Anti-angiogenic peptide as well as screening method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to an anti-angiogenic peptide as well as a screening method and application thereof. The anti-angiogenesis peptide is polypeptide 1 or polypeptide 2; the amino acid sequence of the polypeptide 1 is as shown in SEQ ID NO. 1; the polypeptide 2 is a polypeptide which is derived from the polypeptide 1 through substitution and / or deletion and / or addition of one or two amino acid residues and has the same glycosyl transferase function. The anti-angiogenesis peptide provided by the invention has good targeting property and anti-angiogenesis activity, so that the anti-angiogenesis peptide has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to an anti-angiogenic peptide and a screening method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The theoretical basis of anti-angiogenesis therapy for tumors is the "starving tumor" theory, which means that in the early stages of tumor growth, the tumor mainly relies on diffusion to obtain nutrients from the surrounding area. At this time, the tumor grows slowly and is not easy to metastasize. When the tumor grows to a certain extent and the nutrients obtained cannot meet its own growth needs, the tumor and its surrounding stromal cells will express a variety of angiogenic growth factors to promote the growth of endothelial cells to the tumor tissue site, eventually generating new tumor microvessels and promoting the growth and invasion of tumor cells.

[0004] After decades of research, people have found that vascular endothelial growth factor (VEGF) is the main factor promoting angiogenesis. In a variety of tumor cells, VEGF will be overexpressed when significantly stimulated, thereby promoting new blood vessel formation and tumor growth. A large number of studies have shown that VEGF binds to its receptor VEGFR-2, which can induce tyrosine phosphorylation of VEGFR-2, thereby triggering a series of downstream signaling events, thereby achieving the effect of promoting angiogenesis. Based on this, blocking the signaling pathway of vascular endothelial growth factor and its receptor (VEGF / VEGFR), thereby blocking angiogenesis, is an important direction for tumor treatment. Therefore, it is necessary to use this as a target to try to find and develop more anti-angiogenic active substances. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide an anti-angiogenic peptide and a screening method and application thereof. The anti-angiogenic peptide provided by the present invention has good targeting and anti-angiogenic activity, and therefore has good practical application value.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, an anti-angiogenic peptide is polypeptide 1 or polypeptide 2;

[0008] The amino acid sequence of the polypeptide 1 is shown in SEQ ID NO.1;

[0009] The polypeptide 2 is a polypeptide derived from polypeptide 1 in which one or two amino acid residues are substituted and / or deleted and / or added and which has the same glycosyltransferase function.

[0010] In a second aspect, a method for screening the above-mentioned anti-angiogenic peptides comprises using a phage random twelve-peptide library to screen specific targeting polypeptides using VEGF as a target protein, using phage to perform affinity screening in at least one adsorption-elution-amplification process to obtain a monoclonal phage that can specifically bind to the specific targeting polypeptide, and obtaining the amino acid sequence of the specific targeting polypeptide through the monoclonal phage; and then using ELISA detection and surface plasmon resonance (SPR) in turn to further screen peptides with affinity activity.

[0011] In a third aspect, a method for preparing the above-mentioned anti-angiogenic peptide comprises:

[0012] The anti-angiogenic peptide is prepared by a microbial fermentation method; or,

[0013] The anti-angiogenic peptide is prepared by genetic engineering; or,

[0014] The anti-angiogenic peptides were synthesized by chemical methods.

[0015] In some embodiments, the chemical method is solid phase synthesis.

[0016] In a fourth aspect, a use of the anti-angiogenic peptide described above in the preparation of a drug, wherein the drug is an anti-angiogenic drug or an anti-tumor drug.

[0017] In some embodiments, the medicament is a composition.

[0018] Specifically, the composition comprises one or more of the anti-angiogenic peptides.

[0019] Specifically, the composition includes pharmaceutical excipients. More specifically, the pharmaceutical excipients can be pharmaceutical carriers, such as natural polymer materials (lipids, sugars, proteins, etc.), synthetic polymer materials (polycyanoacrylates, polylactic acid, etc.), small molecule organic materials (such as stearic acid, etc.), etc.; they can also be excipients, such as binders (syrup, gum arabic, sodium carboxymethyl cellulose, etc.), fillers (lactose, starch, etc.), disintegrants (sodium carboxymethyl starch, microcrystalline cellulose, cross-linked polyvinylpyrrolidone, etc.), solubilizers (polysorbate, poloxamer, etc.), preservatives (benzyl alcohol, benzoic acid, salicylic acid, etc.), pH adjusters, stabilizers, etc.

