Polypeptide targeting EGFR (epidermal growth factor receptor) protein extracellular domain as well as screening method and application thereof

Abstract

The invention provides a polypeptide targeting an EGFR (epidermal growth factor receptor) protein extracellular domain as well as a screening method and application of the polypeptide. The polypeptide library is synthesized by adopting a one-bead-one-compound (OBOC) combinatorial chemical method, the polypeptide library is combined with the epidermal growth factor receptor (EGFR) protein extracellular domain, the screened polypeptide is effectively combined with the epidermal growth factor receptor (EGFR) protein extracellular domain, the relative molecular weight is small, and the preparation cost is low. The polypeptide has an important application prospect in the aspects of targeted diagnosis and drug therapy of tumors with high EGFR expression.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology and relates to a polypeptide targeting the extracellular domain of an epidermal growth factor receptor EGFR protein and a screening method and application thereof. Background Art

[0002] EGFR (Epidermal Growth Factor Receptor) is a receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. It is one of the four members of the ErbB receptor family and is also known as ErbB-1. EGFR is a glycoprotein, a tyrosine kinase receptor, cell membrane-penetrating, with a molecular weight of 170KDa. It is located on the surface of the cell membrane and is activated by binding to the ligand to activate downstream pathways, including EGF and TGFα (transforming growth factorα). After activation, EGFR is converted from a monomer to a dimer (there is also evidence that dimers exist before activation). Alternatively, EGFR may also be activated by polymerizing with other members of the ErbB receptor family, such as ErbB2 / Her2 / neu. The activated EGFR can activate multiple downstream kinase pathways in the cell, such as activation sites such as Y992, Y1045, Y1068, Y1148 and Y1173, and through the cascade amplification of autophosphorylation, the final result is to induce cell proliferation.

[0003] Studies have found that EGFR is overexpressed or abnormally expressed in many solid tumors. EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis. Its overexpression plays an important role in the evolution of malignant tumors. EGFR is overexpressed in glial cells, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer and other tissues. Mutant EGFR exists in many tumors, and many types of EGFR mutants have been found. The effects of mutant EGFR may include: continuous activation of cells with ligand-independent receptors; destruction of receptor downregulation mechanisms due to the loss of certain domains of EGFR, activation of abnormal signal transduction pathways, inhibition of apoptosis, etc. The generation of mutants is due to the loss, mutation and rearrangement of the EGFR gene. EGFR ligands have a great influence on intracellular signal transduction. EGFR ligands activate EGFR in an autocrine form to promote cell proliferation. Their co-expression often indicates a poor prognosis for tumors. For example, in a study of breast invasive ductal carcinoma, it was found that TGFα was co-expressed with EGFR, and this co-expression was significantly correlated with the patient's survival rate. Kopp et al.'s study on colorectal cancer showed that the autocrine growth of tumors is the result of the combined effect of EGFR overexpression and its ligand expression. In addition, the study on the relationship between EGFR and tumor angiogenesis, high invasiveness and metastasis found that EGFR can affect tumor angiogenesis by regulating the levels of factors such as Ang-1 and VEGF.

[0004] In summary, the development of polypeptide preparations with specific binding to epidermal growth factor receptor EGFR protein and their application in targeted anti-tumor drugs for disease diagnosis and treatment remains one of the important research goals in this field. Summary of the invention

[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides a polypeptide targeting the extracellular domain of the epidermal growth factor receptor EGFR protein and a screening method and application thereof. The polypeptide can specifically bind to the extracellular domain of the epidermal growth factor receptor EGFR protein, and is of great significance in the development of targeted drugs for cancer, tumor diagnosis and treatment, etc.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein, wherein the amino acid sequence of the polypeptide comprises:

[0008] (1) a sequence shown in SEQ ID NO: 1 to SEQ ID NO: 10, or,

[0009] (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1), or,

[0010] (3) An amino acid sequence that has at least 95% sequence homology (e.g., 96%, 97%, 98% or 99%, etc.) with the sequence described in (1) or (2), and has the same or similar function as the sequence described in (1).

[0011] In the present invention, a one-bead-one-compound combinatorial chemistry method is used to synthesize a polypeptide library. The polypeptide library is characterized in that there is only one polypeptide on one resin. The polypeptide library is cultured with the fluorescently labeled EGFR protein extracellular domain to screen out polypeptides targeting the EGFR protein extracellular domain as shown in SEQ ID NO: 1 to SEQ ID NO: 10.

