-β1 and different concentrations of the dual-function fusion protein prepared in Example 1 and HSP088-01 (isotype control protein that only retained the TGF-β fragment activity of the dual-function fusion protein prepared in Example 1) in different wells, and then cultured for 1 hour after the culture medium was replaced. The culture medium supernatant in the 6-well plate was sucked up, and the cells in the well plate were washed with D-PBS and then decomposed with a decomposition solution. The fully decomposed samples were immediately subjected to SDS-PAGE electrophoresis, and the signals of Smad2 / 3 and phosphorylation (p-Smad2 / 3) were detected by Western blotting. The experimental results are shown in Figure 12.

[0096] The results showed that when MDA-MB-231 cells were treated with TGF-β1 alone, p-Smad2 / 3 was detected by Western blot, illustrating that TGF-β1 can induce the canonical Smad signal pathway in cells by binding to TGF-β receptor (TGF-βR) in 231 cells. Simultaneously, TGF-β1 and different concentrations of the dual function fusion protein prepared in Example 1 and HSP088-01 were treated with When HSP088-01 was added to the 231 cells, p-Smad2 / 3 was not detected by Western blot, indicating that both the dual function fusion protein prepared in Example 1 and HSP088-01 neutralized TGF-β1 and blocked the binding of TGF-β1 to TGF-βR in 231 cells, thereby blocking Smad downstream signaling. It was also shown that the dual function fusion protein prepared in Example 1 can effectively neutralize TGF-β1 at a low concentration of 1 nM.

[0097] Example 11 Tumor suppression experiment in mice using dual-function fusion protein 1 × 10 MDA-MB-231 cells resuspended in PBS 7 A total of 100 mice were inoculated subcutaneously into the right flank of BALB / c nude mice at a concentration of 0.1 mL / cell and 0.1 mL (containing 30% Matrigel) / mouse. 22 days after tumor formation, mice without tumors and mice with the largest and smallest tumor volumes were selected, and 70 mice with appropriate tumor volumes were selected. The mice were divided into 10 groups and were randomly selected to have a mean tumor volume of 70 mm. 3 The administration was started on the day of grouping, and the dual-function fusion protein prepared in Example 1 or the control drug was injected twice a week for a total of 4 weeks. During the experiment, the tumor volume and the body weight of the tumor-bearing mice were measured and recorded.

[0098] Here, the grouping is as follows:

[0099] Group 1 (G1): control group (using saline alone).

[0100] Group 2 (G2): Use the control drug bevacizumab alone.

[0101] Group 3 (G3): Control drug M7824 used alone.

[0102] Group 4 (G4): Use the control drug avelumab alone.

[0103] Group 5 (G5): The control drugs bevacizumab and avelumab will be used in combination.

[0104] Group 6 (G6): The dual function fusion protein prepared in Example 1 is used alone.

[0105] Group 7 (G7): Combine the dual-function fusion protein prepared in Example 1 with avelumab.

[0106] The results are shown in FIG. 13. The experimental data of the mice were analyzed and compiled, and a spider plot was created with the administration time as the abscissa and the tumor volume change rate as the ordinate. It can be seen that the use of the dual-function fusion protein prepared in Example 1 alone in G6 and the combination of the dual-function fusion protein prepared in Example 1 + avelumab in G7 showed a clear inhibitory effect compared to the average tumor volume of mice in the control group G1. It can be seen that the use of the dual-function fusion protein prepared in Example 1 alone in G6 significantly improved the inhibitory effect on the tumor volume of mice compared to the use of the control drug bevacizumab alone in G2. The combination of the dual-function fusion protein prepared in Example 1 + avelumab in G7 significantly improved the inhibitory effect on the tumor volume of mice compared to the combination of the control drug bevacizumab and avelumab in G5, and it can be seen that in this group, the tumors in the body of four mice completely disappeared after administration. Overall, the animal experiments in this example showed that the dual-function fusion protein prepared in Example 1 exhibited good inhibitory effect on MDA-MB-231 tumor cells in BALB / c nude mice, and showed great potential in combination with other immune checkpoint inhibitors.

