Anti-VEGF and complement bifunctional fusion protein, and use thereof

Abstract

Provided are an anti-VEGF and complement bifunctional fusion protein, and a use thereof. The fusion protein comprises a VEGF binding domain, a human IgG Fc domain, and a complement binding domain, and has high expression, high activity, and stable properties. The protein can simultaneously effectively bind VEGF factors and complement molecules, exhibits stronger anti-VEGF and anti-complement effects, and has a better treatment effect on age-related macular degeneration.

Description

An anti-VEGF and complement dual-function fusion protein and its application Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to an anti-VEGF and complement bifunctional fusion protein and an application thereof. Background Art

[0002] Age-related macular degeneration (AMD) is the leading cause of vision loss in patients over 65 years old. Worldwide, 196 million people are living with AMD, a number projected to rise to 288 million by 2040. AMD can be divided into two types: dry AMD (nonvascular, dAMD) and neovascular AMD (nAMD). In the early stages of dry AMD, there is chronic low-level inflammation, drusen deposits in the subretinal space, irreversible loss of the retinal pigment epithelium (RPE) and photoreceptors in the macula, and subsequent progressive loss of central vision. In the late stages of dry AMD, RPE degeneration becomes confluent, and vision loss worsens, a condition known as geographic atrophy (GA). In approximately 10% of patients, AMD progresses to the more aggressive nAMD, which is associated with rapid loss of central vision and is characterized by the growth of abnormal choroidal blood vessels into the macula, known as choroidal neovasculation (CNV). These vessels are "leaky" and contain excessive scar tissue.

[0003] The pathogenesis of AMD is complex. According to literature reports, the relevant signaling pathways mainly include angiogenesis-related pathways (VEGF, PDGF, FGF, etc.), complement-related pathways (C3, C5, etc.), fibrosis-related pathways (FGF, TGFβ, etc.) and immune inflammatory factor pathways (IL-6, TNF-α, etc.).

[0004] Vascular endothelial growth factor (VEGF) can induce the regeneration of existing blood vessels (angiogenesis) or the growth of new blood vessels (angiogenesis), and is a key factor in embryonic development and vascular repair. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factors 1 and 2 (PIGF-1 and PIGF-2). VEGF-A is the most potent and extensively studied angiogenesis-inducing factor to date. VEGF family members are encoded by multiple exons, and alternative splicing can produce different isoforms, which affect solubility and receptor binding. For example, VEGF-A has seven isoforms, while VEGF-B has two isoforms. VEGF family members activate signal transduction by binding to VEGF receptors (VEGFRs). VEGFRs are tyrosine kinase receptors with an extracellular region composed of seven immunoglobulin-like (IG) domains. VEGFR-1 (Flt-1) binds to VEGF-A, VEGF-B, and PIGF and can act as a decoy receptor for VEGF or a regulator of VEGFR-2. VEGFR-2 (KDR / Flk-1) binds all VEGF isoforms and is the primary mediator of the VEGF-induced angiogenesis pathway. VEGFR-3 (Flt-4) binds to VEGF-C and VEGF-D, but not VEGF-A, and is the primary mediator of lymphangiogenesis.

[0005] The complement system is a functional effector of the innate immune system, composed of a variety of soluble proteins, membrane-bound proteins, and complement receptors. Complement activation leads to a cascade of protease activation, stimulating the release of cytokines and amplifying the activation cascade. The complement system can be activated through three different pathways: the classical pathway, the alternative pathway, and the lectin pathway. All three pathways cleave the complement molecules C3 and C5, respectively, through the key C3 convertase and C5 convertase complexes. The ultimate results of complement activation are activation of the cell-killing membrane attack complex (MAC), stimulation of immune cells to release inflammatory factors and chemokines, allergic reactions caused by the anaphylatoxins C3a and C5a, and phagocytosis of pathogens.

[0006] Currently, anti-VEGF therapy (such as bevacizumab, ranibizumab, and aflibercept) is the only approved standard drug therapy for the treatment of nAMD. However, a considerable number of patients still have incomplete responses to anti-VEGF drugs in clinical practice, manifested by persistent leakage, bleeding, and poor visual recovery. In addition, among patients with nAMD who receive long-term injections of anti-VEGF drugs, more than 10% will convert to dry AMD. Among patients who use anti-VEGF drugs long-term, more than 40% will experience macular atrophy, which is similar to the geographic atrophy caused by dry AMD. To address the vision loss in patients with dry AMD, Apellis' complement factor 3 inhibitor pegcetacoplan (trade name Syfovre) and Astellas' complement factor 5 inhibitor avacincaptad pegol (trade name Izervay) have been approved by the FDA for marketing. However, both have shown an increased incidence of nAMD in clinical studies, and this risk is specifically noted in the drug instructions. This application intends to further address the problems encountered in the treatment of dry and wet AMD by combining VEGF inhibition with inhibition of the complement pathway. Summary of the Invention

[0007] The purpose of the present invention is to provide a bifunctional fusion protein with high expression, excellent activity, and high purity against VEGF and complement targets. By effectively inhibiting the VEGF and complement pathways, it can treat a variety of VEGF and complement pathway-related diseases, such as wet and dry AMD, thereby resolving the problems encountered in the aforementioned clinical treatment processes. The results showed that the bifunctional fusion protein of the present invention not only exhibits more efficient VEGF antagonism but also significantly enhances complement hemolytic inhibition activity, demonstrating superior efficacy in both dry and wet AMD animal models. Furthermore, the stability of the molecule is significantly improved by modifying the Fc domain within the molecule.

[0008] One of the technical solutions of the present invention is to provide a bifunctional fusion protein comprising a VEGF binding domain and a complement binding domain;

[0009] Furthermore, the bifunctional fusion protein further comprises a human IgG Fc domain;

[0010] Preferably, the bifunctional fusion protein comprises 1-3 VEGF binding domains, more preferably, comprises 1 or 2 VEGF binding domains;

[0011] Furthermore, the VEGF binding domain is selected from any one of the following (1)-(4):

[0012] (1) The VEGF binding domain comprises the extracellular domain 2 of VEGF receptor 1 (VEGFR1D2) and the extracellular domain 3 of VEGF receptor 2 (VEGFR2D3);

[0013] (2) the VEGF binding domain comprises the extracellular domain 2 of VEGF receptor 1 (VEGFR1D2);

[0014] (3) The VEGF binding domain comprises a tandem structure of two VEGF receptor 1 extracellular domains 2 (VEGFR1D2);

[0015] (4) The VEGF binding domain comprises an amino acid sequence derived from the heavy chain (HC) and light chain (LC) of ranibizumab; the heavy chain (HC) and light chain (LC) of ranibizumab are connected by a self-cleaving peptide, including P2A, T2A, E2A and F2A; or the VEGF binding domain comprises an amino acid sequence derived from the heavy chain variable region (VH) and light chain variable region (VL) of ranibizumab; the heavy chain variable region (VH) and light chain variable region (VL) of ranibizumab are connected by a connecting peptide, such as (GGGGS) n 、(Gly) 6-8 and A(EAAAK) n A grade.

