UPAR high-affinity polypeptide, pharmaceutical composition and application of uPAR high-affinity polypeptide

Abstract

The invention belongs to the technical field of biological medicine, and relates to uPAR high-affinity polypeptide, a pharmaceutical composition and application of the uPAR high-affinity polypeptide. The polypeptide is a tumor targeting peptide with an amino acid sequence as shown in SEQ ID NO.1 or an anti-tumor peptide with an amino acid sequence as shown in SEQ ID NO.2. The invention further discloses a preparation method of the tumor targeting peptide. The tumor targeting peptide provided by the invention has good tumor targeting property, and the anti-tumor active peptide provided by the invention has good anti-tumor activity, so that the tumor targeting peptide has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a uPAR high-affinity polypeptide, a pharmaceutical composition and applications 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] Molecular targeted therapy for tumors can enhance the specificity and selectivity of anticancer therapy, reduce the toxic side effects and drug resistance of general chemotherapy drugs, and have good clinical therapeutic effects. Urokinase-type plasminogen activator receptor (uPAR), also known as CD87, mainly binds to its natural ligand urokinase-type plasminogen activator (uPA). The interaction between uPAR and uPA can trigger a series of proteolytic events and degrade the extracellular matrix (ECM). When the zymogen form of uPA (pro-uPA) binds to uPAR, it will be converted into active uPA. The binding of uPA to uPAR will convert inactive plasminogen into active plasmin, activate a series of downstream proteases, such as matrix metalloproteinases, lead to ECM remodeling, and promote tumor cell migration and invasion. In addition to proteolysis, uPAR can also interact with vitronectin and transmembrane receptors, including integrins and receptor tyrosine kinases, thereby activating intracellular signaling pathways such as focal adhesion kinase, mitogen-activated protein kinase, phosphatidylinositol kinase and Janus kinase 1, thereby promoting tumor cell migration, adhesion, proliferation, angiogenesis and epithelial-mesenchymal transition.

[0004] uPAR has been found to be overexpressed in a variety of cancer cells, such as breast cancer, prostate cancer, glioma, colorectal cancer, endometrial cancer, bladder cancer, liver cancer and melanoma, while it is expressed at a low level in normal cells or adjacent tissues. Its overexpression indicates high tumor malignancy and poor prognosis. uPAR has also been found to be significantly highly expressed in stromal cells in the tumor microenvironment, such as vascular endothelial cells, tumor-associated fibroblasts and tumor-associated macrophages. This characteristic makes uPAR an ideal target for cancer treatment. Therefore, it is necessary to provide a new polypeptide targeting uPAR. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a uPAR high-affinity polypeptide, a pharmaceutical composition and its application, wherein the tumor targeting peptide has good tumor targeting, and the anti-tumor peptide has good anti-tumor activity, and thus has good practical application value.

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

[0007] In a first aspect, a uPAR high-affinity polypeptide is a polypeptide or a derivative thereof, wherein the polypeptide is a tumor-targeting peptide with an amino acid sequence as shown in SEQ ID NO.1 or an anti-tumor peptide with an amino acid sequence as shown in SEQ ID NO.2.

[0008] In some embodiments, the derivative is a derivative peptide obtained by amidation, hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification and / or glycosylation based on the side chain group, amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0009] On the other hand, a method for screening the above-mentioned uPA high-affinity polypeptides selects uPAR as the target protein, uses a phage random twelve-peptide library to screen specific targeting polypeptides, and sequentially uses phage affinity screening, ELISA detection and surface plasmon resonance detection to screen.

[0010] In some embodiments, during phage affinity screening, at least one process of phage adsorption, elution, and amplification is performed.

[0011] The third aspect provides a nucleic acid molecule encoding the above uPAR high affinity polypeptide.

[0012] In a fourth aspect, a recombinant vector comprises the above-mentioned nucleic acid molecule.

[0013] The recombinant vector of the present invention is obtained by effectively connecting the above nucleic acid molecule to a vector. The vector may be a viral vector, a plasmid, a bacteriophage, and the like.

[0014] A fifth aspect provides a transformed cell comprising the above-mentioned nucleic acid molecule or recombinant vector.

[0015] The transformed cells of the present invention may be any one or more of bacterial cells, fungal cells or plant cells.

[0016] A sixth aspect provides a pharmaceutical composition comprising the above-mentioned uPAR high-affinity polypeptide.

[0017] In some embodiments, pharmaceutical excipients are included. The pharmaceutical excipients of the present invention are pharmaceutical carriers and / or excipients. Specifically, the pharmaceutical carrier can be serum protein, lecithin, water, physiological saline, buffer solution, etc. Specifically, the excipient can be a binder, a filler, a disintegrant, a solubilizer, a preservative, a stabilizer, etc.

