A bifunctional fusion protein and its preparation method and application

Abstract

The present invention relates to the field of biotechnology, and discloses a bifunctional fusion protein, a preparation method thereof, and an application thereof. The bifunctional fusion protein comprises an antagonistic VEGF fragment and an antagonistic TGF-β fragment, the antagonistic VEGF fragment comprises SEQ ID No.1 and 2 or a polypeptide fragment having more than 90% sequence identity therewith, and the antagonistic TGF-β fragment comprises SEQ ID No.3 or a polypeptide fragment having more than 90% sequence identity therewith. The present invention also provides a preparation method of the bifunctional fusion protein, and its use in the preparation of a drug for treating diseases related to VEGF and / or TGF-β expression. The bifunctional fusion protein provided by the present invention is not prone to breakage during the purification process and has good stability; at the same time, it can improve purification efficiency and reduce production costs during the production process, and has broad application prospects and good commercial value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a bifunctional fusion protein and a preparation method and application thereof. Background Art

[0002] Within the tumor microenvironment, transforming growth factor-β (TGF-β) plays a crucial role in immunosuppression. TGF-β regulates the production and function of many immune cell types. It controls adaptive immunity by directly promoting the proliferation of Treg cells and inhibiting the production and function of effector T cells (Teff) and antigen-presenting dendritic cells (DC). Similarly, TGF-β controls the innate immune system by inhibiting natural killer cells (NK cells) and regulating the complex behavior of macrophages and neutrophils, thereby forming a negative immune regulatory input network. Research has shown that angiogenesis is a key factor in the growth of malignant tumors. Inhibition of vascular endothelial growth factor (VEGF), a highly specific factor that promotes vascular endothelial cell growth, can effectively inhibit tumor growth. Anti-angiogenic drugs, represented by VEGF antibodies, have become a hot area of research and development for targeted anti-tumor drugs due to their high specificity, efficacy, and resistance to drug resistance.

[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), thereby inhibiting the biological effects of VEGF, including affecting vascular permeability, proliferation, and endothelial cell migration and survival, thereby inhibiting tumor angiogenesis, growth, and metastasis. Currently, the combination of various small and large molecule VEGF antagonists with PD-1 or PD-L1 has demonstrated remarkable efficacy in various cancers, including advanced liver cancer, and has become an important cancer combination therapy.

[0004] A negative inhibitory tumor microenvironment is widely recognized as a major factor hindering and limiting the effective clinical efficacy of immune checkpoint drugs, including programmed cell death receptor 1 (PD-1) and programmed cell death ligand 1 (PD-L1). The negative impact of the tumor microenvironment is particularly prominent in solid tumors, making it a key focus of new drug development in terms of both response rate and drug resistance. For example, M7824, a bifunctional fusion protein developed by Merck Serono, a subsidiary of Merck, consists of an anti-PD-L1 monoclonal antibody fused to the extracellular domain of human TGF-β receptor II (TGF-βRII), capable of simultaneously antagonizing PD-L1 and TGF-β. M7824 demonstrated promising activity in early clinical studies but failed to demonstrate superiority in Phase III clinical trials. This suggests that inhibiting TGF-β alone is not sufficient to overcome the negative regulation of the tumor microenvironment.

[0005] Furthermore, existing bifunctional fusion proteins containing the TGF-β receptor, such as Merck's M7824, are subject to fragmentation during purification due to the inherent physiological properties of the TGF-β receptor. These fragments significantly reduce purification efficiency, increase production costs, and ultimately affect the efficacy of the resulting drug. Furthermore, the clinical dose of PD-L1 antibodies is much higher than that of PD-1 antibodies, further increasing the difficulty of M7824's clinical development and limiting its future clinical application. Summary of the Invention

[0006] In view of the shortcomings of the existing theories and technologies described above, the purpose of the present invention is to provide a bifunctional fusion protein and its preparation method and application, which can be used to solve the problems in the existing technology, and at the same time provide a new technical route for efficiently overcoming the tumor microenvironment and enhancing the efficacy of combined treatment with PD-1 or PD-L1.

[0007] One aspect of the present invention is to provide a bifunctional fusion protein comprising an antagonistic VEGF fragment and an antagonistic TGF-β fragment;

[0008] The antagonistic VEGF fragment includes a heavy chain, and the heavy chain includes at least an amino acid mutation at position 453 compared to the original heavy chain; and / or the antagonistic TGF-β fragment includes at least an amino acid mutation at position 8 compared to the original antagonistic TGF-β fragment.

[0009] Another aspect of the present invention is to provide an isolated polynucleotide encoding the bifunctional fusion protein as described above.

[0010] Another aspect of the present invention is to provide a construct comprising the isolated polynucleotide as described above.

[0011] Another aspect of the present invention is to provide an expression system, which comprises the construct as described above or an exogenous polynucleotide as described above integrated into the genome.

[0012] Another aspect of the present invention is to provide a method for preparing the bifunctional fusion protein as described above, comprising: culturing the expression system as described above under appropriate conditions to express the bifunctional fusion protein, and isolating and purifying to provide the bifunctional fusion protein.

[0013] Another aspect of the present invention provides the use of the bifunctional fusion protein described above in the preparation of a medicament. The medicament is used to treat diseases associated with VEGF and / or TGF-β expression, preferably for treating tumors. The tumor treatment includes inhibiting the formation, growth, and metastasis of tumor blood vessels; inhibiting the formation, proliferation, metastasis, and infiltration of tumor cells; and promoting tumor cell apoptosis.

[0014] Another aspect of the present invention is to provide a pharmaceutical composition comprising the bifunctional fusion protein as described above, or the expression system as described above, or the culture of the expression system as described above.

[0015] In the present invention, the bifunctional fusion protein and the biomaterials related to the bifunctional fusion protein can be artificially synthesized, or the encoding gene can be synthesized and then biologically expressed. In one specific embodiment, the bifunctional fusion protein and the biomaterials related to the bifunctional fusion protein are produced by introducing the gene into Escherichia coli, yeast, or mammalian cells such as CHO cells or HEK293 cells, and expressing the gene in the Escherichia coli, yeast, or mammalian cells.