[0107] As described above, the present invention effectively overcomes various shortcomings in the existing technology and achieves high productivity. It has industrialization and commercial value.

[0108] Sequence information: SEQ ID NO:1 Light chain: JPEG2025508334000001.jpg31159

[0109] SEQ ID NO:2 Heavy Chain: JPEG2025508334000002.jpg62160

[0110] SEQ ID NO:3 JPEG2025508334000003.jpg25156

[0111] SEQ ID NO:4 Heavy Chain: JPEG2025508334000004.jpg81161

[0112] SEQ ID NO:5 JPEG2025508334000005.jpg10161

[0113] SEQ ID NO:6 Heavy Chain: JPEG2025508334000006.jpg64161

[0114] SEQ ID NO:7 Heavy Chain: JPEG2025508334000007.jpg21162

[0115] SEQ ID NO:8 Heavy Chain: JPEG2025508334000008.jpg83162

[0116] The above examples are merely illustrative and are intended to explain the principles and effects of the present invention, and are not intended to limit the present invention. Those familiar with the art may modify and alter the above examples as long as they do not deviate from the spirit and scope of the present invention. Therefore, those with ordinary skill in the art may make modifications or alterations that have equivalent effects within the scope of the spirit and technical ideas disclosed in the present invention, and these modifications or alterations are included in the scope of the claims of the present invention.

Claims

1. a VEGF antagonistic fragment and a TGF-β antagonistic fragment, A dual function fusion protein, characterized in that the VEGF antagonist fragment comprises a heavy chain that includes at least an amino acid mutation at position 453 compared to the original heavy chain, and / or the TGF-β antagonist fragment comprises at least an amino acid mutation at position 8 compared to the original TGF-β antagonist fragment.

2. The dual function fusion protein described in claim 1, characterized in that the amino acid sequence of the original heavy chain is shown in SEQ ID NO: 6 and / or the amino acid sequence of the original TGF-β antagonistic fragment is shown in SEQ ID NO:

7.

3. The dual function fusion protein of claim 1, characterized in that the 453rd amino acid mutation comprises an amino acid deletion or substitution, and / or the 8th amino acid mutation comprises an amino acid deletion or substitution.

4. The dual function fusion protein of claim 3, wherein the mutation at amino acid 453 is a deletion mutation and the mutation at amino acid 8 is a K→A mutation.

5. The heavy chain of the VEGF antagonistic fragment a-1) a polypeptide fragment having an amino acid sequence represented by SEQ ID NO: 2, or b-1) a polypeptide fragment having an amino acid sequence having 90% or more sequence identity with SEQ ID NO:2 and including a deletion mutation at amino acid position 453, and having the function of the polypeptide fragment defined in a-1); The dual function fusion protein of claim 4, comprising:

6. The TGF-β antagonistic fragment is c) a polypeptide fragment having the amino acid sequence set forth in SEQ ID NO:3, or d) a polypeptide fragment having an amino acid sequence having 90% or more sequence identity with SEQ ID NO:3 and including a K→A mutation at the 8th amino acid, and having the function of the polypeptide fragment defined in c); The dual function fusion protein of claim 4, comprising:

7. The VEGF antagonistic fragment comprises a light chain, the light chain comprising a-1) a polypeptide fragment having an amino acid sequence set forth in SEQ ID NO: 1, or b-1) a polypeptide fragment having an amino acid sequence having 90% or more sequence identity with SEQ ID NO:1 and having the function of the polypeptide fragment defined in a-1); The dual function fusion protein according to any one of claims 1 to 6, comprising:

8. The dual function fusion protein of claim 1, further comprising a linked peptide fragment, preferably the linked peptide fragment is rich in G, S and / or A, more preferably the linked peptide fragment comprises a polypeptide fragment having an amino acid sequence set forth in SEQ ID NO:

5.