[0018] A seventh aspect is the use of the uPA high-affinity polypeptide or pharmaceutical composition described above in the preparation of anti-tumor drugs.

[0019] 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.

[0020] The beneficial effects of the present invention are:

[0021] The tumor targeting peptide provided by the present invention has a stronger affinity for uPAR; in vitro tumor targeting experiments show that the tumor targeting peptide has good tumor targeting to mouse breast cancer cells; in vitro cell experiments show that the anti-tumor peptide provided by the present invention has good anti-tumor activity to mouse breast cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 The sensorgrams are analytical graphs of the interaction between different concentrations of U44 polypeptide (A) and U45 polypeptide (B) and uPAR as described in Example 1 of the present invention.

[0024] Figure 2 This is a graph showing the affinity fitting results of the interaction between the U44 polypeptide (A) and the U45 polypeptide (B) described in Example 1 of the present invention and uPAR.

[0025] Figure 3 This is a graph showing the results of an in vitro tumor targeting experiment of the U44 polypeptide and the U45 polypeptide described in Example 2 of the present invention.

[0026] Figure 4 This is a graph showing the experimental results of the U44 polypeptide and the U45 polypeptide described in Example 3 of the present invention inhibiting the proliferation of mouse breast cancer cells in vitro. DETAILED DESCRIPTION

[0027] 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.

[0028] Embodiment 1:

[0029] 1. Four rounds of panning, DNA extraction and sequencing

[0030] (1) uPAR was prepared into a 10 μg / mL solution 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.

[0031] (2) Discard the coating solution, fill up with BSA buffer blocking solution, and incubate at 4°C for 90 minutes.

[0032] (3) Discard the buffer and wash six times with TBST 0.1% Tween 20 for 1 min each time, tapping vigorously.

[0033] (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.

[0034] (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.

[0035] (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.

[0036] (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.

[0037] (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.

[0038] (9) The overnight cultured ER2738 was diluted 1:100 and inoculated into LB medium. 10 mL was aliquoted into a sterile culture bottle.

[0039] (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.

[0040] (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.

[0041] (12) Transfer 80% of the supernatant above into a new EP tube, which is the amplified phage storage solution.

[0042] (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.

[0043] (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.

[0044] (15) Add 70% ethanol to wash the precipitate, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant.

[0045] (16) Add 30 μL TE buffer and sequence the monoclonal phage.

[0046] (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.

[0047] 2. Molecular Docking

[0048] 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:

[0049] (1) The protein structure of uPAR 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.

[0050] (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.

[0051] (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.

[0052] (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 PointPlot. 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.

[0053] The absolute values ​​of ZDock Score were sorted from large to small, and the top 30 screening peptides were selected for the next experiment.

[0054] 3. ELISA experiment

[0055] 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:

[0056] (1) When amplifying plaques for DNA sequencing, store the remaining plaque-containing supernatant at 4°C.

[0057] (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.

[0058] (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.

[0059] (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.

[0060] (5) Centrifuge at 10,000 rpm for 15 min at 4°C to precipitate the cell, discard the supernatant, centrifuge briefly again, and aspirate the remaining supernatant.

[0061] (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.

[0062] (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.

[0063] (8) Resuspend the pellet in 50 μL TBS and measure the phage titer. Store at 4°C.

[0064] (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.

[0065] (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.

[0066] (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.

[0067] (12) 1×10 9 Add phages to the wells coated with target molecules and shake at room temperature for 1 hour.

[0068] (13) Wash the plate 6 times with TBST 0.5% Tween 20. Dilute HRP-labeled anti-M13 antibody at a ratio of 1:5,000 in blocking solution. Add 100 μL of diluted antibody to each well and shake at room temperature for 1 hour.

[0069] (14) Wash the plate 6 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.

[0070] (15) Record the absorbance at 405-415 nm using a plate reader.

[0071] Results The phages whose OD value was more than 2 times higher than that of the negative control group, had lower affinity with the blocking solution and higher affinity with the target protein were regarded as positive phages specifically binding to the target protein.

[0072] IV. SPR Experiment

[0073] (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. If all are passed, proceed to the next step;

[0074] (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.

[0075] (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, 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.

[0076]

[0077] (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.

[0078] The results are as follows Figure 1-2 As shown, Figure 1 The sensorgrams for analyzing the interaction between different concentrations of U44 and U45 and uPAR are shown in Figure 2. Figure 2 The affinity fitting result diagram of U44 and U45 interacting with uPAR, where 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 U44 and U45 and uPAR. 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 U44 is 1.013E-9 and the KD value of U45 is 1.402E-6, which indicates that the U44 polypeptide has a stronger affinity with uPAR than the U45 polypeptide.

