Bispecific fusion proteins targeting vegf-a and ang-2 and uses thereof

Abstract

This invention discloses a bispecific fusion protein targeting VEGF-A and ANG-2 and its applications. The fusion protein adopts a "VHH-Linker-ScFv" conformation, consisting of an anti-VEGF-A nanobody (VHH), a flexible linker peptide (Linker), and an anti-ANG-2 single-chain antibody (ScFv). Experimental results show that this fusion protein can bind to both VEGF-A and ANG-2 with high affinity, significantly inhibiting HUVEC cell proliferation and tube formation at the cellular level, with an inhibition rate exceeding 90%. In a mouse CNV model, this fusion protein significantly reduces fluorescein leakage from fundus vessels and substantially inhibits neovascularization. This invention overcomes the compensatory proliferation and efficacy limitations of single-target therapy through a dual-target synergistic blocking mechanism, demonstrating high clinical application potential in the treatment of fundus neovascular diseases such as wet age-related macular degeneration (nAMD).

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and genetic engineering. Specifically, it relates to a method for constructing, expressing, and purifying a bispecific fusion protein targeting vascular endothelial growth factor A (VEGF-A) and angiopoietin 2 (ANG-2), and its application in the preparation of drugs for treating neovascular diseases of the fundus. Background Technology

[0002] With the increasing aging of the global population, neovascularization of the fundus, such as wet age-related macular degeneration (nAMD), has become one of the leading causes of vision loss. In the pathological process of these diseases, VEGF-A, as a core pro-angiogenic factor, has long been a major target for clinical treatment. However, studies have found that simply inhibiting VEGF-A often induces compensatory increases in other angiogenic factors such as ANG-2, leading to a plateau in the effectiveness of anti-angiogenic therapy, and even relapse and incomplete vascular barrier repair.

[0003] Current clinical treatments largely rely on single-target antibodies, which, while alleviating some leakage symptoms, have significant limitations in improving the long-term stability of choroidal neovascularization (CNV) and reducing compensatory proliferation. Therefore, developing a bispecific drug that can simultaneously block the VEGF-A and ANG-2 signaling pathways, exert a synergistic anti-angiogenic effect, and effectively repair the vascular barrier has become a pressing technical challenge in the field of ophthalmic biopharmaceuticals. This invention aims to construct a high-affinity, structurally stable bispecific fusion protein through molecular engineering, with the goal of improving clinical treatment efficacy and providing better treatment options for fundus vascular diseases. Summary of the Invention

[0004] To address the aforementioned problems, this invention first provides a bispecific fusion protein targeting VEGF-A and ANG-2. This fusion protein adopts a molecular configuration of "VHH-Linker-ScFv". The anti-VEGF-A portion is a nanobody (VHH), whose amino acid sequence is shown in SEQ ID NO:1; the anti-ANG-2 portion is a single-chain antibody (ScFv), which includes a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:3.

[0005] In some embodiments, in the anti-ANG-2 ScFv, the heavy chain variable region and the light chain variable region are spliced ​​together by a (Gly4Ser)3 flexible linker peptide, the full sequence of which is shown in SEQ ID NO:4. The VHH domain and the ScFv domain are linked by a flexible linker consisting of 35 amino acids, the sequence of which is shown in SEQ ID NO:5.

[0006] In some embodiments, the full sequence of the fusion protein is shown in SEQ ID NO:7. This design was optimized through molecular dynamics simulations to ensure a molecular weight between 45-50 kDa, reducing steric hindrance between structural domains while ensuring full exposure of the binding pocket in the CDR region, thereby achieving high-affinity binding to both targets.

[0007] This invention also provides a preparation method comprising the following steps: first, codon optimization of the full sequence for the *E. coli* expression system, adjusting GC content and eliminating internal splicing sites; cloning the synthesized target gene into a high-level expression vector such as pET-28a(+) to construct an expression plasmid; transforming the plasmid into *E. coli* host cells (such as BL21(DE3)); inducing expression at 16°C to improve folding accuracy; and finally obtaining a fusion protein product with a purity higher than 95% through Ni-NTA affinity chromatography and dialysis displacement.

[0008] Finally, the present invention provides an application in the preparation of a medicament for treating neovascular diseases of the fundus.