[0016] Preferably, the bifunctional fusion protein comprises a human IgG Fc domain;

[0017] Furthermore, the human IgG Fc domain is selected from any one of the following (1)-(2):

[0018] (1) The human IgG1 Fc domain comprises 10-15 amino acids of the human IgG1 Hinge region, a CH2 domain, and a CH3 domain (with or without a terminal K);

[0019] (2) The human IgG Fc domain is a modified mutant human IgG Fc domain.

[0020] Preferably, the bifunctional fusion protein comprises 1-2 complement binding domains, more preferably, comprises 1 complement binding domain;

[0021] Furthermore, the complement binding domain is selected from any one of the following (1)-(5):

[0022] (1) The complement binding domain comprises a soluble CD59 (sCD59) domain;

[0023] (2) The complement binding domain comprises amino acid sequences derived from the heavy chain variable region (VH) and light chain variable region (VL) of eculizumab, wherein the VH and VL sequences are connected by a (GGGGS)3 linker peptide;

[0024] (3) the complement binding domain comprises an amino acid sequence derived from pexelizumab;

[0025] (4) the complement binding domain comprises a Factor H-like protein 1 (FHL-1) domain;

[0026] (5) The complement binding domain comprises a Factor I protein (FI) domain.

[0027] Preferably, the VEGF binding domain and the human IgG Fc domain, the complement binding domain and the human IgG Fc domain, and the VEGF binding domain and the complement binding domain are directly connected in series or connected via a connecting peptide;

[0028] Preferably, the connecting peptide is (GGGGS) n , preferably, n=3-6, more preferably n=3 or 4;

[0029] Preferably, the connecting peptide is KESGSVSSEQLAQFRSLD or (Gly) 6-8 ;

[0030] Preferably, the connecting peptide is A(EAAAK) n A, preferably, n=1-4, more preferably n=4; preferably, the connecting peptide is (XP) n , X is A or E or K, n = 5-10;

[0031] More preferably, the bifunctional fusion protein comprises a VEGF binding domain, a modified human IgG Fc domain, and a complement binding domain. The VEGF binding domain comprises the extracellular domain 2 of VEGF receptor 1 (VEGFR1D2) and the extracellular domain 3 of VEGF receptor 2 (VEGFR2D3); the modified human IgG Fc domain comprises amino acid mutations at 1-3 sites, more specifically, the point mutations employed are L234A / L235A / H435A (amino acid numbering is indicated by the EU index in Kabat); the complement binding domain comprises a soluble CD59 domain structure (sCD59). The VEGF binding domain is directly linked to the modified human IgG Fc domain (mhIgG Fc), and the modified human IgG Fc domain and complement binding domain (sCD59) are linked via a connecting peptide (GGGGS) 3. The bifunctional fusion protein structure is: VEGFR1D2-VEGFR2D3-mhIgG Fc-(GGGGS)3-sCD59, and the amino acid sequence is shown in SEQ ID NO.1:

[0032] The bold amino acids above are mutation sites in the human IgG Fc domain;

[0033] Furthermore, the gene encoding the bifunctional fusion protein VEGFR1D2-VEGFR2D3-mhIgG Fc-(GGGGS)3-sCD59 is shown in SEQ ID NO.7.

[0034] More preferably, the bifunctional fusion protein comprises a VEGF binding domain, a modified human IgG Fc domain, and a complement binding domain; the VEGF binding domain comprises the extracellular domain 2 of VEGF receptor 1 (VEGFR1D2); the modified human IgG Fc domain comprises 1-3 amino acid mutations, more specifically, the point mutations employed are L234A / L235A / H435A (amino acid numbering is indicated by the EU index in Kabat); the complement binding domain comprises a soluble CD59 domain structure sCD59; the VEGF binding domain is directly linked to the modified human IgG Fc domain (mhIgG Fc), and the modified human IgG Fc domain and complement binding domain (sCD59) are linked by a connecting peptide (GGGGS) 3. The bifunctional fusion protein structure is: VEGFR1D2-mhIgG Fc-(GGGGS) 3-sCD59, and the amino acid sequence is shown in SEQ ID NO.2:

[0035] The bold amino acids above are mutation sites in the human IgG Fc domain;

[0036] Furthermore, the gene encoding the bifunctional fusion protein VEGFR1D2-mhIgG Fc-(GGGGS)3-sCD59 is shown in SEQ ID NO.8.

[0037] The second technical solution of the present invention is to provide a gene encoding the bifunctional fusion protein described in the first technical solution.

[0038] A third technical solution of the present invention is to provide an expression cassette or recombinant vector for expressing the bifunctional fusion protein of the first technical solution, wherein the expression cassette comprises the structure of Formula I from the 5' to 3' end:

[0039] E1-E2-E3-E4 (Formula I)

[0040] in:

[0041] E1 is the promoter;

[0042] E2 is a signal peptide;

[0043] E3 is the nucleotide sequence encoding the bifunctional fusion protein described in Technical Solution 1;

[0044] E4 is a Poly A sequence.

[0045] Preferably, the promoter is a DNA sequence that can initiate transcription of the target gene, which can be recognized by RNA polymerase and initiate transcription and synthesis of RNA. The promoter includes but is not limited to natural, optimized or combined promoters;

[0046] Furthermore, the promoter is preferably CMV, CBA, EF1a, SV40, PGK1, Ubc, CAG or miniCAG;

[0047] Preferably, the signal peptide is Human OSM, Gaussia luc, Human IL-2 or Albumin (HSA);

[0048] Preferably, the Poly A sequence is selected from bGH polyA, SV40 polyA, HSV-TK polyA or hGH polyA;

[0049] Furthermore, the above-mentioned expression cassette also includes regulatory elements, and the expression regulatory elements include but are not limited to regulatory elements with the following functions: (1) enhancers, which can be derived from SV40 virus, CMV virus or adenovirus, etc.; (2) regulatory elements for expressing miRNA and siRNA sequences; (3) introns; (4) the regulatory element can also be a partial kozak sequence, and the kozak sequence is GNCNCN, such as GCCACC, etc.; (5) the regulatory element can be WPRE.