[0016] The beneficial effects of the present invention include:

[0017] 1) The present invention adjusts the antagonist VEGF fragment by deleting and mutating the K amino acid residue at position 453 of its heavy chain, and adjusts the sequence of the antagonist TGF-β fragment by mutating the K amino acid residue at position 8 of its amino acid sequence to A (K→A mutation). The resulting bifunctional fusion protein has good stability, is less likely to break during the purification process, can improve purification efficiency, and reduce production costs during the production process; the bifunctional fusion protein purified by the present invention has a molecular weight of approximately 179 kD and a purity greater than 98%.

[0018] 2) The bifunctional fusion protein of the present invention has very good affinity and can bind to recombinant human TGF-β1 / 2 / 3 with an affinity substantially equivalent to that of M7824; the bifunctional fusion protein can bind to recombinant human VEGF with an affinity substantially equivalent to that of Avastin; in addition, the bifunctional fusion protein of the present invention can simultaneously bind to recombinant human TGF-β1 / 2 / 3 and Avastin; therefore, the bifunctional fusion protein of the present invention can be used to treat diseases related to VEGF and / or TGF-β expression.

[0019] 3) In vivo anti-tumor efficacy experiments showed that the bifunctional fusion protein of the present invention can effectively inhibit the growth of mouse tumors and can be used for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shown is a schematic diagram of the results of the reduction electrophoresis identification experiment of the bifunctional fusion protein in Example 4 of the present invention.

[0021] Figure 2 Shown is a schematic diagram of the results of the non-reducing electrophoresis identification experiment of the bifunctional fusion protein in Example 4 of the present invention.

[0022] Figure 3 Shown are the results of reducing electrophoresis to identify the stability of the bifunctional fusion protein in Example 4 of the present invention.

[0023] Figure 4 Shown is a schematic diagram of the TGF-β1 ELISA experimental results in Example 5 of the present invention.

[0024] Figure 5 Shown is a schematic diagram of the TGF-β2 ELISA experimental results in Example 5 of the present invention.

[0025] Figure 6 Shown is a schematic diagram of the TGF-β3 ELISA experimental results in Example 5 of the present invention.

[0026] Figure 7 Shown is a schematic diagram of the VEGF ELISA experimental results in Example 6 of the present invention.

[0027] Figure 8 It is a schematic diagram showing the results of the ELISA experiment for simultaneously detecting the binding of TGF-β and VEGF in Example 7 of the present invention.

[0028] Figure 9 Shown is a schematic diagram of the experimental results of inhibiting VEGF-induced HUVEC proliferation in Example 8 of the present invention.

[0029] Figure 10Shown is a schematic diagram of the experimental results of inhibiting TGF-β-induced human breast cancer MDA-MB-231 cell migration in Example 9 of the present invention.

[0030] Figure 11 Shown is a schematic diagram of the statistical results of the experiment on inhibiting TGF-β-induced migration of human breast cancer MDA-MB-231 cells in Example 9 of the present invention.

[0031] Figure 12 Schematic diagram showing the experimental results of the neutralization effect of the bifunctional fusion protein on TGF-β in Example 10 of the present invention, wherein Control is PBS; Control 2 is PBS+TGF-β1; Control 3 is 10nM HSP088-01+TGF-β1; Test 1 is 1nM bifunctional fusion protein+TGF-β1; Test 2 is 10nM bifunctional fusion protein+TGF-β1; Test 3 is 100nM bifunctional fusion protein+TGF-β1.

[0032] Figure 13 Schematic diagram showing the results of the in vivo tumor inhibition experiment in mice using the bifunctional fusion protein in Example 11 of the present invention. The curves in each figure represent the tumor growth curve of each animal in the experimental group. DETAILED DESCRIPTION

[0033] In order to make the purpose of the invention, technical solutions and beneficial technical effects of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. People familiar with this technology can easily understand other advantages and effects of the invention of this application from the contents disclosed in this specification.

[0034] After extensive practical research, the inventors of the present invention used gene-directed mutagenesis technology to mutate the VEGF and TGF-β receptors in the fusion protein, thereby significantly improving the problem of peptide bond cleavage during protein purification and reducing the difficulty of purification. They thus prepared a bifunctional fusion protein that is highly stable and can simultaneously inhibit VEGF and TGF-β, and completed the present invention on this basis.

[0035] In a first aspect, the present invention provides a bifunctional fusion protein comprising an antagonist VEGF fragment and an antagonist TGF-β fragment. The antagonist VEGF fragment in the bifunctional fusion protein is typically a fragment that has undergone genetic mutation, for example, any known antagonist VEGF fragment. The antagonist VEGF fragment may be a VEGF receptor fragment. The antagonist TGF-β fragment may be a fragment that has undergone genetic mutation, for example, any known antagonist TGF-β fragment.

[0036] The antagonistic VEGF fragment comprises a heavy chain, and the heavy chain comprises at least an amino acid mutation at position 453 compared to the original heavy chain.

[0037] The antagonistic TGF-β fragment comprises at least an amino acid mutation at position 8 compared to the original antagonistic TGF-β fragment.

[0038] Preferably, the amino acid mutation at position 453 of the heavy chain of the antagonist VEGF fragment comprises an amino acid deletion or substitution. The substitution refers to replacing the amino acid residue K at position 453 with another non-K amino acid 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.

[0039] Preferably, the amino acid mutation at position 8 of the antagonist TGF-β fragment comprises an amino acid deletion or substitution. The substitution refers to replacing the amino acid residue K at position 8 with another non-K amino acid or its derivative, such as a 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 amino acid mutation at position 8 of the antagonist TGF-β fragment refers to a K to A mutation (K→A).

[0040] Preferably, the bifunctional fusion protein of the present invention comprises at least one of the aforementioned mutations.

[0041] Preferably, the amino acid sequence of the original heavy chain is shown as SEQ ID No.6.

[0042] Preferably, the amino acid sequence of the original antagonistic TGF-β fragment is shown as SEQ ID No.7.

[0043] In some preferred embodiments, the bifunctional fusion protein comprises a deletion mutation of the amino acid at position 453 and a K→A mutation of the amino acid at position 8.