[0009] In some embodiments, the fundus neovascular disease is preferably wet age-related macular degeneration (nAMD) or laser-induced choroidal neovascularization (CNV).

[0010] Compared with the prior art, the present invention has at least the following beneficial effects: Extremely high binding affinity: The affinity constant K of the fusion protein of this invention for VEGF-A D It stabilized at the picomolar (pM) level (averaging approximately 4.82 × 10⁻⁶). -11 M), K for ANG-2 D The value is approximately 1.89 nM, which ensures the stable presence and potent capture of the drug at the target site.

[0011] Significant synergistic activity at the cellular level: In HUVEC cell tube formation assays, the relative inhibition rate of this fusion protein at a concentration of 100 nM was as high as 92.8%, which was significantly better than the individual effects of single-target nanobody (57.5%) or ScFv (37.7%), demonstrating the synergistic effect of dual-target combined blocking.

[0012] Superior in vivo therapeutic performance: In a mouse CNV model, this invention can reduce the proportion of severe fluorescein leakage from 85.0% in the model group to 12.5%, and achieve an 83.2% inhibition rate on neovascularization area, effectively breaking the compensatory feedback mechanism commonly found in single-target therapy.

[0013] This invention is the first to construct a VHH-Linker-ScFv bispecific fusion protein with a specific sequence combination and optimized Linker length. It not only exhibits strong biological activity in vitro, but also demonstrates significant technical advantages in repairing the vascular barrier and inhibiting compensatory proliferation of new blood vessels in in vivo experiments. Attached Figure Description

[0014] Figure 1 A schematic diagram of the molecular model of the bispecific fusion protein (V / A-BiFP) of this invention (left) and an SDS-PAGE electrophoresis identification image (right). Detailed Implementation

[0015] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0016] Example 1: Molecular design, expression, and purification of a bispecific fusion protein targeting VEGF-A and ANG-2 Preparation and Sequence Identification of Anti-VEGF-A Nanobodies: First, recombinant human VEGF-A with a His tag (catalog number JN0245) provided by Beijing BioLab Technology Co., Ltd. was used as an immunogen. After being emulsified with Freund's adjuvant, alpacas were inoculated with multiple booster immunizations to induce high-titer heavy chain antibodies. Once serum titers reached the target, peripheral blood was collected. Lymphocytes were separated using Ficoll density gradient centrifugation, and total RNA was extracted. cDNA was synthesized via reverse transcription. Then, nested PCR technology was used to amplify the heavy chain antibody gene fragment first, and then the VHH gene fragment of approximately 350-400 bp was specifically amplified using this fragment as a template. This fragment was then cloned into a phage display vector and electroporated into TG1 competent cells to construct a library with a capacity greater than 10. 8 The CFU VHH phage display library was then used. Following this, recombinant human VEGF-A (catalog number JN0245) protein was used as bait, and high-affinity clones were enriched through 3-4 rounds of adsorption-elution-amplification biopanning. Strongly positive single clone 1G3 was selected by Phage-ELISA and sequenced. Its amino acid sequence is as follows:

[0017] EVQLVESGGGGSRLSCAASGTFWAYSSHVMGWFRQRQAPKEREGVAAISVDSGSTTAVYYCAARRIDAADFDSLRAEDTATYYAFWSWMYADSVKGRFTISRDNLTLSPETYDYWGQGTQVTVSS (SEQ ID NO: 1).