[0020] In some embodiments, the drug is in the form of tablets, granules, lyophilized powder injections, injections, etc.

[0021] In some embodiments, the drug is administered orally, subcutaneously, intravenously, intramuscularly, or the like.

[0022] In some embodiments, the diseases that anti-angiogenic drugs are used to treat include but are not limited to diabetic retinopathy, coronary heart disease, degenerative arthritis, etc.

[0023] In some embodiments, the tumor includes but is not limited to skin cancer, head and neck cancer, lung cancer, esophageal cancer, cervical cancer, uterine cancer, pancreatic cancer, breast cancer, kidney cancer, ureteral cancer, bladder cancer, squamous cell carcinoma, basal cell carcinoma, melanoma, tongue cancer, pharyngeal squamous cell carcinoma or malignant lymphoma, laryngeal squamous cell carcinoma, lung squamous cell or small cell carcinoma, esophageal squamous cell carcinoma, cervical cancer, digestive tract tumors, reproductive system tumors, lymphomas, bone tumors, head and neck tumors, etc.

[0024] The beneficial effects of the present invention are:

[0025] The anti-angiogenic peptide provided by the present invention has good affinity for VEGF. The tube formation experiment shows that the anti-angiogenic peptide provided by the present invention has good anti-angiogenic activity, which is beneficial to achieve tumor treatment by blocking angiogenesis. In addition, the cell proliferation experiment shows that the anti-angiogenic peptide can also directly inhibit the proliferation of mouse breast cancer cells, thus having good anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is the analytical sensorgram of the interaction between different concentrations of V19 polypeptide and VEGF in Example 1 of the present invention;

[0028] Figure 2 This is a graph showing the affinity fitting results of the interaction between the V19 polypeptide and VEGF in Example 1 of the present invention;

[0029] Figure 3 This is a graph showing the experimental results of the V19 polypeptide screened in Example 2 of the present invention inhibiting endothelial cell lumen formation in vitro.

[0030] Figure 4 This is a graph showing the experimental results of the V19 polypeptide in Example 3 of the present invention inhibiting the proliferation of mouse breast cancer cells in vitro. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0032] Embodiment 1:

[0033] 1. Four rounds of panning, DNA extraction and sequencing

[0034] (1) VEGF was prepared into a solution with a concentration of 10 μg / mL using 0.1 M sodium bicarbonate solution (pH 8.6). 100 μL of the solution was added to a 96-well ELISA plate and shaken at 75 rpm at 4°C overnight.

[0035] (2) Discard the coating solution, fill up with BSA buffer blocking solution, and incubate at 4°C for 90 minutes.

[0036] (3) Discard the buffer and wash six times with TBST 0.1% Tween 20 for 1 min each time, tapping vigorously.

[0037] (4) 1×10 phage virus 11 90 μL TBS and 10 μL phage peptide library stock solution were added into the coated wells and mixed well, and then shaken at room temperature for 60 minutes.

[0038] (5) Discard the phage solution and tap vigorously. To wash away unbound phage, wash six times with TBST (0.1% Tween 20), each time for 1 minute, and tap dry.

[0039] (6) To elute the phages specifically bound to the monoclonal antibody, add 100 μL of glycine buffer and shake slowly and evenly at room temperature for 60 minutes.

[0040] (7) The eluate was transferred into a 1 mL sterile microcentrifuge tube, and 15 μL Tris-HCl buffer was added to obtain the first round of phage eluate. The first round of eluate was used for phage titer determination and amplification.

[0041] (8) Repeat the above process to perform the 2nd to 4th rounds of phage peptide library screening. In the last two rounds of screening, the Tween concentration in the washing step needs to be increased to 0.5% (v / v), and the eluent is changed to a free target molecule solution.

