Bispecific antibody fusion protein simultaneously targeting BMP-2 and VEGF-A and application of bispecific antibody fusion protein in preparation of drugs for treating diabetic retinopathy

Abstract

The invention discloses a bispecific antibody fusion protein simultaneously targeting BMP-2 and VEGF-A. The bispecific antibody fusion protein comprises an anti-VEGF-A antibody part, the amino acid sequence of a heavy chain variable region of the anti-VEGF-A antibody part is shown as SEQ ID NO: 6, and the amino acid sequence of a light chain variable region of the anti-VEGF-A antibody part is shown as SEQ ID NO: 7; the amino acid sequence of a heavy chain variable region of the anti-BMP-2 antibody part is as shown in SEQ ID NO: 8, and the amino acid sequence of a light chain variable region of the anti-BMP-2 antibody part is as shown in SEQ ID NO: 9; the amino acid sequence of the Fc fragment is as shown in SEQ ID NO: 10. The invention also discloses a nucleic acid molecule for coding the bispecific antibody fusion protein, and application of the bispecific antibody fusion protein in preparation of drugs for treating diabetic retinopathy. According to the bispecific antibody fusion protein disclosed by the invention, by simultaneously targeting two key targets, namely VEGF-A and BMP-2, the effusion elimination rate is remarkably increased, the fibrosis area is reduced, the retinal leakage area is reduced, and the pericyte loss rate is reduced, so that the effect of treating diabetic retinopathy is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a bispecific antibody fusion protein targeting BMP-2 and VEGF-A simultaneously and application thereof in preparation of a drug for treating diabetic retinopathy. BACKGROUND

[0002] Diabetic retinopathy (DR) is one of the most common chronic microvascular complications of diabetes, mainly involving the small blood vessels of the eye retina, and its core pathogenesis is closely related to the damage of retinal microvessels caused by long-term high blood sugar: sustained high blood sugar can destroy the function of vascular endothelial cells, leading to increased permeability of microvessel wall and damaged blood-retinal barrier, and then causing retinal ischemia and hypoxia; the body produces pro-angiogenic factors (such as VEGF) to compensate for ischemia, inducing abnormal neovascularization, and the structure of the new blood vessels is fragile and easy to bleed, which may eventually lead to retinal detachment, proliferative lesions and other serious complications.

[0003] At present, the drugs for treating diabetic retinopathy mainly include anti-VEGF drugs, glucocorticoid drugs and anti-platelet drugs. Although these drugs have achieved certain effects in clinical application, they also have some limitations and shortcomings.

[0004] Anti-VEGF drugs mainly inhibit the activity of VEGF to reduce retinal vascular leakage and neovascularization. Common anti-VEGF drugs include ranibizumab, conbercept, aflibercept, etc. These drugs are usually monoclonal antibodies or fusion proteins, which block the interaction of VEGF with its receptors by binding to VEGF, thereby inhibiting angiogenesis and leakage. The production process of anti-VEGF drugs is relatively complex, usually requiring the expression of recombinant proteins in cells through genetic engineering technology, followed by purification, quality control and other steps to prepare the drugs. For example, ranibizumab is a humanized recombinant monoclonal antibody fragment, which is expressed in CHO cells through genetic engineering technology and prepared by multi-step purification process. In clinical application, anti-VEGF drugs can effectively improve the vision and retinal structure of patients, but their therapeutic effect has certain limitations, and some patients do not respond well to the drugs, which is mainly because anti-VEGF drugs only act on a single target point of VEGF, and cannot comprehensively intervene the complex pathological mechanism of diabetic retinopathy. In addition, anti-VEGF drugs need to be injected frequently, usually not more than 3 months, which increases the treatment burden and economic pressure of patients. This frequent treatment frequency has a great impact on the quality of life of patients. Long-term use of anti-VEGF drugs may cause damage to the function of retinal vascular endothelial cells, further affecting the blood perfusion of the retina, and there are significant differences in the response of different patients to VEGF inhibitors, and some patients may be insensitive or resistant to the drugs.