[0018] Preparation and Sequence Identification of Anti-ANG-2 ScFv: In this embodiment, a biotinylated angiopoietin 2 (ANGPT2) protein fragment (tagged with Avi&His) from UABIO (brand name, catalog number UA010902) was used as an immunogen. The ANGPT2 protein fragment (UA010902) was dissolved in sterile PBS, mixed with an equal volume of Freund's complete adjuvant (FCA), and thoroughly emulsified. Balb / c mice aged 6-8 weeks were given a primary immunization at multiple sites in the groin and subcutaneous region, with an immunization dose of 50 μg per mouse. Subsequently, booster immunizations were performed every 3 weeks using the same dose of protein emulsified with Freund's incomplete adjuvant (FIA), for a total of 3 booster immunizations. After the final immunization, the serum titer of the mice was detected by ELISA. Mice with the highest titer were selected, and their spleens were aseptically harvested and ground. Total RNA was extracted using the Trizol method. After reverse transcription to synthesize cDNA, the light chain variable region (VL) and heavy chain variable region (VH) genes of mouse immunoglobulins were amplified by PCR using degenerate primers. Subsequently, using overlap extension PCR (SOE-PCR), a DNA sequence encoding a (Gly4Ser)3 flexible linker peptide was introduced between VH and VL, splicing VH and VL into a single-chain antibody gene fragment (ScFv). This ScFv fragment was then digested with SfiI and NotI restriction endonucleases, ligated into the phage display vector pCANTAB5E, and electroporated into *E. coli* TG1 competent cells to construct a scale of at least 10T. 7 A CFU anti-ANG-2 phage display library was prepared. Using the biotinylated tag of the UA010902 protein, screening was performed using magnetic-activated cell sorting. Biotinylated ANGPT2 protein was bound to streptavidin magnetic beads (M-280) to form an antigen-magnetic bead complex. The phage library and this complex were incubated at 4°C for 2 hours, followed by washing 10-15 times with PBST to thoroughly remove background binding. Specifically bound phages were eluted with 0.1M glycine buffer (pH 2.2) and rapidly neutralized with Tris. This process was repeated three times, decreasing the amount of antigen and increasing the washing pressure in each round to selectively enrich high-affinity single clones. Single clones were randomly selected from the third-round enrichment plate, and positive wells were identified by phage ELISA. Clones with an OD450 value greater than 3 times that of the control were sequenced. Sequence analysis based on the IMGT antibody database determined the amino acid composition of the heavy chain variable region and the light chain variable region, and their amino acid sequences are as follows:

[0019] Heavy chain variable region: QVQLQQSGYDYQSPSSPSEEVEASTYSKITCRASGYFITGDNSSIAYLQLEWIAQIFPADTISGVPPTEESAVYLMDYHPASQGTSVTVPGAVGTKRYWDMGRATFPGAVGMDYWGQGTTVTVSS (SEQ IDNO: 2).

[0020] Light chain variable region: DIQLTQSPASLASISPGERATISCKASQDVSTPPKLLIYDNLVSGLLIYGASSRATGVPRFSGSGSGTDFTLTISIHPVEKVDAATYAQQSAQRFSGSQSYSTPYTFGQGTRLEIK (SEQ ID NO: 3).

[0021] ScFv for ANG-2 resistance: QVQLQQSGYDYQSPSSPSEEVEASTYSKITCRASGYFITGDNSSIAYLQLEWIAQIFPADTISGVPPTEESAVYLMDYHPASQGTSVTVPGAVGTKRYWDMGRATFPGAVGMDYWGQGTTVTVSSGGGGS GGGGSGGGGSDIQLTQSPASLASISPGERATISCKASQDVSTPPKLLIYDNLVSGLLIYGASSRATGVPRFSGSGSGTDFTLTISIHPVEKVDAATYAQQSAQRFSGSQSYSTPYTFGQGTRLEIK (SEQ ID NO:4).

[0022] Construction, expression and purification of bispecific fusion protein: In order to maintain the spatial independence of the two functional domains and achieve synergistic binding, this experiment designed a bispecific fusion protein with the conformation of “VHH-Linker-ScFv” (named: V / A-BiFP).

[0023] A flexible linker consisting of 35 amino acids, (Gly4Ser)7 (SEQ ID NO:5), was introduced between the C-terminus of the 1G3 nanobody and the N-terminus of the 3G7ScFv. This length was optimized using molecular dynamics simulations to ensure good flexibility of the fusion protein, with a molecular weight of approximately 43-45 kDa, within the vitreous body, avoiding steric hindrance between domains. A 6×His tag (SEQ ID NO:6) was tandemly attached to the C-terminus of the ScFv for subsequent affinity chromatography purification. Simultaneously, codon optimization was performed on the full sequence for the *E. coli* expression system, adjusting the GC content to approximately 55% and eliminating hidden internal splicing sites and stop codons.