[0042] (9) The overnight cultured ER2738 was diluted 1:100 and inoculated into LB medium. 10 mL was aliquoted into a sterile culture bottle.

[0043] (10) Pick blue plaques from the fourth round of phage plate (the total number of plaques does not exceed 100) and transfer them to the above culture tube. Incubate on a shaker at 37°C for 4.5 h.

[0044] (11) Transfer the culture into a sterile centrifuge tube and centrifuge at 10,000 rpm for 30 seconds. Take the supernatant and centrifuge again for 30 seconds.

[0045] (12) Transfer 80% of the supernatant above into a new EP tube, which is the amplified phage storage solution.

[0046] (13) After amplifying the selected phage monoclonal, centrifuge and collect 500 μL of phage-containing supernatant. Add 200 μL of PEG / NaCl, invert and mix, and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 10,000 rpm for 10 minutes, and discard the supernatant.

[0047] (14) Add 100 μL of iodide buffer to resuspend, centrifuge briefly, add 250 μL of anhydrous ethanol, and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 10,000 rpm for 10 minutes and discard the supernatant.

[0048] (15) Add 70% ethanol to wash the precipitate, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant.

[0049] (16) Add 30 μL TE buffer and sequence the monoclonal phage.

[0050] (17) The results of monoclonal phage DNA sequencing were analyzed and the insertion sequence was found between the GGTACC Kpn I restriction site and the CGGCCGEag I restriction site. The amino acid sequence of the corresponding inserted polypeptide was then deduced using the genetic code table.

[0051] 2. Molecular Docking

[0052] The target protein and the screened peptide were docked using the ZDOCK module in the Discovery Studio Visualizer client software, and the ZDock Score in the result was used as the scoring function. The larger the absolute value of the ZDock Score, the stronger the binding ability between the receptor and the ligand. The steps are as follows:

[0053] (1) The protein structure of VEGF was retrieved and downloaded from the Protein Data Bank (PDB) database as the receptor protein in the molecular docking experiment. The structure of the screened peptides was predicted from the PEP-FOLD website, and the prediction results were saved and used as the ligand protein in the molecular docking experiment.

[0054] (2) Open the Discovery Studio Visualizer client software, find the target protein and peptide files in File and open them. Select Prepare Protein in the expanded menu bar, click the Clean Protein shortcut tool in the Manual Preparation panel group, and perform pretreatment on the target protein and screened peptides to remove ligand molecules and water molecules in the protein crystal structure, remove protein multiple conformations, add incomplete amino acid residues, and perform protein hydrogenation.

[0055] (3) Use ZDOCK to dock the screened peptide molecules with the processed target protein. Open the receptor and ligand proteins in the same 3D window, click Macromolecules in the Tool toolbar, expand the Dock and Analyze Protein Complexes tool, click Dock Proteins (ZDOCK), and the parameter setting dialog box will open. Click the Input Receptor Protein parameter and select 2ptn:2ptn; click the Input Ligand Protein parameter and select 2ptn:2sta; click the Angular Step Size parameter and select 15. Expand the Clustering parameter group, click the RMSD Cutoff parameter and set the value to 6; click the Interface Cutoff parameter and set the value to 9; click the Maximum Number of Clusters parameter and set the value to 60. Click Run and wait for the results to complete.

[0056] (4) When the calculation is finished, DS will display the Job Completed dialog box. In the Job Explorer, double-click the completed calculation task and DS will open a new Report.htm file. In the Output Files section, click the ZDockResults.dsv link. In the Data Table, click Protein Pose. Click Macromolecule in the Tools toolbar, select the Dock and Analyze Protein Complexes tool, click Browse in Browse Poses, and select Top Poses in Largest Clusters in the drop-down menu. This will display the top 100 highest-scoring conformations in the 10 largest clusters. Select Chart|3D Point Plot. This will open a Choose Plot Axes dialog box. Select ZDock Score on the X-axis, Cluster on the Y-axis, and Density on the Z-axis. Click OK. In the results, select the Pose with a higher ZDock Score. At the same time, the corresponding conformation in the molecule window will be selected. At this time, you can see the ZDock Score corresponding to the selected Pose. This study selected ZDock Score as the scoring function. The larger the absolute value of ZDock Score, the stronger the binding ability between the receptor and the ligand.