[0005] Glucocorticoids reduce retinal vascular leakage and inflammatory cell infiltration by suppressing inflammatory responses. Common glucocorticoids include dexamethasone, triamcinolone, etc. These drugs are usually administered in the form of injections or implants, and can quickly exert anti-inflammatory effects. The production process of glucocorticoids is relatively mature, and is usually prepared by chemical synthesis. For example, dexamethasone is a synthetic glucocorticoid, which is synthesized through a series of chemical reactions, purified and formulated for clinical use. In clinical applications, glucocorticoids also have certain applications in the treatment of diabetic retinopathy, especially for patients with macular edema. Glucocorticoids have strong anti-inflammatory effects, but the direct therapeutic effect on retinal vascular lesions is limited, mainly because glucocorticoids mainly reduce retinal vascular leakage by suppressing inflammatory responses, but cannot directly intervene in angiogenesis and leakage. Long-term use may cause some adverse reactions, such as increased intraocular pressure, increasing the risk of glaucoma, and accelerating the formation of cataracts, affecting the vision of patients.

[0006] Anti-platelet drugs mainly inhibit platelet aggregation and adhesion to reduce the risk of thrombosis. Common anti-platelet drugs include aspirin, clopidogrel, etc. These drugs are usually administered orally and can effectively prevent thrombosis. The production process of anti-platelet drugs is relatively simple and is usually prepared by chemical synthesis. For example, aspirin is a common anti-platelet drug, which is synthesized by acetylation, purified and formulated for clinical use. In clinical applications, anti-platelet drugs are mainly used to prevent thrombosis and reduce the risk of retinal vascular obstruction in the treatment of diabetic retinopathy. However, the direct therapeutic effect of anti-platelet drugs on retinal vascular lesions is limited, and they cannot effectively control retinal vascular leakage and neovascularization, mainly because anti-platelet drugs mainly inhibit platelet aggregation and adhesion to reduce thrombosis, but have limited direct therapeutic effect on retinal vascular lesions. The main side effect is increased risk of bleeding, especially at high doses, anti-platelet drugs may increase the risk of bleeding, especially at high doses, limiting their widespread use in the treatment of diabetic retinopathy. SUMMARY

[0007] In order to overcome the limitations and shortcomings of existing anti-VEGF drugs, glucocorticoids and anti-platelet drugs in the treatment of diabetic retinopathy (DR), the core of the invention is to develop an innovative bispecific antibody fusion protein. This fusion protein targets two key targets, VEGF-A and BMP-2, providing more comprehensive disease intervention, significantly improving treatment effect and reducing treatment frequency, thereby solving the problems existing in the prior art.

[0008] To this end, the present application provides the following technical solutions:

[0009] A bispecific antibody fusion protein simultaneously targeting BMP-2 and VEGF-A, comprising:

[0010] an anti-VEGF-A antibody portion, the amino acid sequence of the heavy chain variable region of which is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of which is shown in SEQ ID NO: 7;

[0011] an anti-BMP-2 antibody portion, the amino acid sequence of the heavy chain variable region of which is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region of which is shown in SEQ ID NO: 9;

[0012] an Fc fragment, the amino acid sequence of which is shown in SEQ ID NO: 10.

[0013] Preferably, the nucleotide sequence encoding the anti-VEGF-A antibody portion is shown in SEQ ID NO: 1.

[0014] Preferably, the nucleotide sequence encoding the anti-BMP-2 antibody portion is shown in SEQ ID NO: 3.

[0015] Preferably, the nucleotide sequence encoding the Fc fragment is shown in SEQ ID NO: 4.

[0016] Preferably, the bispecific antibody fusion protein further comprises a connecting peptide for connecting the heavy chain of the anti-VEGF-A antibody portion and the heavy chain of the anti-BMP-2 antibody portion, respectively.

[0017] More preferably, the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 11.

[0018] Preferably, the nucleotide sequence encoding the connecting peptide is shown in SEQ ID NO: 2.

[0019] The present application also provides a nucleic acid molecule encoding the bispecific antibody fusion protein described in the present application.

[0020] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 5.

[0021] The present application also provides a recombinant vector comprising the nucleic acid molecule described in the present application.

[0022] The present application also provides the use of the bispecific antibody fusion protein described in the present application in the preparation of a medicament for treating diabetic retinopathy.