[0012] It can be understood that the present application screens for polypeptides targeting the extracellular domain of the EGFR protein. Therefore, the polypeptides obtained by replacing, deleting or adding one or at least two amino acid residues to the sequences shown in SEQ ID NO:1 to SEQ ID NO:10 using general technical means in the art, and having the same or similar functions as the original polypeptide, should also be within the scope of protection of the present application.

[0013] SEQ ID NO: 1: LFIIIGQ;

[0014] SEQ ID NO:2:ITGNNIS;

[0015] SEQ ID NO:3: KKKKYVV;

[0016] SEQ ID NO:4: FIITKPN;

[0017] SEQ ID NO:5: RVGGKKP;

[0018] SEQ ID NO:6: FRITKQK;

[0019] SEQ ID NO:7: IRAAWKQ;

[0020] SEQ ID NO:8: IFIIKKS;

[0021] SEQ ID NO:9: IVGGKKP;

[0022] SEQ ID NO: 10: DKQKAIG.

[0023] In a second aspect, the present invention provides a nucleic acid molecule encoding the polypeptide targeting the extracellular domain of the epidermal growth factor receptor EGFR protein as described in Aspect 1.

[0024] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0025] In a fourth aspect, the present invention provides a recombinant cell, characterized in that the recombinant cell expresses the polypeptide targeting the extracellular domain of the epidermal growth factor receptor EGFR protein as described in the first aspect.

[0026] In a fifth aspect, the present invention provides a method for screening a polypeptide targeting the extracellular domain of the epidermal growth factor receptor EGFR protein according to the first aspect, the method comprising the following steps:

[0027] (1) Synthesizing peptide libraries using the one-bead-one-compound method;

[0028] (2) Incubating the polypeptide library of step (1) with the extracellular domain of EGFR protein having a fluorescent label to screen and obtain the polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein.

[0029] According to the present invention, the one-bead-one-compound method is a combinatorial chemistry method. The established peptide library is mainly used to screen peptides with protein targeting function. The peptide library has a high throughput of 10 6～8, short screening time, low false positive rate and other advantages; the present invention utilizes a polypeptide library and the extracellular domain of the epidermal growth factor receptor EGFR protein for screening, and finally obtains a polypeptide resin that can specifically bind to the extracellular domain of the epidermal growth factor receptor EGFR protein, and after cleavage, the amino acid sequence of the polypeptide is obtained by sequencing; the method adopted by the present invention establishes the connection between the protein and the polypeptide, provides a simple and easy method for discovering polypeptides that bind to the epidermal growth factor receptor EGFR protein, provides a basis for further developing polypeptide targeting molecules, and provides a molecular basis for further developing targeted polypeptide drugs for imaging and treatment.

[0030] Preferably, the method for synthesizing the polypeptide library in step (1) specifically comprises:

[0031] (1-1) synthesizing the leader sequence on resin microbeads by solid phase synthesis;

[0032] (1-2) dividing the resin beads equally, activating the amino acid and mixing it with the equally divided resin beads, and performing a condensation reaction with the first deprotected amino acid;

[0033] (1-3) mixing the resin microbeads obtained in step (1-2) and performing Fmoc deprotection;

[0034] (1-4) Repeat steps (1-2) and (1-3) until the last amino acid is synthesized, and then deprotect the Fmoc and side chain to obtain the polypeptide library.

[0035] In the present invention, during the solid phase synthesis process, the resin microbeads are mixed and divided multiple times so that each resin microbead carries a unique random polypeptide sequence, thereby constructing a polypeptide library.

[0036] Preferably, the length of the leading sequence in step (1-1) is 2 to 4 aa.

[0037] Preferably, the leader sequence includes methionine (M).

[0038] In the present invention, a leader sequence containing methionine is first synthesized on the resin microbeads, and its main function is that the cyanogen bromide ethanol solution cleaves methionine to generate homoserine, so that the functional polypeptide falls off the resin microbeads.

[0039] According to the present invention, the leader sequence further contains any one or more of the other 19 amino acids, preferably glycine (G), which is used to space the resin microbeads from the functional polypeptide.