[0024] Flexible Linker (Gly4Ser) 7: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5); 6×His tag: HHHHHH (SEQ ID NO:6); Full sequence of the bispecific fusion protein (V / A-BiFP): EVQLVESGGGGSRLSCAASGTFWAYSSHVMGWFRQRQAPKEREGVAAISVDSGSTTAVYYCAARRIDAADFDSLRAEDTATYYAFWSWMYADSVKGRFTISRDNLTLSPETYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGYDYQSPSSPSEEVEASTYSKITCRASGYFITGDNSSIAYLQLEWI AQIFPADTISGVPPTEESAVYLMDYHPASQGTSVTVPGAVGTKRYWDMGRATFPGAVGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPASLASISPGER ATISCKASQDVSTPPKLLIYDNLVSGLLIYGASSRATGVPRFSGSGSGTDFTLTISIHPVEKVDAATYAQQSAQRFSGSQSYSTPYTFGQGTRLEIKHHHHHH (SEQ ID NO:7); The fusion protein was assembled using [1G3VHH]+[Linker(SEQ5)]+[3G7ScFv(SEQ4)]+[6×His(SEQ6)], and its two-dimensional structure was analyzed using the Swissmodel online website. (See attached image.) Figure 1 .

[0025] The results showed that the two globular functional domains, VHH and ScFv, were spatially distinct and independently distributed, effectively separated by a flexible linker extending 35 aa in the middle. This extended conformation formed by the linker significantly reduced the steric hindrance between the domains, ensuring full exposure of the binding pockets in their respective CDR regions, enabling high-affinity binding to both VEGF-A and ANG-2 simultaneously. The overall QMEAN score indicated good model quality, stable folding, and no anomalous polymerization tendency, validating the rationality of the molecular design.

[0026] The optimized fusion protein full-gene sequence (SEQ ID NO:7) was artificially synthesized by GenScript Biotech Co., Ltd. Using NdeI and XhoI double restriction sites, the target gene was cloned into the high-level expression vector pET-28a(+). The cloning accuracy was verified by bidirectional DNA sequencing, and the constructed expression vector was named pET-28a-V / A-BiFP. The plasmid was transformed into *E. coli* expression host BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and cultured overnight at 37°C. Single colonies were selected and inoculated into 500 mL TB medium, cultured at 37°C with shaking until the OD600 reached 0.6-0.8. IPTG (final concentration 0.5 mM) was added, and the temperature was switched to 16°C for 18 hours for induction. Low-temperature induction helps improve the folding accuracy of the ScFv moiety and reduces inclusion body formation. The cells were then collected by centrifugation at 4°C and 8000 rpm for 20 min. The bacterial pellet was resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM Imidazole, pH 8.0) and sonicated. The supernatant was passed through a Ni-NTA affinity chromatography column (HisTrapHP) using a gradient elution with elution buffers containing different concentrations of imidazole. The 250 mM imidazole eluent was collected and dialyzed through a HiTrapDesalting column to PBS (pH 7.4) buffer. The purified protein sample was then subjected to boiling denaturation with reducing loading buffer and SDS-PAGE electrophoresis. (See attached image). Figure 1 .

[0027] The results showed a clear, single protein band at approximately 43 kDa, consistent with the theoretical molecular weight. Protein concentration was determined using the BCA method, ultimately yielding a fusion protein product with a purity exceeding 95%.

[0028] Example 2: Determination of antigen-binding affinity of the bispecific fusion protein for VEGF-A and ANG-2 This experiment used a Biacore T200 system (Cytiva) to determine the binding kinetics parameters of the fusion protein (SEQ ID NO:7) and the antigen; recombinant human VEGF-A from Beijing Bio-Lab (JN0245) and biotinylated ANG-2 from Youai Bio (UA010902) were used. Streptavidin (SA) was coupled to the surface of the CM5 sensor chip using an amino-coupled assay kit. The experiment was performed in HBS-EP+ buffer (pH 7.4). The antigen was diluted to 5 μg / mL and injected into the channel at a flow rate of 10 μL / min, with the capture level controlled at 100-200 RU to eliminate mass transfer limitation effects. Simultaneously, the fusion protein (V / A-BiFP) prepared in Example 1 was serially diluted to multiple concentration groups of 0.125, 0.25, 0.5, 1, 2, 4, and 8 nM; the fusion protein was flowed through the chip surface at a flow rate of 30 μL / min, with a binding time of 180 s. The dissociation process was switched to continuous rinsing with buffer, and the dissociation signal was recorded for 600 seconds. Regeneration was performed using 10 mM Glycine-HCl (pH 1.5) to ensure that the active groups on the chip surface were restored to baseline levels. Global fitting was performed using Biacore T200 Evaluation software with a 1:1 Langmuir binding model.