[0023] The application also provides a bispecific antibody fusion protein microsphere preparation for simultaneously targeting BMP-2 and VEGF-A, which comprises a polylactic acid degradable shell and the bispecific antibody fusion protein uniformly distributed therein.

[0024] The bispecific antibody fusion protein of the application uses the CrossMAb technology to integrate two independent antibodies together, simultaneously targeting VEGF-A and BMP-2. VEGF-A is the vascular endothelial growth factor mainly responsible for vascular neovascularization and leakage; BMP-2 (bone morphogenetic protein 2) promotes vascular leakage and neovascularization in diabetic retinopathy. By double inhibiting these two targets, the fusion protein can more effectively reduce fluid accumulation and neovascularization. Compared with traditional single-path anti-VEGF drugs, the fusion protein of the application not only inhibits the activity of VEGF-A, but also simultaneously inhibits the activity of BMP-2, thereby providing more comprehensive therapeutic effect. This double inhibition strategy significantly improves the efficacy of the drug, reduces the formation of fluid accumulation, and prolongs the treatment interval, reducing the treatment burden of patients.

[0025] The Fc fragment of the application is improved by amino acid modification to eliminate the binding domain of Fc receptors (FcRn and FcyR), accelerate the clearance rate of the drug in the body, and reduce systemic exposure. This improvement not only reduces the inflammatory response and thrombosis risk caused by the drug, but also eliminates the effector function of the Fc segment, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The improved Fc fragment design significantly improves the safety and tolerability of the drug while maintaining the ocular therapeutic effect. This design makes the fusion protein of the application safer in treating diabetic retinopathy, reduces potential side effects, and improves patient treatment compliance.

[0026] Through experiments on animal models such as mice, rabbits, and African green monkeys, the significant effect of the fusion protein of the application in inhibiting vascular proliferation and removing fluid accumulation is verified. The experimental results show that the fusion protein can significantly reduce subretinal vascular proliferation and fluid accumulation, and the effect is better than that of injecting two single-path drugs at the same time. Preclinical studies not only verify the efficacy of the fusion protein of the application, but also demonstrate its safety and effectiveness in different animal models. These research results provide a solid scientific basis for the clinical application of the fusion protein of the application, and prove its significant advantages in treating diabetic retinopathy.

[0027] The treatment regimen of the fusion protein of the present application allows adjustment of the treatment interval according to the patient's response, up to 16 weeks once. This flexible treatment regimen not only reduces the patient's treatment burden, but also improves the compliance and safety of the treatment. Compared with the traditional once a month or once every two months treatment regimen, the treatment interval of the fusion protein of the present application is significantly prolonged, reducing the number of injections and economic burden of the patient, while maintaining good treatment effect. This optimized treatment regimen significantly improves the patient's treatment experience and quality of life.

[0028] The bispecific antibody fusion protein of the present application significantly improves the effect of treating diabetic retinopathy by simultaneously targeting two key targets of VEGF-A and BMP-2. Specifically, it significantly improves the elimination rate of effusion, reduces the area of fibrosis, reduces the area of retinal leakage, and reduces the rate of pericyte loss. The bispecific antibody can significantly reduce the risk of blindness in patients with diabetic retinopathy, reduce visual impairment and blindness caused by diabetic retinopathy, and improve the quality of life of patients.

[0029] In addition, the bispecific antibody fusion protein of the present application can use biodegradable microspheres as a sustained-release preparation to prolong the release time of the drug in the eye, reduce the injection frequency, and improve the treatment compliance of the patient. This sustained-release preparation can continuously release the drug, maintain the effective concentration of the drug in the eye, significantly reduce the number of injections and the frequency of treatment of the patient, reduce the consumption of medical resources caused by diabetic retinopathy, and reduce the economic burden of the patient. Compared with existing anti-VEGF-A drugs, the annual treatment cost of the bispecific antibody is expected to be reduced by more than 30%. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a schematic diagram of the molecular structure of the bispecific antibody fusion protein of the present application.

[0031] Figure 2 is a schematic diagram of the sustained-release microsphere structure of the bispecific antibody fusion protein of the present application.

[0032] Figure 3 is the result of an animal treatment experiment.

