Polypeptide for blocking HGF-HGFR binding and its application

Abstract

The present invention relates to the field of biomedicine, and specifically to a polypeptide that blocks HGF-HGFR binding and its application. The amino acid sequence of the polypeptide contains any one of a first amino acid sequence as shown in SEQ ID NO: 1 and a second amino acid sequence composed of a plurality of consecutive amino acids in the first amino acid sequence. The polypeptide provided by the present invention can effectively block the binding of HGF-HGFR, prevent HGFR from being overactivated and causing excessive cell proliferation to form tumors, cause tumor cell apoptosis, and inhibit tumor cell proliferation and tumor cell metastasis, thereby preventing and / or treating diseases related to its signaling pathway.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide for blocking HGF-HGFR binding and an application thereof. Background Art

[0002] Hepatocyte growth factor (HGF, also known as scatter factor (SF)) is a heterodimeric paracrine growth factor that exerts its activity by interacting with the hepatocyte growth factor receptor (HGFR), mediating a series of characteristic biological functions, including cell proliferation, motility, differentiation and survival. HGFR is the product of the c-Met oncogene and is also known as MET. MET is a receptor tyrosine kinase composed of a transmembrane β chain connected to an extracellular α chain by a disulfide bond. The binding of HGF to MET activates the kinase catalytic activity of MET, leading to the phosphorylation of Tyr 1234 and Tyr 1235 of the β chain and the subsequent activation of downstream signaling pathways, such as PI3K-Akt, Ras-MAPK, STAT and Wnt / β-catenin, thereby promoting cell proliferation, cell growth, cell migration, vascular invasion and angiogenesis, and playing a key role in normal tissue development and tumor progression.

[0003] Abnormal c-Met activation by HGF, one of the representative carcinogenic mechanisms, is known to be associated with tumor proliferation, apoptosis inhibition, neoangiogenesis, invasion, metastasis, etc. In addition, it has been reported that abnormal c-Met activation by c-Met mutation and amplification is associated with various cancers such as lung cancer, colon cancer, gastric cancer, breast cancer, etc., and is also associated with increased tumor invasiveness and its unfavorable prognosis. Therefore, HGF / c-Met can be used as a target for the design of antitumor drugs.

[0004] As the c-Met-specific small molecule tyrosine kinase inhibitors known so far, there are tivatinib, crizotinib, capmatinib, tepotinib, sirotinib, gumetinib, cabozantinib, gletinib, etc. In addition, rilotumumab, ficlatuzumab, etc. have been developed as HGF-specific monoclonal antibodies, which are ligands of c-Met. In addition, as antagonist monoclonal antibodies targeting c-Met, there are onartuzumab in clinical phase III, emibetuzumab in clinical phase II, SAIT-301 and ABT-700 in clinical phase I, etc.

[0005] As mentioned above, the HGF / c-Met signaling pathway is associated with the emergence and progression of various cancers, so the development of new therapeutic agents that prevent and treat cancer by targeting HGF and / or c-Met is a continuous requirement. Given the advantages of peptide drugs such as good selectivity, low toxicity and side effects, and low production costs, the development of peptide drugs that inhibit tumor cell growth and metastasis has important practical significance and application value. Summary of the invention

[0006] The purpose of the present invention is to provide a pharmaceutical polypeptide capable of blocking HGF-HGFR binding, wherein the pharmaceutical polypeptide has a high affinity with HGF and / or HGFR.

[0007] In order to achieve the above object, the first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof for blocking HGF-HGFR binding, wherein the amino acid sequence of the polypeptide comprises any one of the following amino acid sequences:

[0008] (1a) the first amino acid sequence as shown in SEQ ID NO: 1;

[0009] (1b) an amino acid sequence having at least 90% sequence identity with the first amino acid sequence;

[0010] (1c) a tag protein or a modified amino acid sequence is linked to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in (1a) and (1b);

[0011] (1d) An amino acid sequence having the same function obtained by processing the amino acid sequences shown in (1a), (1b) and (1c); the processing is at least one of replacing at least one amino acid, deleting at least one amino acid and adding at least one amino acid.

[0012] The second aspect of the present invention provides a gene, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that blocks HGF-HGFR binding as described in the first aspect.

[0013] The third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0014] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect.