[0050] Preferably, the bifunctional fusion protein expression cassette is connected to a transient transfection vector; the vector is introduced into a host cell via a transfection reagent to express the bifunctional fusion protein.

[0051] Furthermore, the transient transfection vector includes but is not limited to PTT5, pCDNA3.1(-), pCDNA3.1(+), pPICZαA, pGAPZαA and PYES2.0; preferably, the transient transfection vector is PTT5.

[0052] Furthermore, the transfection reagents used include Lipo2000, Lipo3000, PEI, 293fectin TM , Cellfectin, calcium phosphate, etc.; preferably, the transfection reagent is PEI.

[0053] Furthermore, the present invention also provides a recombinant host cell for expressing the bifunctional fusion protein described in Technical Solution 1, wherein the recombinant host cell comprises the encoding gene described in Technical Solution 2, or the expression cassette or recombinant vector described in Technical Solution 3, and the host cell used for the recombinant host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the host cell is selected from Escherichia coli, yeast cells or mammalian cells; more preferably, the host cell is an Expi293 cell.

[0054] The fourth technical solution of the present invention is an AAV virus comprising the bifunctional fusion protein encoding gene described in the second technical solution or the expression cassette described in the third technical solution. The encoding gene or expression cassette is cloned between the two terminal repeat sequences ITR in the adeno-associated virus AAV skeleton to construct a target gene plasmid GOI, which is applied to the AAV packaging vector system. The packaging vector system includes: the target gene plasmid GOI, a vector carrying the AAV rep and cap genes, and an auxiliary vector. The above-mentioned vector is packaged into an AAV virus by transiently transfecting the production cells with the three plasmids.

[0055] The fifth technical solution of the present invention is to provide a pharmaceutical composition, which contains the bifunctional fusion protein described in technical solution one, or the encoding gene described in technical solution two, or the expression cassette or recombinant vector or recombinant host cell described in technical solution three, or the AAV virus described in technical solution four.

[0056] A sixth technical solution of the present invention is to provide a use of the bifunctional fusion protein described in technical solution one, or the encoding gene described in technical solution two, or the expression cassette or recombinant vector or recombinant host cell described in technical solution three, or the AAV virus described in technical solution four;

[0057] Further, it is used in inhibiting VEGF and complement pathways;

[0058] Furthermore, the invention relates to the use of the present invention in the preparation of a preparation, formula or pharmaceutical composition for treating vascular endothelial growth factor (VEGF) and complement-related diseases, including eye diseases, inflammatory diseases, autoimmune diseases or tumors, and in particular, in the preparation of drugs for treating age-related macular degeneration and diabetic retinopathy.

[0059] Furthermore, the preparation, formulation or drug may be in any dosage form, including but not limited to injection and ointment dosage forms;

[0060] Furthermore, the preparation, formula or medicine contains the above-mentioned bifunctional fusion protein, expression cassette or AAV virus as the only active ingredient.

[0061] Furthermore, the administration method is subretinal injection of AAV virus;

[0062] Furthermore, a single lifetime dose of 1×10 8 -1×10 11 Viral genomes / eye. Beneficial effects:

[0063] The present invention provides an anti-VEGF and complement bifunctional fusion protein with high expression, high activity and stable properties, particularly VEGFR1D2-VEGFR2D3-mhIgGFc-(GGGGS)3-sCD59 and VEGFR1D2-mhIgGFc-(GGGGS)3-sCD59, through molecular combination optimization and molecular modification.

[0064] The bifunctional fusion protein using artificially modified hIgG Fc can significantly improve the protein aggregation problem caused by the use of natural IgG Fc fragments in the preparation of bifunctional fusion proteins. On the one hand, it avoids the reduction of the activity of the fusion protein, and on the other hand, it prevents the cause of immunogenicity problems. Therefore, the modification of the Fc fragment in the present invention is conducive to improving the stability of the bifunctional fusion protein and avoiding aggregation, and is safer and more effective.

[0065] The bifunctional fusion protein can simultaneously and efficiently bind to VEGF factors and complement molecules, especially significantly improving the activity of sCD59. Compared with sCD59, the bifunctional fusion protein can inhibit complement hemolysis activity by 4.6 times and 10 times, respectively.

[0066] Furthermore, the bifunctional fusion protein was effective in both dry and wet AMD models. In the dry AMD model (sodium iodate-induced dry AMD mouse model in Example 13), it demonstrated significant inhibition of ERG amplitude reduction. In the wet AMD model (laser-induced CNV mouse model in Example 9), CNV was effectively inhibited, as measured by leakage area and leakage score, and the bifunctional protein exhibited superior inhibitory activity compared to the monomeric molecule.

[0067] Therefore, the bifunctional fusion protein provided by the present invention is expected to have a significant therapeutic effect on the treatment of age-related macular degeneration, especially dry age-related macular degeneration complicated by fundus angiogenesis, or wet age-related macular degeneration accompanied by macular atrophy. Description of the drawings:

[0068] Figure 1 BFP6 plasmid map.

[0069] Figure 2 BFP10 plasmid map.

[0070] Figure 3. Determination of the expression level of bifunctional fusion protein.

[0071] Figure 4 Purity detection of bifunctional fusion protein by SDS-PAGE

[0072] 4A is a SDS-PAGE electrophoresis diagram of protein G affinity-purified BFP6, BFP10, BFP18, and BFP19; FIG4B is a SDS-PAGE electrophoresis diagram of BFP6, BFP10, BFP18, and BFP19 before and after reduction.

[0073] FIG5 : Determination of the binding activity of the bifunctional fusion protein to VEGF165.

[0074] FIG6 Determination of the inhibitory activity of bifunctional fusion protein on HUVEC proliferation.

[0075] FIG7 shows the inhibitory activity of the bifunctional fusion protein on VEGF-KDR reporter gene cells.

[0076] FIG8 Inhibition of complement hemolytic activity by bifunctional fusion proteins.

[0077] Figure 9. Representative images of the bifunctional fusion protein inhibiting FFA in the laser-induced mouse CNV model.

[0078] FIG10 shows the effect of AAV8 virus carrying target gene in inhibiting MAC deposition in vitro.

[0079] Figure 11 Fluorescence quantitative results of the inhibition of MAC deposition by AAV8 virus carrying the target gene in vitro.

[0080] FIG12 In vivo transgene expression by AAV8 virus carrying target gene.

[0081] Figure 13 Changes in ERG dark-adapted b-wave amplitude in the dry AMD mouse model induced by sodium iodate. Specific implementation method:

[0082] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not intended to limit the present invention.