[0033] Figure 4 is the drug concentration-time curve. DETAILED DESCRIPTION

[0034] The technical solutions of the present application will be further described below in combination with the drawings and specific embodiments.

[0035] Unless otherwise specified, the instruments or reagents used in the examples are conventional instruments or reagents in the art, which are conventional products that can be purchased on the market, and the specific experimental operations involved in this document are understood or known by those skilled in the art according to their mastery of common knowledge or conventional technical means.

[0036] like Figure 1 As shown, the bispecific antibody fusion protein of the present invention has a "YY" shaped overall configuration. Its molecular structure consists of an anti-VEGF-A antibody binding site 1, an anti-BMP-2 antibody binding site 2, and a modified Fc fragment 3. The front end is formed by the anti-VEGF-A antibody moiety and the anti-BMP-2 antibody moiety connected end-to-end by a flexible linker peptide, forming the core structure of the bispecific antibody fusion protein. The rear end is fused with a point-mutated Fc fragment. The spatial relationship of the three is as follows: the N-terminus of the linker peptide is covalently linked to the C-terminus of the anti-VEGF-A antibody heavy chain via a peptide bond, and its C-terminus is covalently linked to the N-terminus of the anti-BMP-2 antibody heavy chain via another peptide bond; the C-termini of the two heavy chains are then connected to the modified Fc fragment via a pair of disulfide bonds, forming a complete molecular backbone. The modified Fc uses a combination of L234F / L235E / P331S mutations to eliminate the FcγR complement-binding domain, retaining only the FcRn binding site, thereby prolonging the serum half-life and reducing the risk of systemic immune activation. The anti-VEGF-A antibody binding site and the anti-BMP-2 antibody binding site target VEGF-A and BMP-2 respectively, achieving dual inhibition. A modified Fc fragment 3, through special design, is linked to the end of the bispecific antibody fusion protein to enhance antibody stability and half-life while reducing immunogenicity. This structural design enables the bispecific antibody fusion protein to simultaneously inhibit the activity of VEGF-A and BMP-2, significantly improving the efficacy of treatment for diabetic retinopathy.

[0037] The mechanism of action of the bispecific antibody fusion protein molecule of the present invention is as follows: Anti-VEGF-AFab specifically binds to vascular endothelial growth factor-A, blocking the VEGF-VEGFR2 signaling pathway and reducing vascular leakage and angiogenesis; Anti-BMP-2Fab specifically binds to bone morphogenetic protein-2, inhibiting the BMP2-Smad signaling pathway and alleviating retinal fibrosis and vascular leakage; Modified Fc enhances FcRn affinity, extending the half-life of the molecule in circulation to about 21 days, while avoiding ADCC and CDC effects.

[0038] Example 1: Preparation of bispecific antibody fusion protein

[0039] 1. Expression vehicle

[0040] The plasmid pCHO1.0 (ThermoFisherScientific, catalog number A13696) was used, containing the CMV promoter and glutamine synthase selection marker.

[0041] 2. Gene construction and plasmid preparation

[0042] The nucleotide sequences encoding anti-VEGF-AVH-CH1 (SEQ ID NO: 1), flexible linker peptide (G4S)3 (SEQ ID NO: 2), anti-BMP-2VH-CH1 (SEQ ID NO: 3), and mutant Fc (L234F / L235E / P331S) (SEQ ID NO: 4) were sequentially fused to obtain the full-length fusion gene SEQ ID NO: 5. 2 μg of pCHO1.0 plasmid was digested with EcoRI and XhoI, and the linear fragment was recovered. This fragment was then ligated with 150 ng of the fusion gene fragment overnight at 16°C using T4 DNA ligase. The ligation product was transformed into DH5α competent cells, screened with ampicillin, and the plasmid was extracted and verified by EcoRI / XhoI double digestion and sequencing.

[0043] 3. CHO-S cell transfection and pressure selection

[0044] Resuscitate CHO-S cells (Thermo Fisher, catalog number R80007) to passages 3-5 and adjust the density to 2×10⁻⁶. 7 cells / mL. Take 5 μg of linearized plasmid and 1×10 7 Cells were mixed and transfected using a Neon electroporation system at 1400V, 20ms, and a single pulse. Immediately after transfection, cells were transferred to CDCHO medium (Thermo Fisher, catalog number 10743029) containing 8 mL of glutamine, and 50 μg / mL puromycin was added for pressure selection. Culture conditions were 37°C, 5% CO2, and 125 rpm, with medium changes every 48 hours for 7-10 days until a stable expression pool was formed.