[0029] The binding process of V / A-BiFP to VEGF-A was monitored using a Biacore T200. The antigen capture volume was set to approximately 120 RU, and the dilution concentration of V / A-BiFP in the mobile phase was set to range from 8 nM to 0.125 nM.

[0030] Table 1: Detailed data on the binding kinetics of V / A-BiFP and VEGF-A The results showed that the binding curve for VEGF-A exhibited extremely high fitting accuracy (Chi). 2 (All less than 0.5), its affinity constant K D Stable at the pimolecular (pM) level, demonstrating the extremely strong capture capability of the VHH segment.

[0031] Biotinylated ANGPT2 (UA010902) was captured using an SA chip, with a capture amount set to approximately 200 RU. The concentration gradient remained constant, taking into account the binding characteristics of the ScFv moiety.

[0032] Table 2. Detailed data on the binding kinetics of V / A-BiFP and ANG-2. The results showed that the binding assay for ANG-2 exhibited a good concentration-dependent effect, with K2... DThe value is approximately 1.89 nM. Although the binding rate K... a Slightly lower than VEGF-A arms, but with a dissociation rate K D It remained at a low level, ensuring the stable presence of the antibody at the target site.

[0033] Example 3: Evaluation of the inhibitory activity of the bispecific fusion protein at the cellular level This embodiment evaluates the inhibitory activity of the bispecific fusion protein V / A-BiFP on the synergistic induction effect of VEGF-A and ANG-2 through human umbilical vein endothelial cell (HUVEC) proliferation and tube formation assays.

[0034] Anti-VEGF-A nanobody: The 1G3VHH gene sequence (SEQ ID NO: 1) was cloned into the pET-28a(+) expression vector using NdeI and XhoI restriction endonucleases, and a 6×His tag was fused to its C-terminus. The constructed recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and cultured in kanamycin-containing medium to the logarithmic growth phase, followed by IPTG induction of expression. The bacterial cells were collected, sonicated, and purified using a Ni-NTA affinity chromatography column via the His tag. Finally, the purified cells were dialyzed to PBS buffer for later use.

[0035] Preparation of anti-ANG-2 ScFv antibody: The 3G7ScFv gene sequence (SEQ ID NO:4) was cloned into the pET-28a(+) vector and tagged with a C-terminal His tag using the same method described above. After transformation into E. coli BL21(DE3), a low-temperature induction strategy (16°C) was employed to improve protein folding efficiency. Single-chain antibody protein with a purity greater than 95% was obtained by nickel column affinity chromatography for subsequent cell viability comparison experiments.

[0036] HUVEC cell proliferation inhibition assay: HUVEC cells were seeded in 96-well plates, approximately 5 × 10⁶ cells per well. 3 Cells were cultured in ECM medium containing 10% FBS for 24 hours to allow them to adhere. They were then starved for 12 hours in serum-free medium. The experiment was divided into the following groups:

[0037] Blank group: only culture medium was added, without any stimulation.

[0038] Model group: Recombinant human VEGF-A (50 ng / mL) and recombinant human ANG-2 (200 ng / mL) were added for combined stimulation.

[0039] Control group 1: Model group stimulating factor + anti-VEGF-A antibody VHH (100nM).

[0040] Control group 2: Model group stimulating factor + anti-ANG-2 antibody ScFv (100nM).

[0041] V / A-BiFP group: Model group stimulating factor + different concentrations of V / A-BiFP (10nM, 100nM).

[0042] After 48 hours of drug treatment, 10 μL of LCK-8 reagent was added to each well and incubated for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the cell proliferation inhibition rate was calculated.

[0043] Table 3: Effects of each group on HUVEC cell proliferation The results showed that V / A-BiFP exhibited a significant concentration-dependent inhibitory effect. At a concentration of 100 nM, the inhibition rate of V / A-BiFP (93.7%) was significantly higher than that of equimolar concentrations of single-target nanobodies or ScFv antibodies, indicating that the combined blocking of the two targets had a significant synergistic effect.