[0044] In some preferred embodiments, the heavy chain of the antagonist VEGF fragment may specifically include:

[0045] a-2) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 2; or

[0046] b-2) A polypeptide fragment having an amino acid sequence with greater than 90% sequence identity to SEQ ID No. 2, comprising 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) refers to a polypeptide fragment obtained by substituting, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids from the amino acid sequence of SEQ ID No. 2, or by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, and having the function of the polypeptide fragment of SEQ ID No. 2, for example, 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 more than 99% with SEQ ID No. 2.

[0047] In some preferred embodiments, the antagonist TGF-β fragment may specifically include:

[0048] c) a polypeptide fragment whose amino acid sequence is shown in SEQ ID No. 3; or

[0049] d) a polypeptide fragment having an amino acid sequence with greater than 90% sequence identity to SEQ ID No. 3, comprising a K8A mutation (K to A) at amino acid residue 8, and having the function of a polypeptide fragment as defined in c). Specifically, the polypeptide fragment in d) refers to a polypeptide fragment obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the amino acid sequence as set forth in SEQ ID No. 3, or by addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, and having the function of a polypeptide fragment as set forth in SEQ ID No. 3, for example, antagonizing TGF-β. The amino acid sequence in d) may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% with SEQ ID No. 3.

[0050] In some preferred embodiments, the antagonist VEGF fragment further comprises a light chain. The light chain may specifically include:

[0051] a-1) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 1; or

[0052] b-1) A polypeptide fragment having an amino acid sequence with more than 90% 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) refers to a polypeptide fragment obtained by substituting, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids from the amino acid sequence of SEQ ID No. 1, or by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, and having the function of the polypeptide fragment of the amino acid sequence of SEQ ID No. 1, for example, antagonizing VEGF. The amino acid sequence in b) may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% with SEQ ID No. 1.

[0053] The bifunctional fusion protein provided by the present invention may further include a connecting peptide fragment. Preferably, the connecting peptide fragment is rich in G, S, and / or A, wherein "rich in G, S, and / or A" means rich in G, S, and A, or rich in G and S, or rich in G and A. More preferably, the connecting peptide fragment includes a polypeptide fragment whose amino acid sequence may be as shown in SEQ ID NO. 5.

[0054] In the bifunctional fusion protein provided by the present invention, the bifunctional fusion protein may include an antagonistic VEGF fragment and an antagonistic TGF-β fragment in sequence from the N-terminus to the C-terminus.

[0055] In a specific embodiment of the present invention, the amino acid sequence of the bifunctional fusion protein may include the heavy chain sequence shown in SEQ ID NO.4 and the light chain sequence shown in SEQ ID NO.1.

[0056] The second aspect of the present invention provides an isolated polynucleotide encoding the bifunctional fusion protein provided by the first aspect of the present invention.

[0057] A third aspect of the present invention provides a construct comprising the isolated polynucleotide of the second aspect of the present invention. Suitable methods for providing the above-mentioned construct should be known to those skilled in the art. For example, the above-mentioned construct can be obtained by inserting the above-mentioned isolated polynucleotide into a suitable vector.

[0058] A fourth aspect of the present invention provides an expression system comprising the construct provided in the third aspect of the present invention or the polynucleotide provided in the second aspect of the present invention integrated into its genome, thereby expressing the fusion protein. The expression system can typically be a host cell, which can be one or more of bacteria, fungi, or mammalian cells.

[0059] The bacteria are selected from one or more of Escherichia coli, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffii, Fusobacterium nucleatum, Bacteroides johnsonii, Bacteroides thaliana, Lactobacillus rhamnosus, Bacteroides massiliense, Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgaris, Bacteroides monomorpha, Bacteroides faecalis, Fusobacterium mortis and Bifidobacterium breve.

[0060] The fungus is selected from yeast and / or mold. The yeast is selected from one or more of Saccharomyces cerevisiae, Candida dunovica, Candida glabrata, Candida guilliermondii, Candida kefir, Candida krusei, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis, Schizosaccharomyces pombe, Candida albicans, Candida portugalensis, Candida merlinii, Candida oleophilus, Candida parapsilosis, Candida tropicalis, and Candida utilis; the mold is selected from one or more of Aspergillus clavatus, Aspergillus glaucus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus pyrophorus, and Aspergillus versicolor.

[0061] Wherein, the mammalian cell is selected from one or more of CHO cells, HEK 293 human embryonic kidney cells, Bowes melanoma cells, COS-7 cells, C127 cells, HeLa cells, BHK cells, SP2 / 0 mouse plasma cells, NS0 mouse plasma cells, COS monkey kidney cells, CHO-S, R1 mouse embryonic cells, 3T3 mouse fibroblasts, BHK21 Syrian hamster fibroblasts, MDCK dog epithelial cells, PtK1 mouse kangaroo epithelial cells, E14.1 mouse embryonic cells, H1 human embryonic cells, H9 human embryonic cells and PER C.6 human embryonic cells. In some preferred embodiments, the mammalian cell is selected from CHO cells and / or HEK293 human embryonic kidney cells. In other preferred embodiments, the host cell is a CHO-K1 cell.

[0062] A fifth aspect of the present invention provides a method for preparing the bifunctional fusion protein provided in the first aspect of the present invention. A person skilled in the art may select an appropriate method to prepare the fusion protein. For example, the method for preparing the multifunctional fusion protein may include: culturing the expression system provided in the fourth aspect of the present invention under appropriate conditions to express the fusion protein, collecting the culture containing the fusion protein, and then isolating and purifying the culture to provide the fusion protein.

[0063] A sixth aspect of the present invention provides the use of the bifunctional fusion protein provided in the first aspect of the present invention in the preparation of a medicament for treating diseases associated with VEGF and / or TGF-β expression, preferably selected from one or more of tumors, autoimmune diseases, and ophthalmic diseases. The bifunctional fusion protein provided by the present invention not only has good stability but also has excellent affinity for both TGF-β and VEGF. Compared with the original drug Avastin and the control drug M7824, the binding ability to the antigen is similar, thereby enabling the bifunctional fusion protein to be used in the treatment of diseases associated with VEGF and / or TGF-β expression.