[0045] 4. Fermentation

[0046] The stable expression pool is 3×10 5 Cells / mL were inoculated into 1.5 L of CDCHO medium and loaded into a 2 L Artorius BIOSTATQplus bioreactor. The pH was maintained at 7.0 ± 0.1 throughout fermentation, automatically regulated by CO2 and sodium bicarbonate; dissolved oxygen was set at 40% air saturation; the temperature was controlled at 37℃ for 0-72 h, then reduced to 34℃ after 72 h. From day 3 onwards, 10% v / v Efficient Feed C+ (catalog number A2503101) was added daily. When cell viability dropped below 80%, the supernatant was collected by centrifugation at 4000g, 20 min, and 4℃, and filtered through a 0.22 μm filter membrane.

[0047] 5. Purification

[0048] Protein A affinity chromatography: MabSelect SuRe packing, loading buffer pH 7.2 PBS, elution buffer 0.1 M glycine-HCl pH 3.5; immediately after elution, pH 7.2 was adjusted by 1 M Tris.

[0049] Low pH virus inactivation: 0.1 M glycine-HCl pH 3.5 for 60 min at room temperature.

[0050] Cation exchange chromatography: SP Sepharose FF was used, equilibration buffer 20 mM sodium acetate pH 5.0, elution with 0-300 mM sodium chloride gradient, 150-200 mM salt peak was collected.

[0051] Anion exchange chromatography: Q Sepharose FF was used, equilibration buffer 20 mM Tris-HCl pH 7.5, elution with 0-150 mM sodium chloride gradient, flow-through peak was collected.

[0052] Nanofiltration virus removal: Planova 20N nanofilter was used, pressure no more than 0.3 bar, 4°C operation, virus removal log reduction value ≥ 6. The final product was detected by CE-SDS, purity ≥ 98%, SEC-HPLC monomer ≥ 99%.

[0053] 6. Formulation

[0054] The purified protein was replaced by 30 kDa MWCO tangential flow ultrafiltration into 20 mM histidine-acetic acid buffer (pH 5.8) containing 5% w / v sucrose. Sterile filtration was performed using a 0.22 μm PES membrane (Millipore Stericup GP). Nitrogen protection was used to fill into BD Hypak 1 mL pre-filled syringes, 0.05 mL per syringe containing 2 mg of protein. The finished product was stored at 2-8°C in the dark, and the shelf life was not less than 24 months. After accelerated storage at 40°C, 75% RH for 6 months, the monomer decreased by less than 2%.

[0055] In clinical application, the drug was administered by intravitreal injection: the initial dose was 2 mg / eye, and the subsequent administration interval was adjusted according to the OCT effusion thickness at weeks 4 and 8, and the longest could be extended to 16 weeks; if combined with macular edema or inflammation, 0.1% sodium fenamate eye drops could be used locally, and the dose and course of treatment could be adjusted by the doctor according to the retinal thickness and inflammation index, and the intraocular pressure and retinal morphology were monitored throughout the course.

[0056] Example 2: Preparation of bi-specific antibody fusion protein sustained-release microspheres

[0057] Figure 2The structure of the bi-specific antibody fusion protein sustained-release microspheres of the present application is shown. The drug core 4 is located in the center of the microspheres, containing the bi-specific antibody fusion protein. The biodegradable shell 5 is wrapped outside the drug core, composed of biodegradable materials such as PLGA, for controlling the release of the drug. The bi-specific antibody fusion protein 6 is also uniformly distributed inside the microspheres, ensuring that the drug can be released continuously, maintaining the effective concentration of the drug in the eye.

[0058] 1. Material preparation:

[0059] The bi-specific antibody fusion protein (concentration of 30 mg / mL) is the core drug component, which is prepared by the method described in Example 1.

[0060] The biodegradable shell material is polylactic acid (PLA) with a molecular weight of 5000-10000 Dalton, purchased from Sigma-Aldrich Company.

