VEGF (vascular endothelial growth factor) combined oligopeptide and application thereof in preparation of anti-angiogenesis therapeutic drugs

Abstract

The invention discloses VEGF (Vascular Endothelial Growth Factor) combined oligopeptide and application thereof in preparation of an anti-angiogenesis therapeutic drug. Compared with the existing anti-VEGF monoclonal antibody and fusion protein, the oligopeptide inhibitor prepared by the recombinant protein expression system has the remarkable advantages of small molecule drugs while keeping the stability of traditional biological products. The invention creates a novel VEGF inhibitor which has binding capacity and is easy to accurately regulate and control through bioengineering, and provides an innovative solution with both transformation efficiency and clinical potential for next-generation anti-angiogenesis treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to VEGF-binding short peptides and their application in the preparation of anti-angiogenic therapeutic drugs. Background Technology

[0002] Vascular endothelial growth factor (VEGF, especially its main subtype VEGF-A) is a core pivotal molecule regulating angiogenesis and vascular permeability. Under physiological conditions, such as embryonic development, tissue repair, and the female reproductive cycle, VEGF specifically binds to high-affinity tyrosine kinase receptors (mainly VEGFR-2) on the surface of vascular endothelial cells, activating multiple downstream signaling pathways, including MAPK / ERK and PI3K / Akt. This precisely drives endothelial cell proliferation, migration, survival, and lumen formation, which is crucial for maintaining tissue blood flow and homeostasis.

[0003] However, dysregulation of this pathway is closely related to the pathological processes of many major diseases. In solid tumors, tumor cells trigger angiogenesis by over-secreting VEGF, inducing the formation of a structurally disordered and functionally dysfunctional neovascular network, providing a material basis for rapid tumor growth and distant metastasis. In age-related macular degeneration, diabetic retinopathy, and other fundus diseases, abnormally high expression of intraocular VEGF leads to pathological angiogenesis and vascular leakage, directly causing irreversible visual impairment. Given the core driving role of VEGF in these diseases, targeting VEGF / VEGFR has become a highly valuable therapeutic strategy.

[0004] Despite the landmark successes achieved by anti-VEGF macromolecular drugs, such as monoclonal antibodies (e.g., bevacizumab) and receptor fusion proteins (e.g., aflibercept), in the treatment of cancer and retinal diseases, their clinical translation still faces multiple limitations. First, these biologics have limited tissue permeability, making it difficult to effectively cross the dense stroma of solid tumors or the blood-retinal barrier, resulting in insufficient inhibitory effects on the core lesion region. Second, their production costs are high, relying on complex eukaryotic expression systems and purification processes, significantly increasing medical costs and limiting accessibility in resource-constrained areas. Furthermore, as exogenous proteins, their potential immunogenicity risks may induce neutralizing antibodies, affecting long-term efficacy and increasing the incidence of adverse reactions.

[0005] From a clinical practice perspective, existing therapies also have significant shortcomings. For example, the treatment of intraocular diseases requires repeated intravitreal injections. This invasive procedure not only imposes a physical and psychological burden on patients but also carries cumulative risks such as infection, bleeding, and retinal detachment, severely impacting treatment adherence and quality of life. More critically, because existing drugs primarily target the single VEGF-A signaling pathway, the tumor microenvironment can escape by activating or upregulating other VEGF family members (such as VEGF-C and PIGF) through bypass signaling, leading to drug resistance. Simultaneously, large molecule drugs cannot inhibit the intracellular kinase domain activity of VEGFR, resulting in insufficient blocking of the signaling pathway. Summary of the Invention

[0006] The purpose of this invention is to provide VEGF-binding short peptides and their application in the preparation of anti-angiogenic therapeutic drugs.

[0007] To achieve the above objectives, the present invention provides a VEGF-binding short peptide, selected from any of the following: VE1: MSPEAWETLRLLAKEGCKDPEEAVRLALEFSPGPLVVEIDGEEVTVSLDEDGEIVLETEKGVKEEEKPELIKKIFEKAAE, as shown in SEQ ID NO.1; VE4: SLEEDFYEDFERSINEILESTPEEKREEVLKKILEDLEKVAKDDPLLAKILKRYKEKR, as shown in SEQID NO.2.

[0008] This invention also provides the application of the aforementioned VEGF-binding short peptide in the preparation of therapeutic drugs for angiogenesis-related diseases.

[0009] As one of the preferred technical solutions, the angiogenesis-related diseases include, but are not limited to: solid tumors, age-related macular degeneration, and diabetic retinopathy.

[0010] This invention also provides the application of the aforementioned VEGF-binding short peptide in the preparation of anti-angiogenic therapeutic drugs.

[0011] As one of the preferred technical solutions, VEGF-binding short peptides are used as VEGF inhibitors in the preparation of anti-angiogenic therapeutic drugs.

[0012] The present invention also provides pharmaceutical compositions comprising VEGF-binding short peptides.