[0040] Preferably, the activation reagent in step (1-2) comprises any one of N,N-dimethylformamide, methanol, dichloromethane, N-methylmorpholine, N,N'-diisopropylcarbodiimide or 1-hydroxybenzotriazole, or a combination of at least two thereof, preferably a mixture of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT).

[0041] Preferably, the amino acid in step (1-2) is an Fmoc-protected amino acid or a side chain conventionally protected amino acid.

[0042] Preferably, the Fmoc deprotection reagent in step (1-3) comprises a DMF solution containing hexahydropyridine or a DMF solution containing piperazine and 1,8-diazabicycloundec-7-ene, preferably a DMF solution containing 20% ​​hexahydropyridine, or a DMF solution containing 5% piperazine and 2% 1,8-diazabicycloundec-7-ene.

[0043] Preferably, the side chain deprotection reagent in step (1-3) contains trifluoroacetic acid, water and benzene.

[0044] Preferably, the length of the polypeptides in the polypeptide library in step (1) is 7 to 10 aa.

[0045] In the present invention, a peptide library with a length of 7-10 aa is synthesized, and the 18 natural amino acids (except cysteine ​​and methionine) theoretically contain 18 7 ~18 10 It can screen out peptides with the function of binding to epidermal growth factor receptor EGFR protein, and ensure the efficiency of screening functional peptides. It is helpful to screen out peptides with the function of binding to epidermal growth factor receptor EGFR protein efficiently. At the same time, the peptides are short in length and small in molecular weight, and have the advantages of weak immunogenicity and high activity.

[0046] Preferably, the amino acid sequence of the extracellular domain of the EGFR protein in step (2) includes the sequence shown in SEQ ID NO:11.

[0047] SEQ ID NO:11:

[0048] .

[0049] Preferably, the fluorescent label in step (2) includes Cy5 fluorescent label.

[0050] Preferably, the incubation time in step (2) is 1 to 4 hours, for example, 1 hour, 2 hours, 3 hours or 4 hours, preferably 2 hours.

[0051] Preferably, the fluorescently labeled EGFR protein extracellular domain described in step (2) uses water as solvent.

[0052] Preferably, the screening in step (2) includes screening based on fluorescent signals and obtaining polypeptide sequences through analysis.

[0053] Preferably, after step (2), the method further includes synthesizing the screened polypeptides on resin microbeads and incubating them with the extracellular domain of EGFR protein with a fluorescent marker for secondary screening.

[0054] As a preferred technical solution, the method for screening polypeptides targeting the extracellular domain of epidermal growth factor receptor EGFR protein comprises the following steps:

[0055] S1. Firstly, a leader sequence with a length of 2 to 4 aa containing methionine is synthesized on resin microbeads;

[0056] S2. After the resin beads are evenly divided, an Fmoc-protected amino acid is activated and added to the resin beads to undergo a condensation reaction with the previously deprotected amino acid;

[0057] S3. The resin beads were mixed and Fmoc deprotection was performed using a DMF solution containing hexahydropyridine;

[0058] S4. Repeat steps S2 and S3 until the last amino acid is synthesized to obtain a polypeptide library, wherein the length of the polypeptides in the polypeptide library is 7 to 10 aa;

[0059] S5. Add the fluorescently labeled EGFR protein extracellular domain with an amino acid sequence as shown in SEQ ID NO: 11 to the polypeptide library, incubate for 2 to 4 hours, screen the resin microbeads with specific binding to the EGFR protein according to the fluorescence signal, and analyze the polypeptide sequence;

[0060] S6. The polypeptide sequence obtained by screening is synthesized on resin microbeads and co-incubated with the fluorescently labeled EGFR protein extracellular domain having an amino acid sequence as shown in SEQ ID NO: 11 for secondary screening.

[0061] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a cytotoxic drug or a contrast agent connected to the polypeptide targeting the extracellular domain of the epidermal growth factor receptor EGFR protein as described in the first aspect.

[0062] In the present invention, the cytotoxic drug connected to the polypeptide described in the first aspect uses the polypeptide to target EGFR-positive tumor cells, so that the cytotoxic drug exerts the efficacy of molecular targeted therapy, specifically kills EGFR-positive tumor cells, and avoids affecting surrounding normal tissue cells. The pharmaceutical composition also includes a contrast agent for imaging, etc., which is enriched in EGFR-positive tumor cells by the targeting effect of the polypeptide for diagnostic imaging, thereby achieving specific imaging.