[0083] Unless otherwise defined herein, scientific and technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0084] In certain embodiments, the present invention relates to bifunctional fusion proteins that simultaneously target vascular endothelial growth factor (VEGF) and complement C3b, C5, C8, or C9. Because VEGF and complement signaling pathways are implicated in numerous diseases, including age-related macular degeneration (AMD), proteins with dual specificity for inhibiting both complement and VEGF may offer superior therapeutic benefits compared to proteins that inhibit either complement or VEGF separately.

[0085] In certain embodiments, the bifunctional fusion protein may be VFC, where V represents a VEGF-binding domain, F represents a human IgG1 Fc domain, and C represents a complement-binding domain. This is produced by fusing a VEGF-binding domain to a human IgG Fc domain at the N-terminus and a complement-binding domain sequence at the C-terminus. In another aspect, the bifunctional fusion protein may be CFV, produced by fusing a complement-binding domain to a human IgG Fc domain at the N-terminus and a VEGF-binding domain sequence at the C-terminus. In other aspects, the bifunctional fusion protein may be a combination of VCF or CVF, where the protein sequences are fused from the N-terminus to the C-terminus in the order of VEGF-binding domain, complement-binding domain, Fc domain, or complement-binding domain, VEGF-binding domain, Fc domain.

[0086] Furthermore, the VEGF binding domain can be a tandem domain of VEGFR1 extracellular domain 2 and VEGFR2 extracellular domain 3; on the other hand, it can also be a single VEGFR1 extracellular domain 2 or two tandem VEGFR1 extracellular domains 2; in other aspects, it can also be derived from the ranibizumab Fab domain; or derived from the ScFv fragment of ranibizumab.

[0087] Furthermore, the complement binding domain can be a scFv fragment derived from eculizumab; or a scFv fragment introduced into pexelizumab; on the other hand, it can also be a domain of factor H-like protein 1 (FHL-1); or a domain of factor I (CFI); in other aspects, it can also be a soluble CD59 (sCD59) fragment.

[0088] Furthermore, the human IgG Fc domain is derived from the human IgG1 Fc domain, or the human IgG1 CH2 and CH3 domains; furthermore, the Fc domain is mutated to improve the stability of the fusion protein.

[0089] Furthermore, in certain embodiments, the human IgG Fc domain significantly enhances the in vitro activity of sCD59 after being linked to the complement binding domain sCD59.

[0090] As used herein, the term "Fab" refers to the antigen-binding region of an antibody, which is composed of the variable and constant domains of the light chain (i.e., the aforementioned LC) and the variable domain and first constant domain of the heavy chain (i.e., the aforementioned HC).

[0091] As used herein, the term [scfv] refers to a single-chain fragment consisting of the variable regions of the heavy chain (VH) and the light chain (VL), which are linked together by a flexible linker peptide, which can be easily expressed in Escherichia coli in a functional form and can be protein engineered to improve the properties of scfv, such as increasing affinity and changing specificity.

[0092] In the present invention, the bifunctional fusion protein was transiently expressed in Expi293 cells and purified from the transfected cell culture supernatant using recombinant protein G affinity chromatography or immobilized metal ion affinity chromatography. A further molecular sieve purification step yielded a product with a purity exceeding 90%, demonstrating that all fusion proteins were correctly formed and expressed.

[0093] In certain embodiments, the binding ability of the bifunctional fusion protein to VEGF was verified by ELISA assay, and the bifunctional fusion protein showed strong binding ability to VEGF165.

[0094] The bifunctional fusion protein provided by the present invention can be used to treat vascular endothelial growth factor (VEGF) and complement-related diseases, as well as to prepare a pharmaceutical composition for treating vascular endothelial growth factor (VEGF) and complement-related diseases; related diseases include eye diseases, inflammatory diseases, autoimmune diseases or tumors.

[0095] Related eye diseases include age-related macular degeneration, diabetic retinopathy, diabetic retinedema, diabetic macular edema, posterior retinal fibrosis, central retinal occlusion, retinal vein occlusion, ischemic retinopathy, hypertensive retinopathy, uveitis (e.g., anterior, middle, posterior, or panuveitis), Behçet's disease, Bietti crystalline corneal retinal dystrophy, blepharitis, open-angle glaucoma, neovascular glaucoma, corneal neovascularization, choroidal neovascularization (CNV), subretinal neovascularization, corneal inflammation, and corneal transplant complications. Age-related macular degeneration includes wet age-related macular degeneration or dry age-related macular degeneration, especially dry age-related macular degeneration complicated by fundus angiogenesis, or wet age-related macular degeneration accompanied by macular atrophy.

[0096] Related inflammatory diseases include rheumatoid arthritis, psoriasis or ankylosing spondylitis.

[0097] Related tumor diseases include colorectal cancer, non-small cell lung cancer, glioblastoma, metastatic renal cancer, breast cancer, colorectal cancer, lung cancer, gastric cancer or retinoblastoma, etc.

[0098] The present invention will be further explained below with reference to specific embodiments.

[0099] Example 1 Design of bifunctional fusion protein structure

[0100] In this example, 19 bifunctional fusion proteins and 12 monomeric proteins were constructed, and their structures are shown in Table 1 below.

[0101] VBD indicates VEGF binding domain protein; CBD indicates complement binding domain protein; BFP indicates bifunctional fusion protein;

[0102] In the VBD, VEGFR1D2 represents VEGF receptor 1 extracellular domain 2 (SEQ ID NO. 3), flanked by 22 amino acids at the N-terminus and 16 amino acids at the C-terminus of the native VEGF receptor 1 extracellular domain 2; VEGFR2D3 represents VEGF receptor 2 extracellular domain 3 (SEQ ID NO. 4), flanked by 7 amino acids at the C-terminus of the native VEGF receptor 2 extracellular domain 3; VEGFR1D2D2 represents two tandem domains of VEGF receptor 1 extracellular domain 2; Rani HC represents the Ranibizumab heavy chain domain; Rani LC represents the Ranibizumab light chain domain; Rani VH represents the Ranibizumab heavy chain variable domain; Rani VL represents the Ranibizumab light chain variable domain;

[0103] In the CBD, Eculi-Scfv1 and Eculi-Scfv2 represent the Scfv fragments formed by the heavy chain variable region VH and light chain variable region VL of eculizumab connected by a (GGGGS)3 linker peptide. Eculi-Scfv1 represents VH-(GGGGS)3-VL, and Eculi-Scfv2 represents VL-(GGGGS)3-VH. Pexe represents pexelizumab. FHL represents factor H-like protein 1. FI represents factor I. sCD59 represents soluble CD59 protein (SEQ ID NO. 5), which is the mature form of the natural full-length human CD59 protein, excluding the N-terminal 25 signal peptide amino acid sequence and the C-terminal GPI-anchor sequence.