[0064] The above-mentioned diseases related to VEGF and / or TGF-β expression can be tumors, etc., specifically solid tumors or blood 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), glioma (GBM), prostate cancer (PCA), pheochromocytoma and paraganglioma (PPGL), gastric cancer (GC), esophageal cancer (EC), renal cell carcinoma (RCC), bladder urothelial carcinoma (BLCA), pancreatic cancer (PAC), breast cancer (BRCA), lymphoma (Lymphoma), sarcoma (SAR), acute or chronic leukemia, etc.

[0065] The above-mentioned disease associated with VEGF and / or TGF-β expression may be an autoimmune disease, and the autoimmune disease is selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjögren's syndrome, and polymyositis.

[0066] The above-mentioned diseases associated with VEGF and / or TGF-β expression can be ophthalmic diseases, including but not limited to dry AMD, wet AMD or choroidal neovascularization (CNV); for example, they can be selected from age-related macular degeneration (AMD), choroidal neovascularization (CNV), choroidal neovascular membrane (CNVM), cystoid macular edema (CME), epiretinal membrane (ERM) and macular hole; myopia-related CNVM, angioid streaks, retinal detachment, diabetic retinopathy, diabetic macular edema (DME), Atrophic or hypertrophic lesions of the retinal pigment epithelium (RPE), retinal vein occlusion, chorioretinal vein occlusion, macular edema; corneal angiogenesis 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, refractory abnormalities, keratoconus, retinopathy of prematurity, angiogenesis of the anterior segment of the eye, corneal angiogenesis after keratitis, corneal transplantation, or keratoplasty.

[0067] The seventh aspect of the present invention provides a pharmaceutical composition comprising the bifunctional fusion protein provided in the first aspect of the present invention or the expression system or culture of the expression system provided in the fourth aspect of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The carrier may comprise various excipients and diluents, which are not necessarily active ingredients themselves and are not excessively toxic after administration. Suitable carriers should be well known to those skilled in the art. For example, a full discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0068] In the present invention, in the above-mentioned medicine or composition, the bifunctional fusion protein provided in the first aspect of the present invention can be used as a single active ingredient or in combination with other active ingredients.

[0069] In an eighth aspect, the present invention provides a treatment method comprising administering to an individual a therapeutically effective dose of the bifunctional fusion protein provided in the first aspect of the present invention, or the composition provided in the seventh aspect of the present invention. The treatment method provided by the present invention can be used to treat diseases associated with VEGF and / or TGF-β expression, specifically tumors.

[0070] In the antibodies, applications or pharmaceutical compositions of the present invention, the bifunctional fusion protein can also be used in combination with other means or drugs for tumor treatment (including standard tumor treatment methods / means), including but not limited to surgery, radiotherapy, chemotherapy, tumor immunotherapy, etc. When the bifunctional fusion protein is used in combination with other drugs for tumor treatment, it can be prepared into a pharmaceutical composition, or administered separately with other drugs for tumor treatment. When prepared into a pharmaceutical composition, the active ingredients of the pharmaceutical composition include, in addition to the bifunctional fusion protein of the present invention, other drugs for tumor treatment. The mass ratio of the bifunctional fusion protein to other drugs for tumor treatment can be any suitable ratio, such as 1: (1-99), specifically 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, 1:64 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.The mass ratio of the other tumor treatment drugs to the bifunctional fusion protein can be any suitable ratio, such as 1:(1-99), specifically 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, 1:5 6, 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. The separate administration can be the administration of other drugs for tumor treatment before, simultaneously with, or after the administration of the bifunctional fusion antibody of the present invention.

[0071] For tumor radiotherapy, common radiation types include, but are not limited to, X-rays, gamma rays, and charged particles such as electrons, protons, and heavy ions.

[0072] For tumor chemotherapy, common chemotherapy drugs include: ① cytotoxic drugs: alkylating agents such as nitrogen mustard, carmustine (carmustine), cyclophosphamide, busulfan (Myleran), lomustine (cyclohexyl nitrosourea), etc.; ② antimetabolites: such as fluorouracil, methotrexate, cytarabine, mercaptopurine, tegafur (furan fluorouracil), etc.; ③ antibiotics: such as actinomycin D (dactinomycin), mitomycin, bleomycin, doxorubicin, bleomycin , daunorubicin, mithramycin, etc.; ④ Alkaloids: such as vincristine, vinblastine, hydroxytoxin and podophyllotoxin etoposide (VP-16), teniposide (VM-26); ⑤ Hormones, such as tamoxifen (tamoxifen), diethylstilbestrol, progesterone, testosterone propionate, thyroxine, prednisone and dexamethasone, etc.; ⑥ Others: such as methylbenzylhydrazine, hydroxyurea, L-asparaginase, cisplatin, carboplatin, anticancer antimony, triazine imidazole amine, etc.

[0073] Tumor immunotherapy mainly includes immunomodulators, adoptive cell transfer therapy (ACT), tumor-specific vaccines, small molecule immune drugs, etc.

[0074] Among them, immunomodulators include activators of co-stimulatory molecules, immune checkpoint inhibitors (ICBs), immune checkpoint activators, etc.

[0075] In certain embodiments, the immunomodulator is an activator of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule 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, an agonist of B7-H3 or CD83 ligand (such as an agonist antibody or its antigen-binding fragment or a soluble fusion protein).

[0076] In certain embodiments, the immunomodulator is an inhibitor (ICB) or activator of an immune checkpoint molecule, which is a molecule of the immune checkpoint stimulation pathway and the inhibition pathway. Immune checkpoint inhibitors are monoclonal antibodies targeting the corresponding immune checkpoints, whose main function is to block the interaction between tumor cells expressing immune checkpoints and immune cells, thereby blocking the inhibitory effect of tumor cells on immune cells, thereby affecting the immune response. In one embodiment, the monoclonal antibody molecule is a whole antibody or a fragment thereof (such as Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In other embodiments, the monoclonal antibody molecule has a heavy chain constant region (Fc), selected from, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE. In one embodiment, the constant region is changed (such as mutation) to modify the characteristics of the antibody molecule (for example, increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function).