[0013] The present invention also provides the use of the aforementioned pharmaceutical composition in the preparation of a medicament for treating angiogenesis-related diseases.

[0014] The present invention also provides the use of the aforementioned pharmaceutical composition in the preparation of anti-angiogenic therapeutic drugs.

[0015] The present invention also provides a therapeutic drug for angiogenesis-related diseases, the active ingredient of which is the aforementioned VEGF-binding short peptide, or a pharmaceutical composition comprising the aforementioned.

[0016] The present invention also provides an anti-angiogenic therapeutic drug, the active ingredient of which is the aforementioned VEGF-binding short peptide, or a pharmaceutical composition comprising the aforementioned.

[0017] The present invention has the following beneficial effects: This invention discloses a VEGF-binding short peptide and its application in the preparation of anti-angiogenic therapeutic drugs. Compared with existing anti-VEGF monoclonal antibodies and fusion proteins, the short peptide inhibitor (<15kDa) prepared by this invention through a recombinant protein expression system retains the stability of traditional biological products while also possessing the significant advantages of small molecule drugs.

[0018] The specific analysis is as follows: First, in terms of production controllability and cost-effectiveness, this design eliminates the complex structure of full-length antibody proteins and enables large-scale soluble expression using efficient expression systems such as prokaryotes or yeast. This avoids the high cost and long cycle of mammalian cell culture, and the purification process is simpler, significantly reducing production costs and process barriers.

[0019] Secondly, in terms of drug permeability and distribution characteristics, its smaller molecular size significantly enhances tissue penetration ability, and is expected to reach the interior of solid tumors or deep retinal lesions more effectively, overcoming the limitations of large molecule drug distribution.

[0020] Third, in terms of structural design flexibility, short peptides based on expression systems are easy to engineer at the gene level. By fusing tags and introducing non-natural modifications, their affinity, stability and pharmacokinetic properties can be precisely optimized, while immunogenicity can be effectively controlled through humanized design.

[0021] In summary, this invention creates a new class of VEGF inhibitors with binding capacity and easy precise regulation through bioengineering, providing an innovative solution with both translational efficiency and clinical potential for next-generation anti-angiogenic therapy.

[0022] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0023] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is the CD result for VE1; Figure 2 This is the CD result for VE4; Figure 3 This is the Tm result for VE1; Figure 4 This is the Tm result for VE4; Figure 5 This is the SPR result for VE1; Figure 6 This is the SPR result for VE4. Detailed Implementation

[0024] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, the present invention can be implemented in many different ways as defined and covered by the claims.

[0025] Example 1 Construction of recombinant VEGF combined with short peptide vector 1. The encoded amino acid sequence is as follows: VE1: MSPEAWETLRLLAKEGCKDPEEAVRLALEFSPGPLVVEIDGEEVTVSLDEDGEIVLETEKGVKEEEKPELIKKIFEKAAE, as shown in SEQ ID NO.1; VE4: SLEEDFYEDFERSINEILESTPEEKREEVLKKILEDLEKVAKDDPLLAKILKRYKEKR, as shown in SEQID NO.2.

[0026] 2. Construction of recombinant luciferase vector: The biotechnology company (Qingke Biotechnology) was commissioned to synthesize the gene for VEGF-binding short peptides, and the company provided the plasmids in return.

[0027] Example 2 Expression and purification of recombinant VEGF-binding short peptides 4 μg of lyophilized plasmid was dissolved in 100 μL of sterile water to obtain a plasmid solution. 2 μL of the plasmid solution was added to 50 μL of competent BL21(DE3) cells (Qingke Biotechnology), and incubated on ice for 20 min. The cells were then heat-shocked at 42℃ for 90 s and cooled on ice for 2 min. 500 μL of LB medium was added to the competent cells in a clean bench, and the cells were incubated at 37℃ for 40 min. 10 μL of kanamycin was added to 5 mL of LB medium, followed by 100 μL of the incubated competent cells, and the cells were incubated overnight at 37℃ with shaking.

[0028] LB medium: Each 1 liter contains 5g yeast extract (Thermo Fisher), 10g tryptone (Thermo Fisher), and 10g NaCl (Sangon Biotech (Shanghai) Co., Ltd.).

[0029] Add 300 μL of kanamycin and 3 mL of bacterial culture to 250 mL of LB medium (stored in a 500 mL Erlenmeyer flask) and incubate at 37 °C and 220 rpm for 4 h.

[0030] Add 150 μL of IPTG (isopropyl-β-D-thiogalactoside) to 250 ml of culture medium and incubate overnight (14-16 h) at 16 °C and 220 rpm.

[0031] After connecting the UV spectrophotometer and setting it to OD600, pipette 2 mL of LB culture medium into a cuvette and zero the instrument. Remove the cuvette, rinse it once with ddH2O, once with 70% ethanol (v / v), and once with ddH2O, then air dry. Pipe 2 mL of bacterial culture into the cuvette and record the readings.