[0057] The absolute values ​​of ZDock Score were sorted from large to small, and the top 30 screening peptides were selected for the next experiment.

[0058] 3. ELISA experiment

[0059] The first 30 phage clones from the molecular docking results were subjected to ELISA test to identify the binding ability between the phage displayed peptide and the target protein. The steps are as follows:

[0060] (1) When amplifying plaques for DNA sequencing, store the remaining plaque-containing supernatant at 4°C.

[0061] (2) Dilute the overnight cultured ER2738 into 20 mL LB medium at a ratio of 1:100. Add 5 μL of phage supernatant to each tube of ER2738 culture medium and culture at 37°C with aeration for 4.5 h.

[0062] (3) The above culture was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a fresh centrifuge tube and centrifuged again.

[0063] (4) Take 80% of the supernatant and place it in a fresh centrifuge tube, add 1 / 6 volume of PEG / NaCl, and precipitate at 4°C overnight.

[0064] (5) Centrifuge at 10,000 rpm for 15 min at 4°C to precipitate the solution, discard the supernatant, centrifuge briefly again, and aspirate off the remaining supernatant.

[0065] (6) The pellet was resuspended in 1 mL of TBS, the suspension was transferred to a microcentrifuge tube, and centrifuged at 4°C for 5 min to remove residual cells in the pellet.

[0066] (7) Transfer the supernatant to a fresh microcentrifuge tube and add 1 / 6 volume of PEG / NaCl for reprecipitation. Incubate on ice for 15-60 min. Centrifuge at 4°C for 10 min, discard the supernatant, centrifuge briefly again, and remove the remaining supernatant.

[0067] (8) Resuspend the pellet in 50 μL TBS and measure the phage titer. Store at 4°C.

[0068] (9) Coat each well of the ELISA plate with 100 μL of 1 μg / mL target molecule, with 3 coated wells for each clone to be identified. Coating was carried out overnight at 4°C in a sealed humidified box.

[0069] (10) Shake off excess target molecule solution and invert the plate on a paper towel to remove residual liquid. Fill each well with blocking solution and block at 4°C for 1 h. In addition, add blocking solution to each uncoated well of the clone to be identified to detect the binding affinity of the selected sequence to the BSA-coated plastic plate.

[0070] (11) Discard the blocking solution and wash the plate six times with TBST 0.5% Tween 20. Each time, invert the plate on a clean paper towel and tap it to remove the solution.

[0071] (12) 1×10 9 Add phages to the wells coated with target molecules and shake at room temperature for 1 hour.

[0072] (13) Wash the plate 6 times with TBST 0.5% Tween 20. Dilute HRP-labeled anti-M13 antibody at a ratio of 1:5000 in blocking solution. Add 100 μL of diluted antibody to each well and shake at room temperature for 1 hour.

[0073] (14) Wash the plate six times with TBST 0.5% Tween 20. Add 200 μL of HRP substrate solution to each well and incubate at room temperature for 1 h.

[0074] (15) Record the absorbance at 405-415 nm using a plate reader.

[0075] The results showed that the phages with OD values ​​more than 2 times higher than that of the negative control group, lower affinity to the blocking solution and higher affinity to the target protein were regarded as positive phages specifically binding to the target protein.

[0076] IV. SPR Experiment

[0077] The peptides of the top three phage clones in the ELISA test results were synthesized, and the surface plasmon resonance (SPR) technique was used to determine the peptide with the strongest affinity to VEGF.

[0078] (1) System test: Replace the buffer with 1.05× PBS and the chip with a new one. In the Tools module of the Biacore X100plus program control software, select the System Check and Pump Calibration option and perform a system test with BIAtest solution and ultrapure water according to the prompts. Proceed to the next step after all results are passed.

[0079] (2) Find the optimal pH for coupling protein on the chip: Use the FindImmobilization pH module in the Kinetics / Affinity module to determine the optimal pH for coupling the target protein. According to the system instructions, dilute the protein to 50 μg / mL with 10 mM sodium acetate buffer solutions at pH 4.0, 4.5, 5.0, and 5.5, respectively, use 50 mM NaOH solution as the cleaning solution, and inject the sample for 180 seconds in sequence. After the end, the result graph pops up. According to the response value and curve shape of the sample under each pH condition, select the appropriate pH as the pH condition for the final coupling protein.