[0061] The organic solvent dichloromethane is of analytical purity, purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0062] The emulsifier polyvinyl alcohol (PVA) has an average polymerization degree of 1750±50, purchased from Aladdin Reagent Company.

[0063] 2. Preparation process:

[0064] (1) Organic phase preparation: weigh 100 mg of PLA and dissolve it in 1 mL of dichloromethane, stir until completely dissolved, to prepare the organic phase. At this time, the concentration of PLA in the organic phase is 100 mg / mL.

[0065] (2) Primary emulsion formation: take 0.1 mL of the above bi-specific antibody fusion protein solution (30 mg / mL) and slowly add it to the prepared organic phase, and use an ultrasonic cell disruptor (power 190 kW) for ultrasonic treatment for 3-5 minutes to form a uniform and stable primary emulsion. In the primary emulsion, the concentration of the fusion protein is calculated to be about 2.73 mg / mL (considering the total volume of 1.1 mL).

[0066] (3) Multiple emulsion preparation: prepare 8 mL of 1% (w / v) PVA aqueous solution, slowly pour the primary emulsion into the PVA aqueous solution, and stir at a speed of 1500 rpm for 10-15 minutes to form a multiple emulsion. Then add 7 mL of 1% (w / v) PVA aqueous solution, and place the mixture on a mechanical stirrer, stirring at a speed of 600 rpm for 6-8 hours, during which the dichloromethane gradually volatilizes.

[0067] (4) Microsphere collection and purification: After the dichloromethane is completely volatilized, the mixed solution is transferred to a centrifuge tube, centrifuged at 3000 rpm for 10 minutes at 4°C, and the precipitated microspheres are collected. The microspheres are washed with distilled water for 3-4 times, and after each washing, the microspheres are centrifuged under the same conditions to remove residual PVA and uncoated fusion proteins. Finally, the washed microspheres are placed in a Buchner funnel and filtered, and then freeze-dried to obtain the dual-specific antibody fusion protein sustained-release microspheres. In the finally prepared sustained-release microspheres, the fusion protein is uniformly distributed inside the degradable shell formed by PLA. In terms of mass proportion, if the final mass of the microspheres is 110 mg (theoretical value, actual value is subject to loss during operation), the fusion protein accounts for about 3 mg, accounting for about 2.73%, PLA accounts for about 100 mg, accounting for about 90.91%, and the rest is a small amount of residual moisture and impurities.

[0068] 3. Sustained-release effect experiment

[0069] (1) Experimental method:

[0070] Experimental animal grouping: 30 healthy adult New Zealand white rabbits were selected and randomly divided into two groups, 15 in each group. The experimental group was given the dual-specific antibody fusion protein sustained-release microsphere preparation prepared by the application, and the control group was given the ordinary dual-specific antibody fusion protein solution preparation (with the same drug concentration).

[0071] Dosing method: After local anesthesia of the eyes of the two groups of rabbits, the experimental group implanted the sustained-release microspheres into the superficial sclera of the equatorial part of the eyeball by subconjunctival injection, and the control group injected the same volume of ordinary solution preparation.

[0072] Sample collection and detection: On the 1st day, 3rd day, 7th day, 14th day, 21st day and 28th day after administration, the eyeballs of the two groups of rabbits were enucleated, and the retinal tissues were quickly separated. The content of dual-specific antibody fusion protein in the retinal tissue was detected by enzyme-linked immunosorbent assay (ELISA), and the retinal tissue of 5 rabbits in each group was detected at each time point, and the average value was taken.

[0073] (2) Experimental result data:

[0074] On the 1st day after administration, the content of fusion protein in the retinal tissue of the control group rapidly increased to (128.6±5.3) μg / mL, and then rapidly decreased to (42.5±2.1) μg / mL on the 7th day, only a trace amount (10.3±0.5) μg / mL was detected on the 14th day, and almost nothing was detected on the 21st day and the 28th day.