[0032] Take a fixed volume of bacterial culture into a 250mL centrifuge bottle and centrifuge at 12000rpm for 5min using a floor centrifuge with a rotor.

[0033] Remove the supernatant, take 5 mL of buffer (1×PBS pH=7.4, TransGen Biotech Ltd.) to resuspend the precipitate, transfer it to a 50 mL centrifuge tube, and wash the 250 mL centrifuge tube 3 times with a certain volume of buffer until the final resuspended liquid volume reaches 11 mL.

[0034] Set the ultrasonic probe power to 100%, with a 7-second rest period followed by a 3-second working period. Perform ultrasonic disruption for 10 minutes, and then store on ice after ultrasonication. Centrifuge at 12000 rpm for 40 minutes, and use a dropper to transfer 10 mL of the supernatant (avoiding the precipitate as much as possible) into a small beaker. Store on ice.

[0035] Remove the supernatant completely and resuspend the precipitate in 10 mL buffer.

[0036] The supernatant was purified by Ni-NTA affinity chromatography, and the impurities were washed with washing buffer to remove the impurities. Then, the target protein was eluted with imidazole elution buffer of different concentrations.

[0037] The total amount of protein eluted at each concentration was determined using nanodrop. The total protein amount was the sum of the proteins eluted by 250 mM and 500 mM imidazole.

[0038] Example 3 Determination of surface plasmon resonance (SPR), circular dichroism (CD), and dissolution temperature (Tm) Data were acquired using a Chirascan plus circular dichroism spectrometer. All samples were diluted to 0.2 mg / mL with PBS (pH=7.4, TransGen Biotech Ltd.) for readings in the 200–260 nm UV spectral range and for thermal denaturation measurements. Thermal melting analysis was performed at 222 nm wavelength, with the temperature increasing from 20 °C to 90 °C at a rate of 2 °C / min, and measurements were recorded at 0.2 °C intervals. All reported measurements were obtained within the instrument's linear range.

[0039] SPR data were acquired at room temperature using a Biacore T200 instrument. The target protein was immobilized onto various channels of the CM5 sensor chip using a standard amine coupling method. A brief summary of the steps is as follows: First, the sensor chip surface was activated for 7 minutes by injecting a mixed solution of 50 mM N-hydroxysuccinimide and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Then, the target protein, at a concentration of 50 μg / mL dissolved in 10 mM sodium acetate buffer, was injected into each channel of the sensor chip at a flow rate of 10 μL / min for 420 seconds. Subsequently, the channels were blocked with 1 M ethanolamine. The binding proteins VE1 and VE4 were dissolved in 1×HBS-EP running buffer (Cytiva) to 200 μM, and then serially diluted to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.56 μM before analysis. Data were analyzed using Biacore T200 evaluation software.

[0040] The results are shown below: Circular dichroism results ( Figure 1 , Figure 2 This indicates that both VE1 and VE4 fold normally into α-helices in solution.

[0041] The result is that the melting temperature is much higher than 60℃. Figure 3 , Figure 4 This indicates that VE1 and VE4 have excellent stability.

[0042] The results of SPR ( Figure 5 , Figure 6 The results showed that the dissociation constant KD of VE1 was 35 μM and that of VE4 was 49.6 μM, indicating that both interacted with the target protein.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A VEGF-binding short peptide, characterized in that, Choose from any of the following: VE1: MSPEAWETLRLLAKEGCKDPEEAVRLALEFSPGPLVVEIDGEEVTVSLDEDGEIVLETEKGVKEEEKPELIKKIFEKAAE, as shown in SEQ ID NO.1; VE4: SLEEDFYEDFERSINEILESTPEEKREEVLKKILEDLEKVAKDDPLLAKILKRYKEKR, as shown in SEQ IDNO.

2.

2. The use of the VEGF-binding short peptide of claim 1 in the preparation of a drug for treating angiogenesis-related diseases.

3. The application according to claim 2, characterized in that, The angiogenesis-related diseases include, but are not limited to: solid tumors, age-related macular degeneration, and diabetic retinopathy.

4. The use of the VEGF-binding short peptide of claim 1 in the preparation of an anti-angiogenic therapeutic drugs.

5. The application according to claim 4, characterized in that, Application of VEGF-binding short peptides as VEGF inhibitors in the preparation of anti-angiogenic therapeutic drugs.

6. A pharmaceutical composition comprising the VEGF-binding short peptide of claim 1.

7. Use of the pharmaceutical composition of claim 6 in the preparation of a medicament for treating angiogenesis-related diseases.

8. Use of the pharmaceutical composition of claim 6 in the preparation of an anti-angiogenic therapeutic medicament.

9. A therapeutic drug for angiogenesis-related diseases, characterized in that, Its active ingredient is the VEGF-binding short peptide as described in claim 1, or it contains the pharmaceutical composition as described in claim 6.

10. An anti-angiogenic therapeutic drug, characterized in that, Its active ingredient is the VEGF-binding short peptide as described in claim 1, or it contains the pharmaceutical composition as described in claim 6.

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