[0077] 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 bifunctional fusion protein is used in combination with an immune checkpoint inhibitor that can inhibit PD-1 and / or PD-L1. The term "inhibit" or "inhibitor" includes inhibiting the activity of a specified molecule, such as the activity of PD-1 or PD-L1, by at least 5%, 10%, 20%, 30%, 40% or more. Thus, inhibition does not necessarily need to be 100%.

[0078] Among them, 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.

[0079] Among them, tumor-specific vaccines include: whole-cell tumor vaccines, dendritic cell vaccines, gene vaccines, RNA vaccines and protein peptide vaccines.

[0080] Whole-cell tumor vaccines are made by processing autologous or allogeneic tumor cells through physical, chemical, biological and other methods to make them lose their tumorigenicity but retain their antigenicity. They are then combined with non-specific stimulating factors (such as BCG) to provide active immunotherapy for tumor patients.

[0081] Dendritic cell vaccine: Dendritic cells are a heterogeneous group of immune cells with the strongest antigen-presenting function. They are the only professional antigen-presenting cells that can activate naive T cells.

[0082] Gene vaccines are products obtained by cloning gene fragments encoding protective antigens of pathogens into expression vectors and using them to transfect cells or eukaryotic and prokaryotic microorganisms, or by deleting the virulence-related genes of pathogens to make gene-deficient vaccines without virulence-related genes. They mainly include viral vector vaccines and bacterial vector vaccines; many viruses are used as vectors for recombinant vaccines, such as adenoviruses and poxviruses; bacterial vectors are mainly Listeria and Salmonella.

[0083] Protein peptide vaccines are prepared through chemical synthesis based on the amino acid sequence of a known or predicted epitope within a pathogen's antigen gene. If a specific antigen within a tumor cell can be identified and used to develop a cancer vaccine, this amino acid sequence could activate immune cells to kill tumor cells bearing the same antigen. Examples include EGF vaccines (Cimavax), MAGE-A3, and TG4010.

[0084] Herein, sequence identity refers to the percentage of identical residues in the compared sequences. Sequence identity between two or more sequences can be calculated using software well known in the art, which can be obtained from channels such as NCBI.

[0085] In the present invention, "individual" generally includes humans, non-human primates, and other mammals such as mice, dogs, cats, horses, sheep, pigs, cattle, etc., which can benefit from treatment with the above-mentioned drugs, compositions, preparations, kits or combined preparations.

[0086] In the present invention, "therapeutically effective dose" generally refers to a dose that can achieve the effect of treating the diseases listed above after an appropriate administration period.

[0087] The bifunctional fusion protein provided by the present invention can simultaneously and efficiently antagonize VEGF and TGF-β. The modified protein significantly improves stability and is less susceptible to spontaneous breakage during the purification process. This significantly reduces the difficulty of protein purification while ensuring efficacy, improves purification efficiency, and reduces production costs. Furthermore, the VEGF / TGF-β bifunctional fusion protein provided by the present invention has the advantages of high efficacy and minimal side effects, and has good prospects for industrialization.

[0088] The invention of the present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0089] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, by Sambrook et al., MOLECULAR CLONING: 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, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0090] Example 1 Preparation of bifunctional fusion protein

[0091] The polynucleotide encoding the heavy chain (SEQ ID NO. 4) and the light chain (SEQ ID NO. 1) of the bifunctional fusion protein were co-transfected into CHO-K1 expression host cells using the same expression vector. High-expressing clones were screened using methionine iminosulfone (MSX) pressure. The screened high-expressing clones were cultured in large quantities in EmCD CHO 104 medium (Eminence) at 37°C and 5% CO2 to induce expression. The cell culture fluid was collected after 12-14 days of culture.

[0092] Example 2 Purification of bifunctional fusion protein

[0093] The cell culture fluid collected in Example 1 was centrifuged and high-speed centrifuged, and then purified by affinity chromatography using Unimab 50HC purification filler from Nanovitamin Technologies and an AKTA (GE Healthcare) protein purification system. The sample was loaded at a dose of 30 mg protein / ml medium. The equilibration buffer was a 50 mM Tris-HCl + 150 mM NaCl solution at pH 7.5, and the elution buffer was a 20 mM Na-Citrater solution at pH 3.0. The flow rate was 5 ml / min. The eluate containing the target protein was collected and the pH was adjusted to 7.0 for detection and identification of physical and chemical properties and biological activity.

[0094] Example 3 Preparation and purification of control fusion protein

[0095] The amino acid sequence of the control fusion protein includes the heavy chain sequence shown in SEQ ID NO.8 and the light chain sequence shown in SEQ ID NO.7.

[0096] The preparation process is the same as that in Example 1; the purification process is the same as that in Example 2.

[0097] Example 4 Identification of bifunctional fusion protein by SDS-PAGE electrophoresis

[0098] 4.1 Determination of molecular weight and purity of bifunctional fusion protein by SDS-PAGE electrophoresis

[0099] Under reducing (β-mercaptoethanol) and non-reducing conditions, the bifunctional fusion protein obtained in Example 2 was subjected to SDS-PAGE electrophoresis identification. The electrophoresis was performed using polyacrylamide gels with separation gel concentrations of 12% and 8%, respectively. The sample load per well was 3 μg of protein. The electrophoresis results are shown in FIG. Figure 1 (Reducing SDS-PAGE), Figure 2 (Non-reducing SDS-PAGE), lanes 1, 2, and 3 are three replicate samples of the bifunctional fusion protein targeting VEGF and TGF-β, respectively. The electrophoresis results show that the target protein has a molecular weight of approximately 179 kD and a purity greater than 98%.