[0104] hIgG1 Fc represents the native human IgG1 Fc domain, comprising the terminal 10 amino acids of the native human IgG1 Hinge region, the CH2 domain and the CH3 domain, i.e., hIgG1 Fc in Table 1; mhIgG Fc represents the engineered human IgG Fc domain (SEQ ID NO. 6), i.e., mhIgG Fc in Table 1.

[0105] P2A represents the porcine teschovirus-1 2A peptide; His tag represents a 6-10-mer histidine tag peptide. The His tag peptide facilitates monomeric protein purification and has minimal impact on protein structure, so removal is generally not necessary.

[0106] Table 1 Fusion protein / monomer protein structure design

[0107] Example 2 Expression and purification of bifunctional fusion protein

[0108] The nucleotide sequences encoding the fusion proteins or monomeric proteins shown in Table 1 above were constructed into the PTT5 vectors (wherein, the recombinant vector plasmid maps containing the BFP6 and BFP10 encoding genes are shown in Figures 1-2, the BFP6 encoding gene is shown in SEQ ID NO.7, and the BFP10 encoding gene is shown in SEQ ID NO.8. During construction, a signal peptide sequence was added to the N segment of the fusion protein: MGVKVLFALICIAVAEA). According to the instructions of the transfection reagent, PEI MAX transfection reagent (purchased from Yisheng Bio) was used to transform the above-constructed plasmid vectors into Expi293 cells. After 5 days of cell culture, the supernatant was collected, 1 ml of the cell supernatant was retained for expression detection by ELISA, and the remaining supernatant was used for protein purification.

[0109] For proteins containing hIgG1 Fc or mhIgG Fc, recombinant Protein G affinity chromatography medium (purchased from GenScript) was used for initial separation and purification. 3-5 ml of recombinant Protein G medium was loaded onto the column and equilibrated with 0.02 M PBS buffer (pH 7.4). Cell culture supernatant was then slowly applied to allow for complete binding of the protein to the medium. Unbound nonspecific contaminants were washed with PBS buffer, and eluted with 0.1 M glycine buffer (pH 3.0). The eluate was adjusted to a neutral pH with Tris buffer (pH 9.0). Fine purification was performed using Superdex 200prep grade gel filtration medium (purchased from Cytiva), following the instructions for use. The purified protein was concentrated using Millipore Amicon Ultra-15 30 kD ultrafiltration concentrators and then desalted using a GE PD-10 desalting column with 20 mM PBS buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4).

[0110] For proteins containing His tags, immobilized metal affinity chromatography (IMAC) was used for purification. Take a nickel NTA agarose gel FF pre-packed column and equilibrate it with 0.02M PBS buffer (pH 7.4, containing 10mM imidazole) until the pH of the effluent is 7.4. Centrifuge the sample, add imidazole to a final concentration of 10mM in the sample, and slowly load the sample to allow the protein to fully bind to the chromatography column. Use the equilibration solution to rinse unbound nonspecific impurities and elute with 0.02M PBS buffer (pH 7.4, containing 200mM imidazole). Filter through a 0.22μm filter membrane to obtain the purified bifunctional fusion protein and monomer protein in Table 1, sterile aliquot, and store at ultra-low temperature.

[0111] Example 3 Determination of the expression level of bifunctional fusion protein

[0112] The protein expression level in the culture supernatant of the plasmid transiently transfected cells was determined by ELISA. VEGF165 (purchased from R&D systems) was diluted to 200 ng / mL with carbonate buffer, 100 μL per well, and coated overnight at 2-8°C. The next day, after washing the plate, 1% BSA was added for blocking. The sample was diluted with 1% BSA, and a standard curve and quality control sample were prepared using VBD1 as a reference standard. After blocking, the plate was washed, and 100 μL of the standard curve, quality control sample, and diluted test sample (cell supernatant prepared in Example 2) were added to each well and incubated at room temperature for 1 hour. The plate was washed, and a 1:4000 diluted HRP-conjugated goat anti-human IgG Fc antibody (purchased from Sigma) was added and incubated at room temperature for 1 hour. The plate was washed, TMB was added for color development, the reaction was terminated after 10 minutes, and the OD value at 450 nm was read using a microplate reader. The standard curve was drawn using software, the concentration of the test sample was calculated, and normalization was performed according to the molecular weight. The results are shown in FIG3 , which indicate that the bifunctional fusion protein molecules BFP5, BFP6, BFP7, BFP10, BFP18 and BFP19 all have high transient expression levels.

[0113] Example 4 Electrophoresis Purity Analysis of the Purified Bifunctional Fusion Protein

[0114] To compare the effect of the modified human IgG Fc fragment on the stability of the fusion protein, SDS-PAGE electrophoresis analysis was performed on BFP6, BFP10, BFP18 and BFP19 (prepared in Example 2) after one-step protein G affinity purification. Unstable fusion proteins have a higher tendency to aggregate. The sample was diluted to 1 mg / ml, 1 μL of the diluted sample was taken, 2.5 μL of 4×LDS Sample Buffer (GenScript M00676) and 6.5 μL of ultrapure water were added, mixed and treated at 95°C for 5 minutes, and centrifuged. Take an SDS-PAGE precast gel (GenScript M00652), tear off the tape at the bottom of the gel plate, install it in a micro vertical electrophoresis tank, and pour Tris-MOPS-SDS Running Buffer into the electrophoresis tank. Pull out the comb smoothly, add 10 μL of the treated sample to each well, cover the electrophoresis tank, set a constant voltage of 140V, and run the electrophoresis for 40 minutes.

[0115] The results are shown in Figure 4A. Lane 1 is BFP18 and lane 2 is BFP6. It can be seen that the band at about 140kd is the main band, which is the bifunctional fusion protein prepared in Example 2. Lane 2 has no other bands except the band at about 140kd, while lane 1 has multiple bands with larger molecular weight above the band at about 140kd. From the molecular weight of the adjacent band above the main band (the molecular weight of the adjacent band above the main band is about 2 times that of the main band), it is speculated that the bands are caused by the formation of aggregates by BFP18. Similarly, lanes 3 and 4 also have the above situation. That is, BFP10 is shown in lane 4, except for the band at about 115kd, and there are no other bands, while BFP19 in lane 3 has multiple bands with larger molecular weight above the band at about 115kd.