[0015] The fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide for blocking HGF-HGFR binding as described in the first aspect or a pharmaceutically acceptable salt thereof.

[0016] The sixth aspect of the present invention provides the use of at least one of the polypeptide blocking HGF-HGFR binding described in the first aspect or a pharmaceutically acceptable salt thereof, the gene described in the second aspect, the vector described in the third aspect, the host cell described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of a drug for preventing and / or treating diseases related to the signal pathway binding to HGF-HGFR.

[0017] The polypeptide provided by the present invention is obtained through screening, and it can effectively block the binding of HGF-HGFR, prevent HGFR from being overactivated and causing excessive cell proliferation to form tumors, cause tumor cell apoptosis, and inhibit tumor cell proliferation and tumor cell metastasis, thereby preventing and / or treating diseases related to its signaling pathway.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a graph showing the ELISA results of the polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 and HGF-HGFR provided by the present invention;

[0020] Figure 2 It is a graph showing the ELISA results of the polypeptides containing the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 14 provided by the present invention and HGF-HGFR, wherein the polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 is a linear peptide;

[0021] Figure 3 It is a graph showing the ELISA results of the polypeptides containing the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 13 provided by the present invention and HGF-HGFR, wherein the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 is a linear peptide;

[0022] Figure 4 It is a graph showing the ELISA results of the polypeptides containing the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO: 53 provided by the present invention and HGF-HGFR, wherein the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 is a cyclic peptide;

[0023] Figure 5It is a graph showing the ELISA results of the polypeptides containing the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 49, and SEQ ID NO: 51 provided by the present invention and HGF-HGFR, wherein the polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 is a cyclic peptide;

[0024] Figure 6 It is a graph of ELISA results of the polypeptides containing the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 22 provided by the present invention and HGF-HGFR, wherein the ring represents a cyclic peptide and the line represents a linear peptide;

[0025] Figure 7 It is a graph showing the ELISA results of the polypeptides provided by the present invention containing the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17 to SEQ ID NO: 21, and SEQ ID NO: 54 and HGF-HGFR;

[0026] Figure 8 It is a graph showing the ELISA results of the polypeptide having the amino acid sequence shown in SEQ ID NO: 23 and HGF-HGFR provided by the present invention;

[0027] Fig. 9 It is a diagram showing the inhibitory effect of the polypeptides containing the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 10 provided by the present invention on the proliferation of tumor cells, wherein the polypeptides containing the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 are all linear peptides;

[0028] Fig.10 It is a graph showing the results of a BLI affinity test between a polypeptide (linear peptide) having an amino acid sequence as shown in SEQ ID NO: 4 provided by the present invention and HGFR-Fc;

[0029] Fig.11 It is a graph showing the results of a BLI affinity test between a polypeptide (linear peptide) having an amino acid sequence as shown in SEQ ID NO: 4 provided by the present invention and EGFR-Fc;

[0030] Fig.12 It is a graph showing the ELISA test results of a polypeptide (linear peptide) containing an amino acid sequence as shown in SEQ ID NO: 4 provided by the present invention and HGFR-Fc;

[0031] Fig.13 It is a graph showing the ELISA test results of the polypeptide (linear peptide) containing the amino acid sequence shown in SEQ ID NO: 4 provided by the present invention and EGFR-Fc. DETAILED DESCRIPTION

[0032] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] It should be noted that, unless otherwise defined herein, the scientific and technical terms used in the present invention should have the meanings commonly understood by those skilled in the art.

[0034] In the present invention, the "sequence identity" refers to the degree to which two sequences (e.g., amino acids) have the same residues at the same position after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity with SEQ ID NO: Y, and is described as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary computer programs for comparing and aligning sequence pairs may be ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN or GCG (Devereux et al., 1984). In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such "conservative" amino acids may be amino acids known in the art.

[0035] As mentioned above, the first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof for blocking HGF-HGFR binding, wherein the amino acid sequence of the polypeptide comprises any one of the amino acid sequences:

[0036] (1a) the first amino acid sequence as shown in SEQ ID NO: 1;

[0037] (1b) an amino acid sequence having at least 90% sequence identity with the first amino acid sequence;

[0038] (1c) a tag protein or a modified amino acid sequence is linked to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in (1a) and (1b);

[0039] (1d) An amino acid sequence having the same function obtained by processing the amino acid sequences shown in (1a), (1b) and (1c); the processing is at least one of replacing at least one amino acid, deleting at least one amino acid and adding at least one amino acid.