[0100] 4.2 Determination of the stability of the bifunctional fusion protein by SDS-PAGE electrophoresis

[0101] Under reducing (β-mercaptoethanol) conditions, the bifunctional fusion protein obtained in Example 2 and the control fusion protein obtained in Example 3 were subjected to SDS-PAGE electrophoresis identification. The electrophoresis was performed using polyacrylamide gels with separation gel concentrations of 12% and 8%, respectively. The sample amount per well was 3 μg of protein. The electrophoresis results are shown in FIG. Figure 3(Reducing SDS-PAGE), lanes 1 and 2 are the control fusion protein obtained in Example 3 and the bifunctional fusion protein obtained in Example 2, respectively. Figure 3 As can be seen, the control fusion protein, i.e., the protein without the K→A mutation, is easily broken (clearly broken protein is present at the location marked by the arrow), while the bifunctional fusion protein containing the K→A mutation has no obvious broken protein at the location corresponding to the arrow in lane 1. This indicates that the bifunctional fusion protein obtained in Example 2 of the present invention has very high stability.

[0102] Example 5 Recombinant human TGF-β1 / 2 / 3 ELISA experiment

[0103] Perform ELISA using standard protocols:

[0104] 5.1 Coat 0.5 μg of 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 on a 96-well plate overnight at room temperature.

[0105] 5.2 On the second day, rinse the 96-well plate four times with TBS (pH 7.4) containing 0.05% Tween-20. Block the plate with TBS (pH 7.4) containing 0.05% Tween-20 and 5% skim milk powder for 1 hour at room temperature. After removing the blocking buffer, add different concentrations of the bifunctional fusion protein or the control drug M7824 to the plate in duplicate and incubate at room temperature for 2 hours.

[0106] 5.3 The plate was then washed four times with a cleaning solution, and anti-human IgG (Fc specific)-peroxidase goat antibody was added and incubated at room temperature in the dark for 30 minutes. The color reaction was then terminated with 1M H2SO4.

[0107] 5.4 Read the absorbance at 450nm using Envision reader and calibrate at 570nm. The detailed results are as follows: Figure 4 , Figure 5 and Figure 6 shown.

[0108] Depend on Figure 4 , Figure 5 and Figure 6As can be seen, the affinity of the bifunctional fusion protein prepared in Example 1 for TGF-β is roughly equivalent to that of M7824, while both have weaker affinities for TGF-β2. The EC50s for the bifunctional fusion protein prepared in Example 1 and M7824 for TGF-β1 are 2.843 nM and 2.076 nM, respectively, and for TGF-β3 are 10.309 nM and 3.323 nM, respectively.

[0109] Example 6 Recombinant human VEGF ELISA experiment

[0110] In order to test whether the bifunctional fusion protein prepared in Example 1 binds to human VEGF, recombinant human VEGF (Novoprotein) was coated in a 96-well plate at room temperature overnight, with a coating amount of 0.5 μg per well. The experiment was carried out using the bifunctional fusion protein prepared in Example 1 or the control drug Avastin. Other experimental methods and steps were the same as in Example 3. The specific results are shown in FIG. Figure 7 shown.

[0111] Depend on Figure 7 It can be seen that the affinity of the bifunctional fusion protein prepared in Example 1 to VEGF is substantially equivalent to that of Avastin, and the EC50 of the affinity to VEGF is 0.0659 nM and 0.0168 nM, respectively.

[0112] Example 7 ELISA assay for simultaneous detection of TGF-β and VEGF binding

[0113] In order to test whether the bifunctional fusion protein prepared in Example 1 binds to recombinant human TGF-β1 / 2 / 3 and VEGF simultaneously, recombinant human TGF-β1 / 2 / 3 and VEGF were coated in a 96-well plate and incubated overnight at room temperature. The amount of TGF-β1 / 2 / 3 and VEGF coated in each well was 0.5 μg, respectively. The experiment was carried out using the bifunctional fusion protein prepared in Example 1 or the control drugs M7824 and Avastin. Other experimental methods and steps were the same as in Example 3. The specific results are shown in FIG. Figure 8 shown.

[0114] Depend on Figure 8It can be seen that the bifunctional fusion protein prepared in Example 1 can simultaneously bind to recombinant human TGF-β and VEGF, with an EC50 of 0.0365 nM. This indicates that the bifunctional fusion protein of the present invention can simultaneously bind to recombinant human TGF-β and VEGF, thereby simultaneously inhibiting the binding of VEGF to VEGF receptors and inhibiting the regulatory effect of TGF-β on the immune system, thereby effectively inhibiting the biological effects of VEGF and TGF-β, including affecting vascular permeability, proliferation, and migration and survival of endothelial cells, and enhancing the production and function of effector T cells and DC cells, thereby inhibiting tumor growth and proliferation and achieving an anti-tumor effect.

[0115] Example 8 Experiment on the inhibition of VEGF-induced proliferation of human umbilical vein endothelial cells (HUVEC) by bifunctional fusion protein

[0116] HUVEC cells were resuspended in complete culture medium and diluted, seeded into 96-well plates at 5,000 cells / well. The plates were then incubated overnight in a 37°C, 5% CO₂, high humidity incubator. The next day, the culture medium was discarded, and ECM + 1% FBS medium was added for 24 hours of starvation. Avastin, the bifunctional fusion protein prepared in Example 1, and HSP088-01 (an isotype control protein in which only the TGF-β fragment activity of the bifunctional fusion protein prepared in Example 1 was retained) were added to the wells containing the cells after a three-fold serial dilution starting at 10 nM. The cells were then incubated in a 37°C, 5% CO₂, high humidity incubator for 72 hours. After the 96-well plate was removed, freshly prepared CCK-8 (10%) detection solution was added and placed in a 37°C incubator for 2 to 4 hours. After gentle shaking, the absorbance at 450 nm was measured on a SpectraMax M5 device, and the absorbance at 650 nm was used as a reference to calculate the inhibition rate (inhibition rate % = [1-(OD drug+VEGF -OD No VEGF ) / (OD Only VEGF -OD No VEGF )]*100), and GraphpadPrism 6 software was used to fit the inhibition curve and calculate the IC 50 Value, see the result Figure 9 .

[0117] The results showed that Avastin and the bifunctional fusion protein prepared in Example 1 could effectively inhibit VEGF-induced HUVEC cell proliferation. 50 0.3869nM and 0.1964nM respectively.