9. The dual-function fusion protein according to claim 1, which comprises, from the N-terminus to the C-terminus, a VEGF antagonistic fragment and a TGF-β antagonistic fragment.

10. The dual-function fusion protein according to claim 1, wherein the amino acid sequence of the dual-function fusion protein comprises a heavy chain sequence shown in SEQ ID NO:4 and a light chain sequence shown in SEQ ID NO:

1. Quality.

11. An isolated polynucleotide encoding the dual function fusion protein of any one of claims 1 to 10.

12. A construct comprising the isolated polynucleotide of claim 11.

13. An expression system containing the construct of claim 12 or having an exogenous polynucleotide of claim 8 integrated into its genome.

14. A method for producing the dual-function fusion protein according to any one of claims 1 to 10, comprising culturing the expression system according to claim 13 under suitable conditions, expressing the dual-function fusion protein, and isolating and purifying the dual-function fusion protein to provide the dual-function fusion protein.

15. Use of the dual function fusion protein according to any one of claims 1 to 10 in the manufacture of a medicament.

16. The use according to claim 15, characterized in that the drug is used for the treatment of a disease associated with the expression of VEGF and / or TGF-β, preferably the disease is one or more selected from tumors, autoimmune diseases and ophthalmological diseases.

17. The use according to claim 15, characterized in that the dual-function fusion protein is combined with other measures of tumor treatment, the other measures of tumor treatment including surgery, radiation therapy, chemotherapy and tumor immunotherapy.

18. The types of radiation in said radiotherapy include X-rays, gamma rays and charged particles; The drugs used in the chemotherapy are: (1) cytotoxic drugs: nitrogen mustard, carmustine, cyclophosphamide, busulfan, lomustine; (2) antimetabolic drugs: fluorouracil, methotrexate, azacytidine, mercaptopurine, tegafur; (3) antibiotics: actinomycin D, mitomycin, bleomycin, doxorubicin, pingyangmycin, daunorubicin, mithramycin; and (4) alkaloids: bin (5) hormones: tamoxifen, diethylstilbestrol, progesterone, testosterone propionate, thyroxine, prednisone and dexamethasone; (6) others: one or more selected from procarbazine, hydroxyurea, L-asparaginase, cisplatin, carboplatin, antimony aminotriacetate and dacarbazine; The tumor immunotherapy is one or more selected from the group consisting of immunomodulators, adoptive cell transfer therapy, tumor-specific vaccines, and small molecule immunopharmaceuticals; Here, the immunomodulatory agent is one or more selected from a costimulatory molecule agonist, an immune checkpoint inhibitor, and an immune checkpoint agonist, and the costimulatory molecule agonist is OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NK p80, CD160, B7-H3, or CD83 ligand agonists, and the immune checkpoint inhibitor is one or more selected from PD-1, PD-L1, PD-L2, PD-L3, CTLA4, TIM-3, LAG3, CEACAM-1, CEACAM-3, CEACAM-5, VISTA, VSIR, BTLA, TIGIT, LAIR1, LMTK3, TIGHT, IDO, CD27L, CD40, CD47, CD137, CD 160, CD244, CD270, GITR, B7-H1, B7-1, 2B4 and / or TGF-βR inhibitors; wherein the adoptive cell transfer therapy is one or more selected from tumor infiltrating lymphocyte therapy, engineered T cell receptor therapy, chimeric antigen receptor T cell therapy, and natural killer cell therapy; The use according to claim 17, characterized in that the tumor-specific vaccine is one or more selected from the group consisting of a tumor whole cell vaccine, a dendritic cell vaccine, a gene vaccine, an RNA vaccine and a protein polypeptide vaccine.

19. A pharmaceutical composition comprising a dual function fusion protein according to any one of claims 1 to 10 or an expression system according to claim 13 or a culture of an expression system according to claim 13.

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