[0040] Preferably, the amino acid sequence of the polypeptide is an amino acid sequence having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity with the first amino acid sequence.

[0041] The first amino acid sequence shown in SEQ ID NO: 1 in the present invention is a polypeptide having the function of blocking HGF-HGFR binding detected from a polypeptide library by high-throughput screening technology. The steps of discovering the polypeptide include: dissolving and diluting the polypeptide library to obtain a mixed solution; then sequentially mixing the mixed solution with a reactant I containing HGFR, a reactant II containing HGF, a reactant III containing an HGFR marker, and a reactant IV capable of undergoing a colorimetric reaction with the marker to obtain a detection solution; finally, using an ELISA instrument to detect the absorbance of the detection solution at a specific wavelength, and obtaining the target polypeptide based on the inhibition rate calculated based on the absorbance.

[0042] It should be noted that the present invention has no particular limitation on the methods of dissolution and dilution, and those skilled in the art may make their selections based on technical means known in the art. A preferred specific implementation is exemplarily provided in the following text of the present invention, which should not be construed as a limitation on the present invention by those skilled in the art.

[0043] In the present invention, the polypeptide library is a polypeptide library containing nearly 73,000 polypeptides of 80 amino acids constructed by Hunan Zhongsheng Quanpeptide Biochemical Co., Ltd. using PICT (Peptide Information Compression Technology) technology. The specific construction method thereof can be found in CN107849737A and CN111727194A.

[0044] It should be noted that the present invention has no particular limitation on the method for synthesizing the polypeptide, and those skilled in the art can select the method according to the technical means known in the art. For example, a preferred method for synthesizing polypeptides is exemplified in the following text of the present invention, which should not be construed as a limitation on the present invention by those skilled in the art.

[0045] It should be noted that the present invention has no particular limitation on the type of the tag protein, and those skilled in the art can select it according to the technical means known in the art. The tag protein will not affect the activity of the polypeptide provided by the present invention. In practical application, those skilled in the art can choose whether to connect the tag protein to the amino terminus and / or carboxyl terminus of the amino acid sequence according to the needs.

[0046] Preferably, the amino acid sequence of the polypeptide further comprises any one of the following amino acid sequences:

[0047] (2a) a second amino acid sequence consisting of 20 to 40 consecutive amino acids in the first amino acid sequence;

[0048] (2b) an amino acid sequence having at least 90% sequence identity with the second amino acid sequence;

[0049] (2c) connecting a tag protein or a modified amino acid sequence to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in (2a) and (2b);

[0050] (2d) An amino acid sequence having the same function obtained by processing the amino acid sequences shown in (2a), (2b) and (2c); the processing is at least one of replacing at least one amino acid, deleting at least one amino acid and adding at least one amino acid.

[0051] More preferably, the amino acid sequence of the polypeptide is an amino acid sequence having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity with the second amino acid sequence.

[0052] Further preferably, the second amino acid sequence is shown in any one of SEQ ID NO: 2-SEQ ID NO: 23, SEQ ID NO: 48-SEQ ID NO: 54.

[0053] Particularly preferably, the second amino acid sequence is shown in any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 23. The inventors of the present invention have found that the polypeptide in this preferred embodiment has a higher affinity with HGF and / or HGFR.

[0054] According to a particularly preferred embodiment, the polypeptide comprising the first amino acid sequence or the amino acid sequence shown in any one of SEQ ID NO: 48 to SEQ ID NO: 54 is a cyclic peptide. The inventors of the present invention have found that the polypeptide in this preferred embodiment has a higher affinity with HGF and / or HGFR.

[0055] According to another particularly preferred embodiment, the polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 6 to SEQ ID NO: 23 is a linear peptide. The inventors of the present invention have found that the polypeptide in this preferred embodiment has a higher affinity with HGF and / or HGFR.

[0056] Preferably, the amino acid sequence in (2d) is shown in any one of SEQ ID NO: 24 to SEQ ID NO: 47.