[0116] In order to further determine whether the miscellaneous bands produced by the above-mentioned BFP18 and BFP19 are aggregates, BFP18 and BFP19 were reduced and then subjected to SDS-PAGE again. The results are shown in Figure 4B. After reduction, there was only one band in lane 2 and lane 4. It can be determined that the miscellaneous band in lane 3 is caused by the formation of aggregates by BFP18, and the miscellaneous band in lane 7 is caused by the formation of aggregates by BFP19.

[0117] The structural differences between BFP6 and BFP18, as well as BFP10 and BFP19, are that BFP6 / BFP10 uses a modified IgG Fc fragment, while BFP18 / BFP19 uses a native IgG Fc fragment. The SDS-PAGE results above show that the monomer purity of the fusion protein containing the modified human IgG Fc fragment is significantly higher than that of the fusion protein containing the native Fc fragment, and the aggregates are significantly reduced. It is generally believed that aggregates can lead to reduced activity of the fusion protein and cause immunogenicity, and unstable fusion proteins have a higher tendency to aggregate. Therefore, the modification of the Fc fragment in the present invention is beneficial to improving the stability of the bifunctional fusion protein, avoiding aggregation, and is safer and more effective.

[0118] Due to the presence of impurities in the purified products of BFP18 and BFP19, the BFP18 and BFP19 proteins will be further purified by molecular sieve before subsequent analytical experiments on the effect of Fc fragment modification on the activity of the bifunctional fusion protein will be conducted.

[0119] Example 5 Determination of Binding Activity of Bifunctional Fusion Protein to VEGF165

[0120] Using VBD as a reference, the binding activity of the bifunctional fusion protein molecule to VEGF165 was investigated. The test method was similar to that described in Example 3, except that the samples investigated were protein samples purified in Example 2. Each protein sample was diluted from 50 nM to 0.00085 nM in a 3-fold gradient. Finally, a four-parameter regression model was used for curve fitting to calculate the binding activity EC of each sample. 50 .

[0121] As shown in Figure 5 and Table 2 below, the results show that the binding ability of the bifunctional fusion protein molecules BFP6 and BFP10 to VEGF165 is not affected, and the Fc fragment modification has no obvious effect on the binding of the fusion molecules to VEGF165; while the binding ability of BFP5 and BFP7 to VEGF165 is affected by the complement binding domain, and the binding ability is significantly reduced (the evaluation standard is that the EC50 ratio is in the range of 80% to 120%, which is considered unaffected).

[0122] Table 2 Binding activity of bifunctional fusion protein to VEGF165

[0123] Example 6: Bifunctional fusion protein inhibits HUVEC cell proliferation activity

[0124] Using VBD as a reference, the biological activity of the bifunctional fusion protein molecule in inhibiting VEGF-dependent HUVEC cell proliferation was investigated. 6×10 cells were seeded per well in a 96-well cell culture plate. 3 HUVEC cells (purchased from Promcell) were grown and allowed to adhere overnight. The next day, purified protein samples were diluted using DMEM + 2% FBS (purchased from Gibco) medium. Each protein sample (purified protein prepared in Example 2) was diluted from 50 nM to 1.3 nM using a 1.5-fold gradient. VEGF165 was diluted to 400 ng / mL using DMEM + 2% FBS medium. Each gradient dilution of protein samples was mixed with VEGF165 in a 1:1 ratio. The original culture medium from the cell wells was aspirated, and 100 μL of the above mixture was added to each well, taking care not to use the edge wells. The treated cells were cultured in a 37°C, 5% CO2 incubator. After 96 hours, the cell plates were removed and 10 μL of CCK-8 colorimetric solution (purchased from Dojindo) was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 3 hours, and the OD values ​​at 450 nm were read using a microplate reader. Finally, the four-parameter regression model was used for curve fitting to calculate the IC value of each sample's biological activity in inhibiting HUVEC cell proliferation. 50 .

[0125] As shown in Figure 6 and Table 3, the results showed that the biological activity of the bifunctional fusion proteins BFP5, BFP6, BFP7 and BFP10 in inhibiting HUVEC cell proliferation was not affected (evaluation was marked as IC50 ratios in the range of 70% to 130%, which was considered unaffected).

[0126] Table 3 Bifunctional fusion protein inhibits HUVEC proliferation activity

[0127] Example 7: Bifunctional fusion protein inhibits VEGF-KDR reporter gene cell activity

[0128] The bifunctional protein has an inhibitory effect on VEGFA-induced transcriptional activation of reporter gene cells. VEGF-KDR reporter gene cells were cultured overnight in a white-walled, transparent-bottomed PDL-coated 96-well cell culture plate. The next day, VEGFA protein (final concentration 10 ng / mL) and a gradient dilution of the protein sample (purified protein prepared in Example 2, starting with a maximum final concentration of 30 nM and diluted 3-fold over 9 concentrations) were added. The 96-well plate was placed in a 37-degree cell culture incubator and incubated for 6 hours. 100 μL / well of Bright-Glo was then added. TM Let the luciferase assay reagent sit for 3 to 5 minutes, then place it in a microplate reader in chemiluminescence mode. Calculate the signal inhibition rate for each concentration gradient, plot a concentration-inhibition rate curve, and calculate the IC50 for each sample.

[0129] As shown in Figure 7 and Table 4, the results showed that BFP6 and BFP10 showed higher inhibitory activity on VEGF-KDR reporter gene cell transcriptional activation, while in general, the addition of a complement domain to a bifunctional fusion protein compared to VBD would increase steric hindrance and reduce activity.

[0130] Table 4 Inhibitory activity of the bifunctional fusion protein on VEGF-KDR reporter gene cells

[0131] Example 8 Bifunctional fusion protein inhibits complement hemolytic activity

[0132] The complex formed by sheep red blood cells and the corresponding antibody (hemolysin) can activate complement in the serum, forming the MAC complex (C5b-9), which causes red blood cell lysis. CBD6 (sCD59) binds to complement C9, preventing the formation of MAC, thereby inhibiting hemolysis.

[0133] First, 2% sheep erythrocytes were mixed with an equal amount of diluted hemolysin (1:2000) and incubated at 37°C for 30 minutes; normal human serum in different proportions was diluted with GVB++ buffer (0.1% gelatin, 5mM Veronal, 145mM NaCl, 0.025% NaN3, pH 7.3), and then the activated sheep erythrocytes were incubated with normal human serum in different proportions at 37°C for 30 minutes to determine the normal human serum dilution that caused 90% lysis of the sheep erythrocytes. The normal human serum dilution that can lyse 90% of the sheep erythrocytes was mixed with 0-20μM CBD6 protein, BFP6, BFP10, BFP18 and BFP19 protein at 37°C for 1 hour, and then the above mixture was incubated with sheep erythrocytes at 37°C for 30 minutes to determine the degree of hemolysis of the sheep erythrocytes. The curve was fitted using a four-parameter regression model to calculate the IC 50 .