[0044] HUVEC cell tube formation assay: Matrigel (50 μL / well) was pre-coated into 96-well plates and incubated at 37°C for 30 minutes to solidify. HUVEC cells were resuspended in culture medium containing different drugs and stimulating factors (VEGF-A 50 ng / mL + ANG-2 200 ng / mL) and seeded onto Matrigel surfaces (2 × 10⁶ cells / well). 4 (Number of cells / well). Grouping method: Same as the HUVEC cell proliferation inhibition assay, V / A-BiFP concentration was set at 100 nM. Detection indicators: After incubation for 8-12 hours, the cells were observed and photographed under a microscope. The number of nodes and total length of the tubular structures were counted using ImageJ software.

[0045] Table 4: Effect of each group on HUVEC tube forming capability This table shows the inhibitory effects of each drug group on the tubular formation ability of endothelial cells in a simulated neovascular microenvironment.

[0046] This experiment objectively evaluated the anti-angiogenic activity of V / A-BiFP by statistically analyzing two key indicators: common tube length and the number of nodes. The results showed that, under the combined induction of VEGF-A and ANG-2, the model group achieved the highest values ​​for both common tube length (5860 μm) and the number of nodes (68), indicating that endothelial cells formed a dense vascular-like network.

[0047] In comparison, the V / A-BiFP group (100 nM) performed significantly better than the single-target control groups, with its total tube length shortened to 1580 μm, the number of nodes reduced to 15, and a relative inhibition rate as high as 92.8%. This level of inhibition is far higher than the simple sum of nanobody (57.5%) and ScFv (37.7%).

[0048] This demonstrates that V / A-BiFP is not simply a product of component stacking, but rather, through a dual-target synergistic blocking mechanism, it almost completely reverses the tube-forming effect induced by VEGF-A and ANG-2 at the cellular level. This fusion protein possesses extremely strong anti-angiogenic potential, providing a solid biological basis for its clinical application in the treatment of retinal vascular diseases.

[0049] Example 4: Study on the therapeutic effect of bispecific fusion protein on a mouse laser-induced CNV model This embodiment establishes a mouse laser-induced choroidal neovascularization (CNV) model to evaluate the in vivo therapeutic effect of the bispecific fusion protein V / A-BiFP on fundus neovascular diseases.

[0050] Fifty male SPF-grade C57BL / 6J mice aged 6-8 weeks were randomly divided into 5 groups of 10 mice each: Normal group: No laser modeling was performed, and no medication was administered.

[0051] Model group: After laser modeling, an equal volume of PBS was injected into the vitreous cavity.

[0052] Control group 1: After laser modeling, anti-VEGF-A antibody VHH (0.5 mg / mL) was injected into the vitreous cavity.

[0053] Control group 2: After laser modeling, anti-ANG-2 antibody ScFv (0.5 mg / mL) was injected into the vitreous cavity.

[0054] V / A-BiFP treatment group: After laser modeling, the bispecific fusion protein V / A-BiFP (0.5 mg / mL) was injected into the vitreous cavity.

[0055] CNV model establishment: Mice were anesthetized intraperitoneally with 1% sodium pentobarbital and mydriatics were dilated using compound tropicamide eye drops. Under slit-lamp guidance, photocoagulation was performed around the optic disc of the mice using a 532nm laser (120mW power, 0.1s duration, 50μm spot diameter), avoiding the major retinal vessels. Four points were targeted per eye. The appearance of tiny bubbles at the photocoagulation site (a sign of Bruch's membrane rupture) was considered a successful model establishment.

[0056] Administration method: On day 1 after modeling, mice in each group were injected intravitreally. The injection volume was 2 μL / eye, and care was taken to avoid damaging the lens during the procedure.

[0057] Fundus fluorescein angiography (FFA): On day 14 after modeling, 5 mice were randomly selected from each group and injected intraperitoneally with 10% sodium fluorescein. Fluorescein leakage at the laser photocoagulation point was dynamically observed using a fundus imaging system. Leakage was scored according to the degree of leakage (0: no leakage; 1: weak leakage; 2: moderate leakage; 3: strong leakage).