[0057] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

[0058] In the present invention, the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 54 are shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062] Preferably, the modification is selected from at least one of amino modification, hydroxylation modification, carboxylation modification, carbonylation modification, amidation modification, alkylation modification, phosphorylation modification, glycosylation modification, cyclization modification, biotinylation modification, acetylation modification, esterification modification, fluorescent group modification, polyethylene glycol modification and immobilization modification.

[0063] As mentioned above, the second aspect of the present invention provides a gene, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that blocks HGF-HGFR binding as described in the first aspect.

[0064] As mentioned above, the third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0065] Preferably, the vector is selected from a plasmid vector, a cosmid vector, and a viral vector.

[0066] More preferably, the vector is selected from a phage vector, an adenovirus vector, and a retrovirus vector.

[0067] As mentioned above, the fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect.

[0068] As mentioned above, the fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide for blocking HGF-HGFR binding as described in the first aspect or a pharmaceutically acceptable salt thereof.

[0069] It should be noted that the "therapeutically effective amount" in the present invention refers to an amount sufficient to prevent and treat related diseases.

[0070] Preferably, the composition further comprises an excipient.

[0071] It should be noted that the present invention has no particular limitation on the type of the excipient, and those skilled in the art can select the excipient according to technical means known in the art, as long as the polypeptide provided in the present invention can target HGF and / or HGFR.

[0072] As described above, the sixth aspect of the present invention provides the use of at least one of the polypeptide or pharmaceutically acceptable salt thereof that blocks HGF-HGFR binding as described in the first aspect, the gene as described in the second aspect, the vector as described in the third aspect, the host cell as described in the fourth aspect, and the pharmaceutical composition as described in the fifth aspect for the preparation of a drug for preventing and / or treating diseases related to the signaling pathway binding to HGF-HGFR.

[0073] Preferably, the disease associated with the signaling pathway bound to HGF-HGFR is selected from at least one of breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, prostate cancer, lung cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, kidney cancer, gastric cancer, liver cancer, bladder cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, thyroid cancer, pancreatic cancer, thymic cancer, Merkel cell carcinoma, lymphoma, leukemia, melanoma, glioma, glioblastoma, gastrointestinal stromal tumor, myeloma, mesothelioma, sarcoma, and hematological malignancies.

[0074] The present invention will be described in detail below by way of examples.

[0075] In the following examples, unless otherwise specified, all raw materials used were commercially available.

[0076] In the following examples, unless otherwise specified, the room temperature refers to 25±5°C.

[0077] Experimental reagents:

[0078] Reagent I: Biotinylated Human HGFR Protein, Avitag, His Tag, manufacturer: Acro, catalog number: MET-H82E1;

[0079] Reagent II: Human HGF Protein, Fc Tag, manufacturer: Acro, catalog number: HGF-H5253;

[0080] Reagent III: Perodoxidase-conjugated AffiniPure Goat anti-Human IgG, FcγFragment, manufacturer: Jackson ImmunoResearch Inc., catalog number 109-035-008;

[0081] Reagent IV: Human EGFR Protein, Fc Tag, manufacturer: Acro, catalog number: EGR-H5252;

[0082] Reagent V: Human HGFR Protein, Fc Tag, manufacturer: Acro, catalog number: MET-H5256.

[0083] Example 1: ELISA screening of polypeptides

[0084] Dissolution of peptide library: Peptides in the peptide library were placed in a 96-deep well plate, centrifuged at 4000 rpm for 3 min, and then 200 μL / well of ultrapure water (peptide concentration was 50 μM) was added; then the plate was sealed with a silicone cap, placed in a 95°C water bath for 5 min, and then centrifuged at 4000 rpm for 3 min;

[0085] Dilution of peptide library: The peptides after centrifugation are transferred to a 384-well plate using a workstation, and the peptide concentration is diluted to the experimental concentration using loading buffer (Tris-HCl buffer, pH=7.4) to obtain peptide samples of different concentrations;