[0118] Example 9: Experiment on the inhibition of TGF-β-induced migration of human breast cancer MDA-MB-231 cells by bifunctional fusion protein

[0119] MDA-MB-231 cells were cultured and passaged in complete medium, and cells in the logarithmic growth phase were collected. After the cells adhered, they were starved for 16 hours with L15 + 1% FBS medium. Matrigel was diluted with serum-free L15 medium at a ratio of 1:2 and added to the Transwell chamber and incubated at 37°C for 30 minutes. The MDA-MB-231 cells after starvation culture were resuspended in serum-free L15 medium and then added to each Transwell chamber containing Matrigel, and blank culture medium, the bifunctional fusion protein prepared in Example 1, and HSP088-01 (an isotype control protein in which the bifunctional fusion protein prepared in Example 1 retains only the activity of the TGF-β fragment) were added respectively. After 1 hour of co-incubation, TGF-β stock solution was added to the upper chamber to a final concentration of 0.23nM (the blank culture medium group only added the same volume of culture medium). The total volume of the chamber was 400μL (including 200μL Matrigel), and the lower chamber contained 600μL of fresh L-15 culture medium. The cells were incubated at 37°C for 24 hours. After incubation, the cells were fixed with paraformaldehyde for 20 minutes and stained with crystal violet for 20 minutes. After washing with PBS, the cells were photographed with an inverted microscope, and the cell number was statistically analyzed using Image J software. The results are shown in Figure 2. Figure 10 and Figure 11 .

[0120] The results showed that TGF-β could induce the migration of MDA-MB-231 cells. After adding the bifunctional fusion protein prepared in Example 1 and HSP088-01, the number of migrating MDA-MB-231 cells was significantly reduced. As the concentration of the bifunctional fusion protein prepared in Example 1 increased, the inhibitory effect on the migration of MDA-MB-231 cells gradually increased. The inhibitory effect of 100nM FS8002 on the migration of MDA-MB-231 cells was significantly different from that of the negative control containing only TGF-β (p < 0.05). ＊＊ ).

[0121] Example 10 Experiment on the neutralization effect of bifunctional fusion protein on TGF-β

[0122] MDA-MB-231 cells were seeded in 6-well plates (1×10 6cells / well), cultured at 37°C for 24 hours, and then replaced with PBS, PBS+TGF-β1, or culture medium containing TGF-β1 and different concentrations of the bifunctional fusion protein prepared in Example 1 and HSP088-01 (the bifunctional fusion protein prepared in Example 1 only retains the isotype control protein of the TGF-β fragment activity) in different wells. After changing the medium, continue to culture for 1 hour. The supernatant of the culture medium in the 6-well plate was aspirated, and the cells in the well plate were washed with D-PBS and lysed with lysis buffer. The samples after sufficient lysis were immediately subjected to SDS-PAGE electrophoresis, and Smad2 / 3 and phosphorylation (p-Smad2 / 3) signals were detected by Western Blot. The experimental results are shown in Figure 12 .

[0123] The results showed that when MDA-MB-231 cells were treated with TGF-β1 alone, p-Smad2 / 3 was detected by Western Blot, indicating that TGF-β1 can induce the classic intracellular Smad signaling pathway by binding to the TGF-β receptor (TGF-βR) on 231 cells. When TGF-β1 and different concentrations of the bifunctional fusion protein prepared in Example 1 and HSP088-01 were added simultaneously, no p-Smad2 / 3 was detected by Western Blot, indicating that the bifunctional fusion protein prepared in Example 1 and HSP088-01 can neutralize TGF-β1, blocking TGF-β1 binding to TGF-βR on 231 cells, thereby blocking Smad downstream signaling. In addition, the bifunctional fusion protein prepared in Example 1 can effectively neutralize TGF-β1 at a concentration as low as 1 nM.

[0124] Example 11 In vivo tumor inhibition experiment of bifunctional fusion protein in mice

[0125] MDA-MB-231 cells resuspended in PBS were cultured at a concentration of 1×10 7 The concentration of 0.1 mL was 0.1 mL (containing 30% Matrigel) per mouse, and the cells were inoculated subcutaneously in the right flank of BALB / c nude mice. A total of 100 mice were transferred. After 22 days of tumor formation, the mice without tumors and the mice with the largest and smallest tumor volumes were eliminated. 70 mice with moderate tumor volumes were selected for grouping, with 10 mice in each group. The average tumor volume reached 70 mm. 3 The mice were dosed starting on the day of grouping, with the bifunctional fusion protein prepared in Example 1 or the control drug injected twice a week for a total of four weeks. The tumor volume and weight of the tumor-bearing mice were measured and recorded during the experiment.

[0126] The grouping is as follows:

[0127] Group 1 (G1): control group (normal saline alone);

[0128] Group 2 (G2): control drug bevacizumab alone;

[0129] Group 3 (G3): control drug M7824 alone;

[0130] Group 4 (G4): control drug avelumab alone;

[0131] Group 5 (G5): control drug bevacizumab combined with avelumab;

[0132] Group 6 (G6): The bifunctional fusion protein prepared in Example 1 was used alone;

[0133] Group 7 (G7): The bifunctional fusion protein prepared in Example 1 + avelumab were used in combination.

[0134] See the results Figure 13 The experimental data from the mice was analyzed and organized using a spider plot with dosing time as the horizontal axis and tumor volume change rate as the vertical axis. It can be concluded that both G6, using the bifunctional fusion protein prepared in Example 1 alone, and G7, using the bifunctional fusion protein prepared in Example 1 in combination with avelumab, showed significant inhibitory effects on the average tumor volume of mice compared to the G1 control group. Compared to G2, using the control drug bevacizumab alone, G6, using the bifunctional fusion protein prepared in Example 1 alone, significantly improved the inhibitory effect on mouse tumor volume. Compared to G5, using the control drug bevacizumab and avelumab in combination, G7, using the bifunctional fusion protein prepared in Example 1 in combination with avelumab, significantly improved the inhibitory effect on mouse tumor volume. In this group, four mice even experienced complete tumor elimination after treatment. Overall, the animal experiments in this example demonstrate the potent inhibitory effect of the bifunctional fusion protein prepared in Example 1 on MDA-MB-231 tumor cells in BALB / c nude mice and demonstrate the significant potential for combination with other immune checkpoint inhibitors.