[0080] (3) Coupling protein: Select the Immobilize option in the Kinetics / Affinity module, and immobilize the target protein solution at the optimal pH selected in the previous step on the CM5 chip by the amino coupling method. Flow Cell 2 is used as the sample channel, and the blank Flow Cell 1 is used as the reference channel. The maximum protein coupling amount Rmax is set to 100RU according to the formula, and the ligand coupling level RL is calculated according to the following formula, where SM is the stoichiometric ratio, which is usually set to 1. The actual coupling amount in the experiment is 1.5 times RL.

[0081]

[0082] (4) Affinity (KD value) determination: Since the peptide drug used in SPR is dissolved in less than 0.5% DMSO, and extremely low concentrations of DMSO have almost no effect on the SPR results, this experiment does not require a calibration curve. Prepare 100 μM of the peptide to be tested, and measure the affinity of a series of peptides with a concentration gradient (half-fold dilution method) to the target protein. Obtain the RU at the binding saturation period, and use the single-site interaction model to fit the binding affinity between the target protein and the peptide.

[0083] Figure 1 The sensorgrams for analyzing the interaction between V19 peptide and VEGF at different concentrations are shown in FIG. Figure 2 The affinity fitting result diagram of the interaction between V19 polypeptide and VEGF, in which the vertical line represents the fitting KD value, which falls within the sample concentration range, indicating that the fitting data is credible. Table 1 is the fitting result data of the interaction between V19 polypeptide and VEGF. The smaller the affinity fitting KD value, the stronger the affinity between the polypeptide and the protein. It can be seen from the results that the KD value of V19 is 9.803E-5, which indicates that the V19 polypeptide has a good affinity with VEGF.

[0084] Table 1 Fitting results of the interaction between V19 polypeptide and VEGF

[0085] KD(M) Rmax(RU) <![CDATA[Chi 2 (RU2)]]> 9.803E-5 26.76 0.710

[0086] The amino acid sequence of the screened polypeptide V19 is RTSPSPRGKHGE, as shown in SEQ ID NO. 1. It was commissioned to Hangzhou Baige Pharmaceutical Technology Co., Ltd. for synthesis.

[0087] Example 2 Effect of V19 peptide on endothelial cell lumen formation

[0088] (1) Use a pre-cooled pipette tip to add the melted Matrigel into a pre-cooled 48-well plate (80 μL / well), being careful to avoid creating bubbles.

[0089] (2) Collect EAhy 926 cells in the logarithmic growth phase and adjust the cell suspension concentration to 11×10 4 pcs / well and added into the well plate covered with matrix gel.

[0090] (3) The wells without drug but with only DMEM medium were used as blank control group, the wells with serum-free DEME medium containing 10 ng / mL VEGF were used as negative control group, the wells with serum-free DEME medium containing 10 ng / mL VEGF and ES2 (an anti-angiogenic peptide) were used as positive control group, and the wells with serum-free DEME medium containing 10 ng / mL VEGF and V19 peptide were used as experimental group. The drug concentrations were set at 500 μg / mL and 1 mg / mL, and 3 replicate wells were set for each group.

[0091] (4) After the addition of drugs, the well plate was cultured for another 6 h, and the formation of the cell vascular network was observed and photographed using an inverted fluorescence microscope. The photos were processed using Image J software.

[0092] The results are as follows Figure 3 As shown, compared with the negative control group, both V19 and ES2 can significantly inhibit the formation of endothelial cell lumens, as shown by the obvious lattice-like structure of endothelial cells in the blank control group and the negative control group, while the endothelial cells in the V19 experimental group and the ES2 positive control group are mostly in a single-layer adherent state, with only a small amount of tubular structures formed. This result shows that the V19 polypeptide has a strong anti-angiogenic ability in vitro.