[0134] As shown in Figure 8 and Table 5, the results showed that compared with CBD6, the bifunctional fusion proteins BFP6 and BFP10 increased their complement hemolytic activity by 4.6 times and 10 times, respectively, and the bifunctional fusion proteins BFP18 and BFP19 without Fc modification showed similar gain effects.

[0135] Table 5 Bifunctional fusion protein inhibits complement hemolytic activity

[0136] Example 9: Bifunctional fusion protein inhibits laser-induced CNV model in mice

[0137] SPF-grade C57BL / 6J male mice, approximately 2 months old, were purchased and housed in the laboratory for 3-5 days.

[0138] Before modeling, animals were anesthetized with Zotai (25-50 mg / kg, ip) and Xylazine hydrochloride (5 mg / kg, ip) injections, and ophthalmoscopy was used to examine the fundus for abnormalities, and normal animals were selected for inclusion in the group.

[0139] The model was established in both eyes, and a YAG laser photocoagulator (VITRA, Quantel Medical) was used to photocoagulate three points around the optic disc at a distance of 1-1.5 PD from the optic disc (wavelength 532 nm, 250 mW, 50 μm, 100 ms). The laser spot was positioned away from the large retinal blood vessels and the injection site.

[0140] Dosing: On the 3rd day after model establishment, 2 μl PBS and 0.15 mM of each test protein (purified BFP6, BFP10, VBD1, VBD2, CBD6 prepared in Example 2) were injected through the vitreous cavity (IVT).

[0141] On the 10th day after administration, fundus fluorescein angiography (FFA) was performed to evaluate the choroidal neovascularization (CNV) score and leakage area. The scoring criteria are as follows:

[0142] The scores for levels I to IV are 1 to 4 points respectively.

[0143] Representative FFA images of each group are shown in Figure 9, and the leakage scores and leakage area statistics are shown in Table 6. The results show that compared with the PBS group, each test protein can effectively inhibit CNV, and the inhibitory activity of the bifunctional protein is better than that of the monomeric molecule.

[0144] Table 6 CNV score and leakage area Note: *P<0.05, **P<0.01 (one-way analysis of variance).

[0145] Example 10 Preparation of AAV8 Virus Carrying Target Protein Gene

[0146] AAV virus was packaged by co-transfecting VPC2.0 cells with three plasmids, including an auxiliary packaging plasmid, an AAV8 rep-cap plasmid, and each transgenic plasmid GOI.

[0147] The pTT5 vector containing the nucleic acid sequences of the VBD1, VBD2, BFP6, BFP10, and CBD6 encoding genes in Example 2 was double-digested with EcoRI / HindIII, and the target gene fragments were recovered by electrophoresis. The target gene fragments were homologously recombined into the pAAV-MCS vector double-digested with EcoRI / HindIII, transformed into E. coli, and clones were picked and sequenced to obtain pAAV-VBD1, pAAV-VBD2, pAAV-BFP6, pAAV-BFP10, and pAAV-CBD6 vectors, in which the VBD1, VBD2, BFP6, BFP10, and CBD6 encoding genes were cloned into the pAAV-MCS vector, respectively, as GOI plasmids.

[0148] The GOI plasmid, AAV8 rep-cap plasmid, and pHelper helper plasmid must be extensively extracted to a concentration greater than 1 μg / μL, with an A260 / 280 ratio between 1.8 and 2.0 for viral packaging. VPC2.0 cells are cultured in a serum-free suspension culture to a cell density of 1E+6 cells / mL. The three extracted plasmids are mixed in a 1:1:1 molar ratio. The plasmid DNA is then mixed with PEIpro transfection reagent in a 1:2 weight ratio based on the total mass. After incubation at room temperature for 20 minutes, the cells are slowly added to the cell suspension (at a cell to plasmid ratio of 1 mL:1 μg). The cells are then cultured in a shaker at 37°C with 8% CO2 for 3 days, and the cell suspension is harvested.

[0149] The cell suspension was centrifuged at 10,000 g for 10 minutes. The resulting supernatant was transferred to a fresh centrifuge tube. The resulting cell pellet was resuspended in a small amount of PBS and repeatedly freeze-thawed to lyse the cells. After freeze-thaw, the cells were centrifuged again at 10,000 g for 10 minutes, and the supernatant was collected. The supernatants from both centrifugations were combined and filtered through a 0.45 μm filter to remove impurities. Half a volume of 1 M NaCl and 10% PEG 8000 solution was added, mixed thoroughly, and incubated overnight at 4°C. Centrifuged at 12,000 rpm for 2 hours, the supernatant discarded, and the viral pellet was dissolved in an appropriate amount of PBS. Once completely dissolved, it was sterilized by filtering through a 0.22 μm filter. Benzonase nuclease was added to digest and remove residual plasmid DNA (final concentration 50 U / ml). The tube was capped and inverted several times to mix thoroughly. Incubated at 37°C for 30 minutes. Filtered through a 0.45 μm syringe filter, the filtrate was the concentrated AAV virus.

[0150] Solid CsCl was added to the viral concentrate until the density reached 1.41 g / ml (refractive index 1.372). The sample was then added to an ultracentrifuge tube, and the remaining space in the tube was filled with a pre-prepared 1.41 g / ml CsCl solution. The tube was centrifuged at 175,000 g for 24 hours to form a density gradient. Samples of varying densities were collected sequentially and titered. Fractions enriched with AAV particles were collected.

[0151] Repeat the above process once. Place the virus into a 100 kDa dialysis bag and dialyze overnight at 4°C for desalination. The dialysis buffer consists of PBS containing 0.001% Pluronic F68, pH 7.2. The resulting dialyzed sample is the purified AAV virus and can be used for in vivo efficacy testing.

[0152] The AAV viruses used in the present invention are all prepared by the above-mentioned method. By packaging different GOI plasmids, AAVs carrying different target genes can be prepared. The above-mentioned method can obtain AAV8-VBD1 virus carrying VBD1, AAV8-VBD2 virus carrying VBD2, AAV8-BFP6 virus carrying BFP6, AAV8-BFP10 virus carrying BFP10, and AAV8-CBD6 virus carrying CBD6.

[0153] Example 11 AAV8 virus carrying target gene inhibits MAC deposition in vitro

[0154] At the terminal end of the complement activation pathway, C5b stably binds to C6 to form C5b6, which then spontaneously binds to C7 to form C5b67. C7 in this complex initially inserts into the lipid bilayer of the target cell membrane. C8 then binds with high affinity to the membrane-inserted C5b67, forming C5b678, which is stable and deeply inserted into the cell membrane. This complex then binds to 12–18 C9 molecules to form C5b6789n, the membrane-attacking complex (MAC). CD59 is a MAC inhibitory protein that prevents C9 from aggregating to the C5b678 complex, thereby preventing MAC formation.