[0063] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent, and the dosage form of the pharmaceutical composition is preferably an injection preparation.

[0064] In a seventh aspect, the present invention provides use of the polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein described in the first aspect or the pharmaceutical composition described in the sixth aspect in the preparation of disease treatment drugs or imaging detection agents.

[0065] Preferably, the disease comprises an EGFR-positive tumor.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) The present invention adopts a one-bead-one-compound method to synthesize a polypeptide library, and uses the polypeptide library and the extracellular domain of the epidermal growth factor receptor EGFR protein to screen and obtain polypeptides having the activity of targeting the extracellular domain of the epidermal growth factor receptor EGFR protein as shown in SEQ ID NO: 1 to SEQ ID NO: 10;

[0068] (2) Compared with antibody-targeted drug molecules, the polypeptides of the present invention have advantages such as small molecular weight, low immunogenicity, high activity, low cost, and easy synthesis and modification;

[0069] (3) The polypeptide screening method of the present invention has the advantages of high throughput and high screening efficiency. The screened polypeptides have strong binding ability with the extracellular domain of the epidermal growth factor receptor EGFR protein, good specificity, small molecular weight, weak immunogenicity, and high activity. The connection between the protein and the polypeptide is established, which provides a simple and easy method for discovering polypeptides that bind to the epidermal growth factor receptor EGFR protein, and provides a basis for further research and development of polypeptide drugs targeting the epidermal growth factor receptor EGFR protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a fluorescence microscopy image of the one-bead-one-compound method OBOC peptide library resin after binding to Cy5-labeled EGFR protein;

[0071] Figure 2A This is a result diagram of the polypeptide sequence FIITKPN (SEQ ID NO: 4) obtained by secondary mass spectrometry analysis;

[0072] Figure 2B The result diagram of the polypeptide sequence ITGNNIS (SEQ ID NO: 2) obtained by secondary mass spectrometry analysis;

[0073] Figure 2C The result diagram of the polypeptide sequence IVGGKKP (SEQ ID NO: 9) obtained by secondary mass spectrometry analysis;

[0074] Figure 3 This is the result of ELISA experiment testing the binding force between peptide and protein;

[0075] Figure 4 The binding constant KD of ITGNNIS and epidermal growth factor receptor EGFR protein was 7.1×10 -7 , showing good binding ability with EGFR protein;

[0076] Figure 5A This is a graph showing the results of a toxicity test of the ITGNNIS polypeptide on cells with high EGFR expression;

[0077] Figure 5B The figure is the result of the toxicity test of ITGNNIS polypeptide on cells with low EGFR expression;

[0078] Figure 5C The figure shows the results of the toxicity test between ten peptides and Hela cells. DETAILED DESCRIPTION

[0079] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0080] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0081] Example 1 Water-soluble Cy5 labeling of epidermal growth factor receptor EGFR protein

[0082] In this example, the epidermal growth factor receptor EGFR protein (SEQ ID NO: 11) is first labeled with Cy5, and the steps are as follows:

[0083] 0.15 mol / L NaCl solution and 0.15 mol / L NaHCO3-Na2CO3 buffer solution with pH=9.0 were mixed at a molar ratio of 9:1, and epidermal growth factor receptor EGFR protein was added to the mixed solution for protein dissolution, so that the final concentration of epidermal growth factor receptor EGFR protein was 10 mg / mL;

[0084] Cy5 was added to the EGFR protein solution, and the solution was placed in an ice bath and magnetically stirred overnight in a light-proof condition;

[0085] On the second day, the mixed solution was placed in a dialysis bag with a molecular weight cut-off of 1000 and dialyzed in deionized water for two days, with the water changed several times;

[0086] Finally, the dialyzed solution was collected and freeze-dried using a freeze dryer to obtain the Cy5-labeled epidermal growth factor receptor EGFR protein.

[0087] Example 2 Establishment of OBOC polypeptide library

[0088] In this example, a peptide library was synthesized on resin beads using a one-bead-one-compound method. The steps are as follows:

[0089] (1) Take 1.8 g of resin microbeads and place them in a synthesis tube. Add a certain amount of DMF to immerse the resin to swell the resin and soak for more than 2 hours.