[0093] Example 3 Effect of V19 peptide on proliferation of mouse breast cancer cells

[0094] 4T1 cells in logarithmic phase were collected, cell suspension was adjusted to an appropriate concentration, and 5×10 3Cells were inoculated into 96-well plates, placed in a carbon dioxide incubator, and cultured overnight at 37°C until the cells adhered to the wall. Then V19 and ES2 (an anti-angiogenic peptide that can inhibit endothelial cell proliferation but has no effect on tumor cells, used as a control group) were added, with drug concentrations of 1μg / mL, 5μg / mL, 10μg / mL, 50μg / mL, 100μg / mL, 500μg / mL, and 1mg / mL, respectively, and 8 replicates were set for each drug. The wells containing only DMEM culture medium were set as blank control groups, and the wells with cells and no drug-containing culture medium were set as negative control groups. The 96-well plate was placed in a carbon dioxide incubator and incubated for 48 h. The culture medium was discarded under light-proof conditions, and CCK-8 solution was added to the 96-well plate, 10 μL per well. The 96-well plate was placed in an incubator and taken out after the culture medium turned orange. The OD value of each well was then detected by a microplate reader at a wavelength of 450 nm, and the cell proliferation rate was calculated as follows: Cell proliferation rate = [(experimental group - blank control group) / (negative control group - blank control group)] × 100%.

[0095] The results are as follows Figure 4 As shown, it can be seen that compared with the control group ES2 polypeptide, with the increase of concentration, the cell proliferation rate of the V19 polypeptide group showed a significant downward trend, which indicates that the V19 polypeptide has a significant inhibitory effect on the proliferation of 4T1 cells and thus has good in vitro anti-tumor activity.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-angiogenic peptide, characterized in that: It is polypeptide 1 or polypeptide 2; The amino acid sequence of the polypeptide 1 is shown in SEQ ID NO.1; The polypeptide 2 is a polypeptide derived from polypeptide 1 in which one or two amino acid residues are substituted and / or deleted and / or added and which has the same glycosyltransferase function.

2. A method for screening anti-angiogenic peptides according to claim 1, characterized in that: A phage random twelve-peptide library is used to screen specific targeting polypeptides with VEGF as the target protein, and phage is used to perform affinity screening in at least one adsorption-elution-amplification process to obtain a monoclonal phage that can specifically bind to the specific targeting polypeptide, and the amino acid sequence of the specific targeting polypeptide is obtained through the monoclonal phage; and then ELISA detection and surface plasmon resonance are used in sequence to further screen peptides with affinity activity.

3. A method for preparing the anti-angiogenic peptide according to claim 1, characterized in that: The method includes: The anti-angiogenic peptide is prepared by a microbial fermentation method; or, The anti-angiogenic peptide is prepared by genetic engineering; or, The anti-angiogenic peptides were synthesized by chemical methods.

4. The method according to claim 3, characterized in that: The chemical method is a solid phase synthesis method.

5. Use of the anti-angiogenic peptide according to claim 1 in the preparation of a drug, wherein the drug is an anti-angiogenic drug or an anti-tumor drug.

6. The use according to claim 5, characterized in that: The medicine is a composition; Preferably, the composition comprises one or more of said anti-angiogenic peptides; Preferably, the composition comprises a pharmaceutically acceptable excipient.

7. The use according to claim 5, characterized in that: The dosage form of the medicine is tablet, granule, freeze-dried powder injection or injection.

8. The use according to claim 5, characterized in that: The drug is administered orally, by subcutaneous injection, intravenous injection or intramuscular injection.

9. The use according to claim 5, characterized in that: Antiangiogenic drugs are used to treat diseases such as diabetic retinopathy, coronary heart disease or degenerative arthritis.

10. The use according to claim 5, characterized in that: The tumor is skin cancer, head and neck cancer, lung cancer, esophageal cancer, cervical cancer, uterine cancer, pancreatic cancer, breast cancer, kidney cancer, ureteral cancer, bladder cancer, squamous cell carcinoma, basal cell carcinoma, melanoma, tongue cancer, pharyngeal squamous cell carcinoma or malignant lymphoma, laryngeal squamous cell carcinoma, lung squamous cell or small cell carcinoma, esophageal squamous cell carcinoma, cervical cancer, digestive tract tumors, reproductive system tumors, lymphomas, bone tumors or head and neck tumors.

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