[0086] Screening of polypeptides: Streptavidin (SA) protein (concentration of 2 μg / mL) was coated in a 384-well plate at 25 μL / well to block excess sites, and reagent I (concentration of 0.125 μg / mL) was transferred to the aforementioned 384-well plate at 12.5 μL / well and incubated at 37°C for 1 h; then, different concentrations of polypeptide samples and reagent II (concentration of 0.01 μg / mL) were added to the aforementioned 384-well plate in sequence at 12.5 μL / well and incubated at 37°C for 1 h; then, reagent III (concentration of 0.1 μg / mL) was added to the aforementioned 384-well plate at 25 μL / well and incubated at 37°C for 2 h. After discarding the liquid in the wells, add 80 μL of 1×TBST washing solution with pH = 7.4 to wash the plate 4 times, 4 minutes each time; control the 384-well plate, add 25 μL of color development solution (manufacturer is Solarbio, item number is PR1210) to each well, and continue to incubate in the incubator at 37°C for 30 minutes; finally, add 25 μL of stop solution (1M HCl) to each well to terminate the reaction. Use an enzyme reader (model cytation5) to read the absorbance of each well at 450nm. After calculating the inhibition rate of each polypeptide concentration according to the formula, GraphPad prism software is used to draw a graph to calculate the IC50 value of each polypeptide, and screen out a target polypeptide, that is, a polypeptide (cyclic peptide) with an amino acid sequence as shown in SEQ ID NO: 1; wherein, the calculation formula for the inhibition rate is:

[0087] Inhibition% = (1-(absorbance of sample - absorbance of background) / (absorbance of positive - absorbance of background)) × 100%; where,

[0088] Sample: refers to adding peptides into the reaction system;

[0089] Positive: refers to the absence of peptide in the reaction system;

[0090] Background: refers to the reaction system without adding peptide and reagent I.

[0091] Example 2: Synthesis of polypeptides

[0092] A polypeptide containing an amino acid sequence of 5-80 amino acids was designed based on the target polypeptide obtained in Example 1 and synthesized according to the following method:

[0093] The polypeptide compound and its derivatives provided by the present invention adopt a solid phase synthesis method to synthesize their linear precursors, and obtain a crude target polypeptide after cutting. The solid phase carrier is 2-Chlotrityl Resin resin. In the synthesis process, the 2-Chlotrityl Resin resin is first fully swollen in N, N-dimethylformamide (DMF), and then the swollen solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (v:v:v:v=90:2.5:2.5:5) is reacted with the solid phase resin to cleave the polypeptide from the solid phase carrier, and then the target polypeptide crude product is obtained after precipitation by frozen methyl tert-butyl ether. The target polypeptide crude product is purified and separated by a C18 reverse phase preparative chromatography column in a 0.1% trifluoroacetic acid acetonitrile / water system to obtain a pure product of the polypeptide and its derivatives.

[0094] The following provides a specific method for synthesizing a polypeptide having an amino acid sequence as shown in SEQ ID NO: 23, wherein Table 2 lists the reagents used in the synthesis.

[0095] Table 2

[0096]

[0097]

[0098] The structure of the polypeptide compound having the amino acid sequence shown in SEQ ID NO: 23 is as follows:

[0099]

[0100] Step 1: Coupling of the first amino acid Fmoc-Pro-OH

[0101] Fully swell 0.1mmol of 2-Chlorotrityl chloride resin in DCM for 1h. Weigh 0.08mmol of Fmoc-Pro-OH and 0.32mmol of DIEA and dissolve them in 5mL of DCM, add the swollen resin and react at room temperature for 2h. After the reaction is completed, add 10mL of blocking solution (DCM: methanol: DIEA (v: v: v = 85: 10: 5) and block at room temperature for 10min. Wash the blocked resin with DCM 5 times and then with DMF 5 times before use.

[0102] Step 2: Synthesis of linear precursor peptide chain

[0103] The linear precursor peptide chain of SEQ ID NO: 23: DELNEIWSNAFTNVIEFVNWK-RSKP.

[0104] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

[0105] (1) Fmoc-deprotect the swollen resin twice with 10 mL of a mixture of piperidine:DMF (v:v=20:80), each time for 8 min;

[0106] (2) Wash the resin with DMF 6-8 times until the pH of the liquid is neutral;

[0107] (3) Dissolve 0.5 mmol of Fmoc-AA, 0.5 mmol of HCTU and 1 mmol of NMM in DMF, then add the washed resin and react at room temperature for 1 h;

[0108] (4) Wash the resin 4-6 times with DMF before coupling the next amino acid;

[0109] After the synthesis of the linear precursor peptide chain, the resin was washed 5 times with DMF and then 5 times with DCM, and the washed resin was dried in a vacuum for later use.