[0090] (2) Add Fmoc-protected methionine (M) to the synthesis tube, and add a mixture of HOBT (1-hydroxybenzotriazole) and DIC (N,N'-diisopropylcarbodiimide), and shake the tube for reaction for more than 2 hours; rinse the tube with methanol and DMF alternately three times, and repeatedly blow up the beads with a pipette during the rinsing process; then, test the beads in a boiling water bath with ninhydrin, and the beads will appear colorless;

[0091] (3) Add deprotection agent (20% hexahydropyridine + 80% DMF) to carry out Fmoc deprotection for 10 min, and rinse with methanol and DMF alternately three times. During the rinsing process, the microbeads are repeatedly blown up with a pipette; then the microbeads are tested in a boiling water bath with ninhydrin, and the color of the microbeads turns dark blue-purple;

[0092] (4) Add Fmoc-protected glycine (G) to the synthesis tube and repeat steps (2) and (3);

[0093] (5) Divide the resin in the synthesis tube into 18 equal parts, add the corresponding dose of Fmoc-protected amino acids (18 natural amino acids except methionine and cysteine) and HOBT (1-hydroxybenzotriazole) / DIC (N,N'-diisopropylcarbodiimide) mixed solution into each synthesis tube, and shake the reaction for more than 6 hours;

[0094] (6) Rinse with methanol and DMF alternately three times. During the rinsing process, the microbeads are repeatedly blown up with a pipette to ensure sufficient mixing and rinsing;

[0095] (7) Check the synthesis efficiency of three of the peptides, take out the beads, soak them in ethanol, and then test them in a boiling water bath with ninhydrin, repeat steps (5) to (7) until the beads become colorless, mix the resins in all the synthesis tubes, and perform Fmoc deprotection;

[0096] (8) Repeat steps (5) to (8) until the last amino acid is synthesized;

[0097] (9) After removing the side chain Fmoc protecting group with a deprotecting agent (20% hexahydropyridine + 80% DMF), rinse with DMF, methanol, and DMF three times in sequence, and dry-pump for 15 min at 25°C in the dark to obtain a relatively dry resin; transfer the dry resin to a small reaction bottle (dried in advance), add a magnetic bar, and use an acidic deprotecting agent (TFA 82.5%, phenol 5%, water 10%, TRIS 2.5%) to remove all acid-responsive side chain protecting groups, react at 25°C for 4 hours, and construct a peptide library.

[0098] Example 3 Screening of polypeptides targeting epidermal growth factor receptor EGFR protein

[0099] The Cy5-labeled epidermal growth factor receptor EGFR protein and the peptide library were cultured in PBS buffer for 2 hours, and the luminescence was observed under a fluorescence microscope (excitation wavelength 670 nm), and the peptide resin with obvious red fluorescence was selected.

[0100] like Figure 1 The picture shown is a fluorescence microscope picture of the interaction between the Cy5-labeled epidermal growth factor receptor EGFR protein and the OBOC peptide library. Red fluorescence can be observed in the microbeads bound to the epidermal growth factor receptor EGFR protein, and the peptides that have the ability to bind to the epidermal growth factor receptor EGFR protein are selected based on the fluorescence.

[0101] The selected peptide resin was washed several times with ultrapure water and dried, cleaved overnight with 30% CNBr3 ethanol solution, centrifuged to obtain the supernatant, and then desalted for peptide sequencing.

[0102] Peptide sequencing mainly uses tandem mass spectrometry to test secondary mass spectrometry, and the peptide sequence is confirmed through analysis and comparison of secondary mass spectrometry. Figure 2A , Figure 2B , Figure 2C As shown, the secondary mass spectra of three polypeptides FIITKPN (SEQ ID NO: 4), ITGNNIS (SEQ ID NO: 2), and IVGGKKP (SEQ ID NO: 9) are illustrated.

[0103] Example 4 ELISA further confirms and screens polypeptides

[0104] The peptide was diluted to 2 μg / mL with ELISA coating buffer, and 100 μL of the diluted peptide was added to each well of the 96-well plate, and coated overnight at 4°C. 300 μL of ELISA washing solution was added to each well, and removed after standing for 20 seconds. This step was repeated three times. 100 μL of ELISA blocking solution containing BSA was added to each well, and blocked at 25°C for 2 hours. After blocking, the plate was washed three times and dried. The biotin-labeled EGFR protein was diluted to 6 concentrations, 200 μL per well, and washed three times with washing solution after incubation at 37°C for 2 hours. The horseradish peroxidase labeled with Avidin was diluted 2000 times, 80 μL was added to one well, and washed three times with washing solution after incubation at 37°C for 1 hour. 100 μL of TMB was added to each well, and after incubation at 37°C in the dark for 30 minutes, 50 μL of terminator was added to each well, and the absorbance was measured at 450 nM.