[0075] The content of fusion protein in the retinal tissue of the experimental group was (85.2±4.8) μg / mL on the first day after administration, which was lower than that of the control group on the first day, indicating that the sustained-release microspheres avoided the burst release phenomenon of the drug. On the seventh day, the content of fusion protein was still maintained at (72.3±3.7) μg / mL, on the fourteenth day was (56.8±2.8) μg / mL, on the twenty-first day was (40.8±2.3) μg / mL, and on the twenty-eighth day was still detectable (22.1±1.8) μg / mL.

[0076] By drawing the drug concentration-time curves of the two groups, it can be obviously seen that the curve of the experimental group is relatively flat, and the drug is in a state of sustained and slow release in the eye, while the curve of the control group fluctuates greatly, and the drug rapidly releases and rapidly decays Figure 4 ) after rapid release. Statistical analysis shows that on the seventh day, the fourteenth day, the twenty-first day and the twenty-eighth day after administration, the content of fusion protein in the retinal tissue of the experimental group is significantly higher than that of the control group (P<0.05), which fully proves that the prepared sustained-release microspheres can gradually release the drug in the eye, prolong the action time of the drug, and have obvious sustained-release advantage compared with ordinary preparations.

[0077] It can be seen that the design of such sustained-release microspheres enables the drug to be gradually released in the eye, prolongs the action time of the drug, reduces the injection frequency, and thus improves the treatment compliance of patients. Through the use of biodegradable materials, the microspheres can gradually degrade after releasing the drug, avoiding the potential impact on the intraocular tissues caused by long-term residue.

[0078] Example 3: Verification experiment of technical effect of bispecific antibody fusion protein

[0079] 1. Effusion elimination rate experiment:

[0080] Animal models and grouping: three animal models were used: C57BL / 6J diabetic mice, New Zealand white rabbits, and African green monkeys. Each model was divided into 5 groups, 10 animals in each group. They were respectively the control group (PBS), the single antibody VEGF-A group, the single antibody BMP-2 group, the single antibody VEGF-A and single antibody BMP-2 combined group, and the bispecific antibody group (the bispecific antibody prepared in Example 1 of the present application).

[0081] Single antibody VEGF-A: commercially available anti-human VEGF-A monoclonal antibody (bevacizumab, Roche Diagnostics, specification 100 mg / 4 mL, product number 50116767) was used after cross-reactivity verification according to the species of animal models in the experiment.

[0082] Monoclonal antibody BMP-2: Commercially available anti-human BMP-2 monoclonal antibody (R&D Systems, item number MAB355, clone number 52C5B1) was used, which cross-reacts with mouse, rabbit, and monkey BMP-2, meeting the requirements of multi-animal model experiments.

[0083] Monoclonal antibody VEGF-A and monoclonal antibody BMP-2 combination group: Commercially available monoclonal antibody VEGF-A and monoclonal antibody BMP-2 were combined in a 1:1 mass ratio.

[0084] Induction of diabetes: All animals were induced to develop diabetes by intraperitoneal injection of streptozotocin (STZ) (50 mg / kg / day for mice, 40 mg / kg / day for rabbits, and 30 mg / kg / day for monkeys, for 5 consecutive days). After 8 weeks, the animals were confirmed to have developed retinal effusion.

[0085] Administration: After randomization, the animals were administered by intravitreal injection. Mice were injected with 1 μL containing 2 μg of antibody; rabbits were injected with 50 μL containing 100 μg of antibody; and African green monkeys were injected with 100 μL containing 200 μg of antibody. The solvent was 20 mM histidine-acetic acid buffer (pH 5.8) containing 5% (w / v) sucrose.

[0086] Detection: Before administration and on day 14 after administration, SD-OCT (Spectralis HRA+OCT) was used to scan along the concentric circles of the optic disc to obtain the height of subretinal effusion within a 1 mm diameter.

[0087] Calculation: Effusion elimination rate = (effusion height before administration - effusion height after administration) / effusion height before administration x 100%.

[0088] The results showed that the effusion elimination rate of the bispecific antibody group reached 80% in the mouse model, which was significantly higher than the 40% of the monoclonal antibody VEGF-A group and the 35% of the monoclonal antibody BMP-2 group; in the rabbit model, the effusion elimination rate of the bispecific antibody group reached 75%, which was significantly higher than the 35% of the monoclonal antibody VEGF-A group and the 30% of the monoclonal antibody BMP-2 group; in the African green monkey model, the effusion elimination rate of the bispecific antibody group reached 70%, which was significantly higher than the 30% of the monoclonal antibody VEGF-A group and the 25% of the monoclonal antibody BMP-2 group.