[0135] In summary, the present invention effectively overcomes the various shortcomings of the prior art and has high industrial and commercial value.

[0136] Sequence information:

[0137] SEQ ID No.1

[0138] Light chain:

[0139] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFS

[0140] GSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK

[0141] SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0142] EKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID No.2

[0143] Heavy chain:

[0144] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPT

[0145] YAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL

[0146] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0147] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0148] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0149] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

[0150] EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

[0151] WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0152] SEQ ID No.3

[0153] GIPPHVQASVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV

[0154] CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN

[0155] DNIIFSEEYNTSNPDSEQ ID No.4

[0156] Heavy chain:

[0157] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPT

[0158] YAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL

[0159] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0160] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0161] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0162] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

[0163] EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

[0164] WQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQA

[0165] SVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

[0166] NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEY

[0167] NTSNPD

[0168] SEQ ID No.5

[0169] AGGGGSGGGGSGGGGSGGGGS

[0170] SEQ ID No.6

[0171] Heavy chain:

[0172] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPT

[0173] YAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL

[0174] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0175] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0176] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0177] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

[0178] EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

[0179] WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID No.7

[0180] Heavy chain:

[0181] GIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV

[0182] CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN

[0183] DNIIFSEEYNTSNPDSEQ ID No.8

[0184] Heavy chain:

[0185] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPT

[0186] YAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL

[0187] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0188] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0189] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0190] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE

[0191] EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

[0192] WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAGGGGSGGGGSGGGGSGGGGSGIPPHVQ

[0193] KSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR

[0194] KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE

[0195] YNTSNPD

[0196] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A bifunctional fusion protein, characterized in that: The bifunctional fusion protein includes an antagonistic VEGF fragment and an antagonistic TGF-β fragment; The antagonistic VEGF fragment comprises a heavy chain, wherein the heavy chain comprises at least an amino acid mutation at position 453 compared to the original heavy chain; and the antagonistic TGF-β fragment comprises at least an amino acid mutation at position 8 compared to the original antagonistic TGF-β fragment; The amino acid sequence of the bifunctional fusion protein includes a heavy chain sequence as shown in SEQ ID NO.4 and a light chain sequence as shown in SEQ ID NO.

1.

2. An isolated polynucleotide encoding the bifunctional fusion protein according to claim 1.

3. A construct comprising the isolated polynucleotide of claim 2.

4. An expression system comprising the construct according to claim 3 or the polynucleotide according to claim 2 integrated into the genome.

5. The method for preparing the bifunctional fusion protein according to claim 1, comprising: The expression system according to claim 4 is cultured under appropriate conditions to express the bifunctional fusion protein, and then separated and purified to provide the bifunctional fusion protein.

6. Use of the bifunctional fusion protein according to claim 1 in preparing a medicament, wherein the medicament is used to treat a disease associated with VEGF and / or TGF-β expression; the disease is selected from one or more of a tumor, an autoimmune disease, an ophthalmic disease, or a tissue fibrosis disease; the tumor is selected from breast cancer, colorectal cancer, liver cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, gastric cancer, esophageal cancer, gastrointestinal stromal tumor, prostate cancer, pancreatic cancer, glioma, melanoma, head and neck cancer, nasopharyngeal carcinoma, and lymphoma; the autoimmune disease is selected from one or more of systemic sclerosis, Sjögren's syndrome, and polymyositis; the ophthalmic disease is selected from dry AMD, wet AMD, or choroidal neovascularization; and the tissue fibrosis disease is selected from any one or more of liver fibrosis, pulmonary fibrosis, and renal fibrosis.

7. The use according to claim 6, characterized in that The bifunctional fusion protein is used in combination with other means of tumor treatment, which include surgery, radiotherapy, chemotherapy, and tumor immunotherapy.

8. The use according to claim 7, characterized in that The types of radiation used in radiotherapy include X-rays, gamma rays, and charged particles; The drugs suitable for the chemotherapy are selected from: ① cytotoxic drugs: nitrogen mustard, carmustine, cyclophosphamide, busulfan, lomustine; ② antimetabolites: fluorouracil, methotrexate, cytarabine, mercaptopurine, tegafur; ③ antibiotics: actinomycin D, mitomycin, bleomycin, doxorubicin, bleomycin, daunorubicin, mithramycin; ④ alkaloids: vincristine, vinblastine, hydroxyproline and podophyllotoxins etoposide, teniposide; ⑤ hormones: tamoxifen, diethylstilbestrol, progesterone, testosterone propionate, thyroxine, prednisone and dexamethasone; ⑥ others: one or more of methylprocarbazine, hydroxyurea, L-asparaginase, cisplatin, carboplatin, anticancer antimony, and triazine imidazole; The tumor immunotherapy is selected from one or more of immunomodulators, adoptive cell transfer therapy, tumor-specific vaccines, and small molecule immune drugs; The immunomodulator is selected from one or more of a co-stimulatory molecule activator, an immune checkpoint inhibitor, and an immune checkpoint activator; the co-stimulatory molecule agonist is selected from OX40, CD2, CD27, 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 C One or more agonists of D83 ligand; the immune checkpoint inhibitor is selected from one or more inhibitors of PD-1, PD-L1, PD-L2, PD-L3, CTLA4, TIM-3, LAG3, CEACAM-1, CEACAM-3, CEACAM-5, VISTA, VSIR, BTLA, TIGIT, LAIR1, LMTK3, IDO, CD27L, CD47, CD244, CD270, B7-H1, B7-1, 2B4 and / or TGF-βR; Wherein, the adoptive cell transfer therapy is selected from one or more of tumor infiltrating lymphocyte therapy, engineered T cell receptor therapy, chimeric antigen receptor T cell therapy and natural killer cell therapy; The tumor-specific vaccine is selected from one or more of tumor whole cell vaccines, dendritic cell vaccines, gene vaccines, RNA vaccines and protein polypeptide vaccines.

9. A pharmaceutical composition comprising the bifunctional fusion protein according to claim 1 or the expression system according to claim 4 or a culture of the expression system according to claim 4.

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