[0105] like Figure 3As shown, the ten polypeptides (amino acid sequences are SEQ ID NO: 1 to SEQ ID NO: 10) can bind to the EGFR protein, and the binding of the polypeptides to the EGFR protein is significantly different from that to BSA, indicating that the polypeptides have the ability to specifically target proteins.

[0106] Example 5: Binding experiment of polypeptide to epidermal growth factor receptor EGFR protein

[0107] In order to quantitatively investigate the binding of peptides and proteins, the binding constant Kd of peptides and proteins was tested by the MST method.

[0108] (1) EGFR protein was prepared into a 1.25 μM protein solution using PBS (pH=7.4), and labeled with the dye N-hydroxysuccinimide (NHS)-ester. After labeling, free dye was removed by gel column filtration;

[0109] (2) 12 peptide solutions with different concentration gradients (5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039, 0.0195, 0.01, 0.005, 0.0025 μM) were mixed with the dye-labeled EGFR protein solution and then tested on the machine;

[0110] For example, Figure 4 As shown, the Kd value of ITGNNIS peptide is 10 -7 This indicates that the peptide can bind well to the epidermal growth factor receptor EGFR protein and has a good targeting effect.

[0111] In summary, the present invention adopts a one-bead-one-compound method to synthesize a polypeptide library, and screens out polypeptides targeting the epidermal growth factor receptor EGFR protein. The polypeptide has a strong ability to bind to the extracellular domain of the epidermal growth factor receptor EGFR protein, and the polypeptide sequence obtained thereby provides support for the research and development of new targeted polypeptide drugs; the polypeptide screening method of the present invention has the advantages of high throughput and high screening efficiency, and provides a simple and easy method for discovering polypeptides binding to the epidermal growth factor receptor EGFR protein.

[0112] Example 6 Cytotoxicity Experiment

[0113] Preparation of single cell suspension: Hela cells were suspended in medium containing 10% fetal bovine serum, counted, inoculated into a 96-well plate (5000-10000 cells / well) with a volume of 100 μL, and cultured overnight in a cell culture incubator at 37°C and 5% CO2;

[0114] After the cells adhered, the ITGNNIS polypeptide solution prepared with culture medium was added at concentrations of 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM. Three replicate wells were made for each concentration, with 100 μL per well. The remaining 1× PBS buffer was used as a blank control group and continued to be cultured in the incubator overnight. At the same time, the lymphoma cell line L-428 that does not express EGFR protein was used as a control group and treated in the same way as above;

[0115] After 24 hours, the culture medium was removed, and the cells were rinsed with PBS for 1 to 2 times. Then, 100 μL of CCK-8 reagent was added to the blank wells of CCK-8 reagent as the control group. The cells were cultured in the incubator for another 2 hours. The absorbance of each well at 450 nm was detected by an enzyme-labeled instrument, and the absorbance at 650 nm was set as the reference. The results were recorded.

[0116] like Figure 5A As shown in Figure 2, the ITGNNIS peptide only showed a certain killing effect on EGFR protein-positive Hela cells, and had no significant effect on 293T cells that did not express EGFR ( Figure 5B ), indicating that the polypeptide of the present invention has a specific binding effect on EGFR protein.

[0117] like Figure 5C As shown, the results of the interaction between the polypeptide and Hela cells at a concentration of 160 micromolar indicate that the ten polypeptides discovered in the present invention all have a certain ability to kill Hela cells.

[0118] In summary, the present invention adopts the one-bead-one-substance method to synthesize a polypeptide library and screens out polypeptides targeting EGFR protein. The polypeptides have strong binding ability with EGFR protein and low cytotoxicity. Compared with antibody-guided drugs, they have the advantages of small molecular weight, weak immunogenicity, high activity, low preparation cost and easy modification. They have important application prospects in the field of tumor treatment and / or detection.

[0119] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein, characterized in that: The amino acid sequence of the polypeptide comprises: (1) a sequence shown in SEQ ID NO: 1 to SEQ ID NO: 10, or, (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1), or, (3) An amino acid sequence that has at least 95% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide targeting the extracellular domain of the epidermal growth factor receptor EGFR protein according to claim 1.

3. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 2.

4. A recombinant cell, characterized in that The recombinant cell expresses the polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein according to claim 1.

5. A method for screening a polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein according to claim 1, characterized in that: The method comprises the following steps: (1) Synthesizing peptide libraries using the one-bead-one-compound method; (2) Incubating the polypeptide library of step (1) with the extracellular domain of EGFR protein having a fluorescent label to screen and obtain the polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein.

6. The method for screening a polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein according to claim 5, characterized in that: The method for synthesizing the polypeptide library in step (1) specifically comprises: (1-1) synthesizing the leader sequence on resin microbeads by solid phase synthesis; (1-2) dividing the resin beads equally, activating the amino acid and mixing it with the equally divided resin beads, and performing a condensation reaction with the first deprotected amino acid; (1-3) mixing the resin microbeads obtained in step (1-2) and performing Fmoc deprotection; (1-4) repeating steps (1-2) and (1-3) until the last amino acid is synthesized, and then removing Fmoc and side chain protection to obtain the polypeptide library; Preferably, the length of the leading sequence in step (1-1) is 2 to 4 aa; Preferably, the leader sequence includes methionine; Preferably, the activation reagent in step (1-2) comprises any one of N,N-dimethylformamide, methanol, dichloromethane, N-methylmorpholine, N,N'-diisopropylcarbodiimide or 1-hydroxybenzotriazole, or a combination of at least two thereof; Preferably, the amino acid in step (1-2) is an Fmoc-protected amino acid or a side chain conventionally protected amino acid; Preferably, the Fmoc deprotection reagent in step (1-3) comprises a DMF solution containing hexahydropyridine or a DMF solution containing piperazine and 1,8-diazabicycloundec-7-ene; Preferably, the side chain deprotection reagent in step (1-3) contains trifluoroacetic acid, water and benzene.

7. The method for screening a polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein according to claim 5 or 6, characterized in that: The length of the polypeptides in the polypeptide library in step (1) is 7 to 10 aa; Preferably, the amino acid sequence of the EGFR protein extracellular domain in step (2) includes the sequence shown in SEQ ID NO: 11; Preferably, the fluorescent label in step (2) includes a Cy5 fluorescent label; Preferably, the incubation time in step (2) is 1 to 4 hours; Preferably, the screening in step (2) includes screening based on fluorescent signals, and obtaining the polypeptide sequence by analysis; Preferably, after step (2), the method further includes synthesizing the screened polypeptides on resin microbeads and incubating them with the extracellular domain of EGFR protein with a fluorescent marker for secondary screening.

8. The method for screening a polypeptide targeting the extracellular domain of epidermal growth factor receptor EGFR protein according to any one of claims 5 to 7, characterized in that: The method comprises the following steps: S1. Firstly, a leader sequence with a length of 2 to 4 aa containing methionine is synthesized on resin microbeads; S2. After the resin beads are evenly divided, an Fmoc-protected amino acid is activated and added to the resin beads to undergo a condensation reaction with the previously deprotected amino acid; S3. The resin beads were mixed and Fmoc deprotection was performed using a DMF solution containing hexahydropyridine; S4. Repeat steps S2 and S3 until the last amino acid is synthesized to obtain a polypeptide library, wherein the length of the polypeptides in the polypeptide library is 7 to 10 aa; S5. Add the fluorescently labeled EGFR protein extracellular domain with an amino acid sequence as shown in SEQ ID NO: 11 to the polypeptide library, incubate for 2 to 4 hours, screen the resin microbeads with specific binding to the EGFR protein according to the fluorescence signal, and analyze the polypeptide sequence; S6. The polypeptide sequence obtained by screening is synthesized on resin microbeads and co-incubated with the fluorescently labeled EGFR protein extracellular domain having an amino acid sequence as shown in SEQ ID NO: 11 for secondary screening.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a cytotoxic drug or a contrast agent connected with the polypeptide targeting the extracellular domain of the epidermal growth factor receptor EGFR protein according to claim 1.

10. Use of the polypeptide targeting the extracellular domain of epidermal growth factor receptor (EGFR) protein according to claim 1 or the pharmaceutical composition according to claim 9 in the preparation of disease therapeutic drugs or imaging detection agents.

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