[0155] 2E5 / well hepa-1c1c7 cells were seeded in a 6-well plate containing a poly-D-lysine coated slide and incubated overnight. The cells were infected with AAV8-BFP6, AAV8-BFP10 and AAV8-CBD6 at an MOI of 2E5 vg / cell, and an uninfected control group was set up. After 72 hours, the cells were washed twice with PBS, 10% normal human serum was prepared with GVB++ buffer, added to the cell wells and incubated at 37°C for 5 minutes. The normal human serum was aspirated, and the cells were immediately washed 3 times with pre-cooled PBS, and then fixed with 4% paraformaldehyde for 15 minutes. After fixation, they were washed 3 times with PBS, and mouse Anti-C5b-9+C5b-8 antibody [aE11] (Abcam, ab66768) was added and incubated for 2.5 hours. After washing, Goat Anti-Mouse IgG H&L was added. Pre-adsorbed (Abcam, ab97035) was incubated in a dark box for 1.5 hours. After washing, DAPI was added for staining for 1 minute, and the slide was washed twice more. A drop of Fluoromount-G fluorescent mounting medium was added, and the slide was inverted on a glass slide. MAC staining results were observed under a fluorescence microscope. Representative images of MAC staining under each condition are shown in Figure 10. Each group of images was processed using ImageJ software, and the total fluorescence intensity was calculated after background subtraction. The statistical results are shown in Figure 11. The results show that AAV8-BFP10 and AAV8-BFP6 exhibited higher inhibitory activity against MAC deposition than AAV8-CBD6.

[0156] Example 12 In vivo transgenic expression of AAV8 virus carrying target gene

[0157] 6-8 week old C57BL / 6 mice were raised in the laboratory for 3-5 days. Animals with no abnormalities in both eyes were screened for subretinal injection and given AAV8-VBD1, AAV8-VBD2, AAV8-BFP6 and AAV8-BFP10, respectively, at a dose of 3E8vg / eye. After the subretinal injection, ofloxacin eye ointment was applied for 2 consecutive days, twice a day. One week, two weeks and four weeks after administration, the animals were euthanized, the eyeballs were taken, 200μl / eye of PBS (containing protease inhibitors) were added for homogenization, and the supernatant was taken by centrifugation. The expression of transgenic proteins was detected using the method of Example 3. As shown in Figure 12, the results show that each AAV molecule can achieve stable expression of transgenic proteins.

[0158] Example 13 AAV8 virus carrying target gene inhibits sodium iodate-induced dry AMD mouse model

[0159] Six- to eight-week-old C57BL / 6J mice were housed in the laboratory for 3-5 days. Groups were divided and subretinally injected with PBS, AAV8-CBD6, AAV8-BFP6, or AAV8-BFP10 at a dose of 3E8 vg / eye in a 1 μl volume. Twenty-one days later, 40 mg / kg sodium iodate was injected via the tail vein to establish the model. Optical coherence tomography (OCT) and retinal membrane potential (ERG) were performed on days 3 and 7 after model establishment to measure changes in retinal outer nuclear layer thickness and ERG amplitude.

[0160] The results are shown in Figure 13. As time goes on, sodium iodate causes changes in mouse retinal function, which is manifested as a decrease in the ERG dark-adapted b-wave amplitude. Compared with the control PBS group, AAV8-BFP6 and AAV8-BFP10 showed a significant inhibitory effect on the decrease in ERG amplitude; and compared with AAV8-CBD6, AAV8-BFP6 and AAV8-BFP10 have obvious advantages in improving the dry AMD mouse model.

[0161] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims.

Claims

1. A bifunctional fusion protein, characterized in that, The bifunctional fusion protein comprises a VEGF binding domain, a modified human IgG Fc domain, and a complement binding domain; The VEGF binding domain comprises extracellular domain 2 of VEGF receptor 1, the complement binding domain comprises soluble CD59 domain structure sCD59, the modified human IgG Fc domain and sCD59 are linked by a linker peptide (GGGGS)3, and the structure of the bifunctional fusion protein is: VEGFR1D2-mhIgG Fc-(GGGGS)3-sCD59, and the amino acid sequence is as shown in SEQ ID NO.2; or, The VEGF binding domain comprises extracellular domain 2 of VEGF receptor 1 and extracellular domain 3 of VEGF receptor 2, the complement binding domain comprises soluble CD59 domain structure sCD59, the modified human IgG Fc domain and sCD59 are linked by a linker peptide (GGGGS)3, and the structure of the bifunctional fusion protein is: VEGFR1D2-VEGFR2D3-mhIgG Fc-(GGGGS)3-sCD59, and the amino acid sequence is as shown in SEQ ID NO.

1.

2. The coding gene of the bifunctional fusion protein according to claim 1.

3. An expression cassette or recombinant vector for expressing the bifunctional fusion protein according to claim 1, characterized in that, The expression cassette comprises the following structure as shown in Formula I from the 5'-3' end: E1-E2-E3-E4 (Formula I) Wherein: E1 is a promoter; E2 is a signal peptide; E3 is the nucleotide sequence encoding the bifunctional fusion protein according to claim 1 or 2; E4 is a Poly A sequence.

4. A recombinant host cell comprising the coding gene according to claim 2, or the expression cassette according to claim 3, or a recombinant vector.

5. An AAV virus comprising the coding gene according to claim 2 or the expression cassette according to claim 3, characterized in that, The gene or the expression cassette is cloned between two terminal repeats ITRs of an adeno-associated virus AAV backbone to construct a plasmid of the target gene GOI, which is applied to an AAV packaging vector system. The packaging vector system comprises: the plasmid of the target gene GOI, a vector carrying AAV rep and cap genes, and an auxiliary vector. The above vectors are transiently transfected into production cells by three plasmids to be packaged into AAV viruses.

6. A pharmaceutical composition, characterized in that, Comprising the bifunctional fusion protein according to claim 1, the coding gene according to claim 2, the recombinant host cell according to claim 4, or the AAV virus according to claim 5.

7. Use of the bifunctional fusion protein according to claim 1, or the encoding gene according to claim 2, or the expression cassette according to claim 3, or the recombinant host cell according to claim 4, or the AAV virus according to claim 5, or the pharmaceutical composition according to claim 6, characterized in that It is used in the preparation of a preparation for treating age-related macular degeneration.

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