[0089] 2. Retinal fibrosis area experiment:

[0090] Sampling: On day 21 after administration, the eyeballs were fixed in 4% paraformaldehyde for 24 hours, paraffin-embedded, and then sectioned at 5 μm.

[0091] Staining: Masson's trichrome staining was used. After staining with Weigert's iron hematoxylin for 10 minutes, acid fuchsin-ponceau for 10 minutes, and aniline blue for 5 minutes, the sections were mounted.

[0092] Image acquisition and analysis: Whole retinal scanning using 40x objective, ImageJ automatic threshold segmentation of fibrosis area.

[0093] The results show that the fibrosis area of the bispecific antibody group is significantly reduced, which is reduced by 36% compared with the single antibody combination group.

[0094] 3. Retinal leakage area experiment:

[0095] Dosing: 14 days after administration, 100 mg / kg FITC-dextran (MW 150 kDa) was injected into the tail vein.

[0096] Imaging: After 10 minutes, retinal flat mounts were photographed using a fluorescence microscope (Zeiss Axio Imager Z2) with an excitation / emission wavelength of 495 / 519 nm.

[0097] Image analysis: Leaking areas were outlined using ImageJ and the fluorescence area was quantified.

[0098] The results show that the retinal leakage area of the bispecific antibody group is significantly reduced, which is reduced by 40% compared with the single antibody combination group.

[0099] 4. Pericyte loss rate experiment:

[0100] Labeling: 14 days after administration, retinal flat mounts were labeled with anti-NG2 antibody (1:200, 4°C overnight) and Alexa Fluor 594 secondary antibody (1:500, room temperature for 2 hours) to label pericytes.

[0101] Imaging: Using a confocal microscope (ZEISS LSM 880) under 40x oil lens, 5 0.5mm x 0.5mm areas were randomly selected to count NG2 + cells.

[0102] Calculation: Pericyte loss rate = (control group pericyte number-experimental group pericyte number) / control group pericyte number x 100%.

[0103] The results show that the pericyte loss rate of the bispecific antibody group is significantly reduced, which is reduced by 15% compared with the single antibody combination group. The determination results of the above experiments are shown in Table 1 below.

[0104] Table 1. Experimental results

[0105]

Claims

1. A bispecific antibody fusion protein simultaneously targeting BMP-2 and VEGF-A, characterized in that, include: The amino acid sequence of the heavy chain variable region of the anti-VEGF-A antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7; The amino acid sequence of the heavy chain variable region of the anti-BMP-2 antibody is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9; The Fc fragment has the amino acid sequence shown in SEQ ID NO:

10.

2. The bispecific antibody fusion protein according to claim 1, characterized in that, The nucleotide sequence encoding the anti-VEGF-A antibody portion is shown in SEQ ID NO:

1.

3. The bispecific antibody fusion protein according to claim 1, characterized in that, The nucleotide sequence encoding the anti-BMP-2 antibody portion is shown in SEQ ID NO:

3.

4. The bispecific antibody fusion protein according to claim 1, characterized in that, The nucleotide sequence encoding the Fc fragment is shown in SEQ ID NO:

4.

5. The bispecific antibody fusion protein according to claim 1, characterized in that, The bispecific antibody fusion protein also includes linker peptides for connecting the partial heavy chains of the anti-VEGF-A antibody and the anti-BMP-2 antibody, respectively.

6. The bispecific antibody fusion protein according to claim 5, characterized in that, The amino acid sequence of the linker peptide is shown in SEQ ID NO:

11.

7. The bispecific antibody fusion protein according to claim 6, characterized in that, The nucleotide sequence encoding the linker peptide is shown in SEQ ID NO:

2.

8. A nucleic acid molecule encoding a bispecific antibody fusion protein as described in any one of claims 1 to 7.

9. The nucleic acid molecule according to claim 8, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

5.

10. The use of the bispecific antibody fusion protein as described in any one of claims 1 to 7 in the preparation of a therapeutic agent for diabetic retinopathy.

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