[0058] Table 5: Distribution of CNV fluorescein leakage scores in each group of mice The fluorescein fundus angiography (FFA) grading system directly reflects the ability of different drugs to improve vascular permeability. In the model group, the proportion of grade 2-3 severe leakage was as high as 85.0%, indicating that laser damage induced severe vascular barrier dysfunction. While blocking VEGF-A (control group 1) or ANG-2 (control group 2) alone reduced the proportion of severe leakage, significant leakage was still observed in approximately 47.5%-60.0% of photocoagulation sites, suggesting limitations in single-target therapy in maintaining vascular stability. In stark contrast, the V / A-BiFP fusion protein group increased the proportion of grade 0-1 mild leakage to 87.5%, while reducing the severe leakage rate to 12.5%. This data demonstrates that V / A-BiFP is not simply a simple additive effect, but rather a dual-target approach that synergistically repairs the damaged barrier, exhibiting significant therapeutic advantages in improving vascular leakage, a core clinical symptom.

[0059] FITC-Dextran perfusion of choroidal spreads: After FFA examination, mice were euthanized and their eyes were enucleated. The retina and choroid-sclera complex were dissected. FITC-Dextran was used for perfusion staining, and choroidal spreads were photographed under a fluorescence microscope. The area (μm2) of CNV at each photocoagulation point was calculated using image analysis software.

[0060] Table 6: Results of CNV area measurement in each group of mice Table 6 provides quantitative analysis data that further validates the inhibitory effect of the fusion protein on angiogenesis from a physical pathology perspective. Based on approximately 18540±2150 typical lesions in the model group, the single-target control group 1 and control group 2 achieved area inhibition of 50.2% and 32.8%, respectively. However, the V / A-BiFP treatment group significantly reduced the CNV area to 3120±480, with an inhibition rate as high as 83.2%; that is, simply inhibiting VEGF often induces compensatory increases in factors such as ANG-2, leading to a plateau in efficacy. V / A-BiFP, by simultaneously severing two major angiogenesis signaling pathways, breaks this compensatory feedback, thereby achieving near-complete blockade of choroidal angiogenesis proliferation in vivo.

[0061] The above results indicate that V / A-BiFP's simultaneous targeting of VEGF-A and ANG-2 produces a synergistic therapeutic effect, not only significantly reducing vascular leakage but also more effectively inhibiting compensatory angiogenesis. These in vivo efficacy results validate the potential clinical value of the bispecific fusion protein of this invention in treating fundus neovascular diseases such as wet age-related macular degeneration (nAMD).

[0062] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bispecific fusion protein targeting VEGF-A and ANG-2, characterized in that, The fusion protein comprises a VHH domain against VEGF-A, a ScFv domain against ANG-2, and a connecting peptide Linker connecting the VHH domain and the ScFv domain.

2. The bispecific fusion protein of claim 1, wherein: The amino acid sequence of the VHH domain against VEGF-A is shown as SEQ ID NO: 1; the ScFv domain against ANG-2 comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO: 2 and a light chain variable region with an amino acid sequence shown as SEQ ID NO: 3; preferably, the amino acid sequence of the ScFv domain against ANG-2 is shown as SEQ ID NO:

4.

3. The bispecific fusion protein of claim 1, wherein, The amino acid sequence of the connecting peptide Linker is shown as SEQ ID NO: 5, and the configuration of the fusion protein is VHH-Linker-ScFv.

4. The bispecific fusion protein of any one of claims 1-3, wherein, The amino acid sequence of the fusion protein is shown as SEQ ID NO: 7, or a sequence further connected with a His tag at the C terminal of the sequence shown as SEQ ID NO:

7.

5. An isolated nucleic acid molecule, comprising, The nucleic acid molecule encodes the bispecific fusion protein of any one of claims 1-4.

6. A recombinant expression vector or host cell comprising, The recombinant expression vector contains the nucleic acid molecule of claim 5; the host cell contains the nucleic acid molecule of claim 5 or the recombinant expression vector.

7. Use of the bispecific fusion protein of any one of claims 1-4 in the preparation of a medicament for treating ocular fundus neovascular disease, preferably wet age-related macular degeneration (nAMD).

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