[0079] Table 1 is the fitting result data of the interaction between U44 polypeptide and U45 and uPAR described in Example 1 of the present invention

[0080]

[0081]

[0082] The amino acid sequence of the screened U44 peptide is PNAEARAISHTM, as shown in SEQ ID NO.1, and the amino acid sequence of the screened U45 peptide is APHKHQTWWRHS, as shown in SEQ ID NO.2, which were commissioned to Hangzhou Baige Pharmaceutical Technology Co., Ltd. for synthesis.

[0083] Example 2 In vitro tumor targeting of U44 peptide and U45 peptide

[0084] 4T1 cells in the logarithmic phase were collected, and cell suspensions of appropriate concentrations were prepared and inoculated into 12-well plates, and placed in a 37°C, 5% CO2 incubator until the cells adhered to the wall. The experiment was set up as a blank group, a uPA pre-incubation group, and a normal administration group. U44 polypeptide and U45 polypeptide were experimental samples, and ES2 was a control sample (no tumor targeting). A certain amount of free uPA protein was added to the uPA pre-incubation group so that the uPA concentration in each well was 1 μg / mL, and the cells were placed in an incubator and incubated overnight. The culture medium in the dish was discarded, and after washing twice with PBS, an appropriate amount of fluorescently labeled drugs (wherein the concentration of FITC was 50 μg / mL) was added to the uPA pre-incubation group and the normal administration group, and the incubation continued for 6 hours. After the incubation, the cells of each group were collected, and the fluorescence intensity of FITC in each group of cells was immediately detected by flow cytometry after washing twice with PBS.

[0085] The results are as follows Figure 3 As shown in the figure, it can be seen from the experimental results that after 4T1 cells were pre-incubated with free uPA, the cellular uptake of U44 peptide was significantly reduced, and the cellular uptake of U45 peptide also decreased to a certain extent, while the uptake of ES2 peptide in the control group was almost unchanged. This is because after pre-incubation with free uPA, the uPAR receptor on the surface of 4T1 cells binds to uPA and reaches a saturated state, which makes U44 and peptide lose the advantage of tumor targeting, and the cellular uptake is also reduced. This result shows that U44 peptide has excellent tumor targeting, and this targeting is mediated by uPAR receptor.

[0086] Example 3 Effect of U44 peptide on proliferation of mouse breast cancer cells

[0087] 4T1 cells growing in the logarithmic phase were collected, the 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 U44, U45, and ES2 were added, and the drug concentrations were 1μg / mL, 5μg / mL, 10μg / mL, 50μg / mL, 100μg / mL, 500μg / mL, and 1mg / mL, respectively. Eight replicate wells were set for each drug. The wells containing only DMEM medium were set as blank control groups, and the wells with cells but no drug-containing 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%.

[0088] The results are as follows Figure 4 As shown, it can be seen that compared with the control group ES2 peptide and tumor targeting peptide U44, the cell proliferation rate of the anti-tumor active peptide U45 group showed a significant downward trend with the increase in concentration, which indicates that the U45 polypeptide has a significant inhibitory effect on the proliferation of 4T1 cells and thus has good anti-tumor activity.

[0089] 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. A uPAR high affinity polypeptide, characterized in that: It is a polypeptide or a derivative thereof, wherein the polypeptide is a tumor targeting peptide with an amino acid sequence as shown in SEQ ID NO.1 or an anti-tumor peptide with an amino acid sequence as shown in SEQ ID NO.

2.

2. The uPAR high affinity polypeptide according to claim 1, characterized in that: The derivative is a derivative peptide obtained by amidation, hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification and / or glycosylation modification based on the side chain group, amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. A method for screening the uPAR high affinity polypeptide according to claim 1, characterized in that: uPAR was selected as the target protein, and a phage random twelve-peptide library was used to screen specific targeting peptides, which were then screened using phage affinity screening, ELISA detection, and surface plasmon resonance detection.

4. The method for screening uPAR high affinity polypeptides according to claim 3, characterized in that: In phage affinity screening, the process of phage adsorption, elution and amplification is carried out at least once.

5. A nucleic acid molecule, characterized in that: It encodes the uPAR high-affinity polypeptide described in claim 1.

6. A recombinant vector, characterized in that: Comprising the nucleic acid molecule of claim 5.

7. A transformed cell, characterized in that: Contains the above nucleic acid molecule or recombinant vector.

8. A pharmaceutical composition, characterized in that: Comprising the uPAR high affinity polypeptide according to claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that: Comprises pharmaceutical excipients; preferably, the pharmaceutical excipients are pharmaceutical carriers and / or excipients.

10. Use of the uPAR high affinity polypeptide according to claim 1 or 2 or the pharmaceutical composition according to claim 8 or 9 in the preparation of anti-tumor drugs; Preferably, 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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