[0110] Step 3: Cleavage of the linear precursor peptide chain

[0111] Add 10 mL of a freshly prepared mixture of trifluoroacetic acid: water: triisopropylsilane: thioanisole (v:v:v:v = 90:2.5:2.5:5) to the resin obtained in step 2 and shake for 2 h at room temperature. After the reaction is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of frozen MTBE to obtain a crude polypeptide. Wash the crude polypeptide with MTBE 3 times and then dry it in a vacuum for later use.

[0112] Step 4: Purification of crude peptide

[0113] The crude polypeptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffer was phase A (0.1% trifluoroacetic acid aqueous solution) and phase B (0.1% trifluoroacetic acid acetonitrile solution). The chromatographic column was a BR-C18 (Saifen Technology) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230 nm, the flow rate was 15 mL / min, and the gradient was that phase B changed from 20% to 50% within 0-40 min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure polypeptide.

[0114] Other polypeptides of the present invention can be synthesized by referring to the synthesis method of SEQ ID NO: 23.

[0115] Example 3: Further ELISA screening of polypeptides

[0116] The polypeptides synthesized in Example 2 were further screened according to the polypeptide screening method in Example 1, and 33 polypeptides that inhibit the binding of HGF and HGFR were screened, namely, polypeptides with amino acid sequences as shown in SEQ ID NO: 2-SEQ ID NO: 23, SEQ ID NO: 48-SEQ ID NO: 54.

[0117] Table 3: IC of peptides inhibiting the binding of HGF and HGFR 50 value

[0118]

[0119] Example 4: Further design of polypeptide

[0120] The target polypeptides obtained by further screening in Example 3 were designed. Specifically, the amino acid sequence shown in SEQ ID NO: 23 was used as the reference sequence, and an alanine was used for substitution to obtain target polypeptides with amino acid sequences as shown in SEQ ID NO: 24-SEQ ID NO: 47. The inhibition rate of the binding between HGF and HGFR was tested according to the method in Example 1. The results are shown in Table 4.

[0121] Table 4: Inhibition rate of peptides on the binding of HGF and HGFR

[0122]

[0123]

[0124] Example 5: Testing of the inhibitory effect of polypeptides on tumor cell proliferation

[0125] BxPC-3 cells were seeded in 96-well plates at 2500 cells / well and cultured overnight at 37°C and 5% CO2. After the cells adhered to the wall, the original culture medium in the well was discarded, and serum-free 1640 basal medium was added at 80 μL / well for starvation treatment for 24 hours. Then, a premix was added at 20 μL / well (HGF solution (1000 ng / mL, 10X solution) and peptide sample solution (0.137-100 μM, 10X solution) were prepared with 1640 basal medium, and the HGF solution and the peptide sample solution were mixed at a volume ratio of 1:1 to obtain a premix), and culture was continued for 48 hours. Then, CellTiter-Blue was added at 20 μL / well, incubated at 37°C for 2 hours, and 560 EX / 590 EM The specific results are shown in Table 5 and Fig. 9 .

[0126] Table 5: IC of peptides for inhibiting tumor cell proliferation 50 value

[0127] Linear peptides SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 7 SEQ ID NO: 10 <![CDATA[IC 50 (μM)]]> 0.26 1.07 0.89 1.56

[0128] Example 6: Using BLI to test the affinity of polypeptides to HGFR or EGFR

[0129] The target polypeptide labeled with biotin was diluted to 2 μg / mL with K buffer (0.02wt% Tween + PBS, pH=7.4), and reagent IV (EGFR-Fc) or reagent V (HGFR-Fc) was diluted to the concentrations of 1000nM, 333nM, 111nM, 37nM, 12.4nM, and 4.12nM, respectively, with K buffer.

[0130] The above reagents IV, V and peptides were placed in a 96-well plate, shaken for 120 seconds at 25°C; the peptide was fixed on the His probe of the biosensor, and then the sensor was immersed in K Buffer and shaken for 120 seconds for equilibrium, and then the sensor was immersed in the diluted reagent IV solution or reagent V solution with different concentrations and shaken for 120 seconds for binding reaction, and finally the sensor was immersed in K Buffer and shaken for 120 seconds for dissociation reaction, and K was calculated. on Value and K off The affinity KD value was calculated by 1:1 fitting method. The specific results are shown in Table 6 and Fig.10 and Fig.11 .

[0131] Table 6: KD values ​​of peptides with HGFR or EGFR

[0132]

[0133] Example 7: Testing the affinity of polypeptides to HGFR or EGFR using ELISA

[0134] Streptavidin (SA) protein (concentration of 2 μg / mL) was coated in a 384-well plate at 25 μL / well to block excess sites, and different concentrations of a polypeptide sample containing an amino acid sequence as shown in SEQ ID NO: 4 labeled with biotin were added to the aforementioned 384-well plate at 25 μL / well and incubated at 37°C for 1 hour; then different concentrations of reagent IV or reagent V were added to the aforementioned 384-well plate at 25 μL / well and incubated at 37°C for 2 hours; after discarding the liquid in the wells, the plate was washed and reagent III (concentration of 0.1 μg / mL) was added to the aforementioned 384-well plate at 25 μL / well and incubated at 37°C for 1 hour. After discarding the liquid in the wells, add 80 μL of 1×TBST washing solution with pH=7.4 and wash the plate 4 times, 4 minutes each time; drain the 384-well plate, add 25 μL of color development solution (manufacturer: Solarbio, item number: PR1210) to each well, and continue to incubate in the incubator at 37°C for 30 minutes; finally, add 25 μL of stop solution (1M HCl) to each well to terminate the reaction. Use an enzyme reader (model: cytation5) to read the absorbance of each well at 450nm. For specific results, see Fig.12 and Fig.13 .

[0135] Figures 1 to 8 The ELISA results of the polypeptides containing the amino acid sequences shown in SEQ ID NO: 1-SEQ ID NO: 23, SEQ ID NO: 48-SEQ ID NO: 54 and HGF-HGFR provided by the present invention are shown in FIG. Antagonist in the figure represents the concentration of the polypeptide, and Inhibition represents the inhibition rate. The IC value of each polypeptide can be calculated from the figure. 50 The specific results are shown in Table 3.

[0136] It can be seen from the results in Table 3 and Table 4 that the polypeptide provided by the present invention can effectively block the binding of HGF-HGFR.

[0137] Fig. 9 It is a graph showing the inhibitory effect of some polypeptides provided by the present invention on tumor cell proliferation. As can be seen from the graph and Table 5, the polypeptides provided by the present invention have a good inhibitory effect on HGF-promoted tumor cell proliferation.

[0138] Fig.10 and Fig.11The following are the BLI affinity test results of the polypeptide (linear peptide) containing the amino acid sequence shown in SEQ ID NO: 4 provided by the present invention and HGFR-Fc and EGFR-Fc. It can be seen from these two figures and Table 6 that the polypeptide provided by the present invention can bind to HGFR-Fc with a high affinity, but cannot bind to EGFR-Fc.

[0139] Fig.12 and Fig.13 The ELISA test results of the polypeptide (linear peptide) containing the amino acid sequence shown in SEQ ID NO: 4 provided by the present invention and HGFR-Fc and EGFR-Fc are shown in Figure 4. It can be seen from the figure that the polypeptide provided by the present invention has a high affinity with HGFR-Fc, but has no obvious affinity with EGFR-Fc.

[0140] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A polypeptide for blocking HGF-HGFR binding or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO: 4-SEQ ID NO: 7, SEQ ID NO: 10-SEQ ID NO: 11; and the polypeptide is a linear peptide.

2. A gene, characterized in that The nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide for blocking HGF-HGFR binding as claimed in claim 1.

3. A carrier, characterized in that The vector contains the gene according to claim 2.

4. A host cell, characterized in that The host cell contains the vector according to claim 3.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the polypeptide for blocking HGF-HGFR binding according to claim 1 or a pharmaceutically acceptable salt thereof.

6. Use of at least one of the polypeptide for blocking HGF-HGFR binding according to claim 1 or a pharmaceutically acceptable salt thereof, the gene according to claim 2, the vector according to claim 3, the host cell according to claim 4, and the pharmaceutical composition according to claim 5 in the preparation of a drug for preventing and / or treating a disease related to a signaling pathway binding to HGF-HGFR; wherein, The disease associated with the signaling pathway binding to HGF-HGFR is pancreatic cancer.

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