USP7 targeting polypeptide and polypeptide-proteolysis targeting chimera

Abstract

The invention relates to the technical field of biomedicine and targeted drug therapy, in particular to a USP7-targeted polypeptide and a polypeptide-proteolysis targeted chimera. The polypeptide targeting the USP7, provided by the invention, is a bioactive peptide obtained by adopting a phage display technology through multiple rounds of screening and high-throughput sequencing, and the polypeptide has relatively good cell membrane penetrability and can target the USP7. The polypeptide obtained through screening is used as a target protein ligand of the USP7, the USP7 target protein ligand is coupled with a specific E3 ligase ligand through a linker, and the polypeptide-proteolysis targeting chimera targeting the USP7 is constructed. The polypeptide-proteolysis targeting chimera not only can efficiently degrade USP7, but also can down-regulate PD-L1 on the surfaces of tumor cells, so that the tumor treatment effect is improved. Therefore, the invention can provide effective technical support for research and development of tumor targeted drugs and treatment of related cancers.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and targeted drug therapy, and in particular to a polypeptide targeting USP7 and a polypeptide-protein hydrolysis targeting chimera. Background Art

[0002] USP7 (Ubiquitin-specific protease 7) is a member of the ubiquitin-specific protease family. Originally named for its interaction with the herpes simplex virus type 1 protein ICP0, it is also known as the herpes virus-associated ubiquitin-specific protease. It is one of the most disease-relevant deubiquitinating enzymes. USP7 is 1102 amino acids long and consists of three main components: an N-terminal tumor necrosis factor receptor-associated factor domain, a central catalytic domain, and five C-terminal ubiquitin-like domains. The tumor necrosis factor receptor-associated factor domain and the ubiquitin-like domain are primarily responsible for substrate recognition, while the catalytic domain is primarily responsible for binding ubiquitin and deubiquitination. Numerous studies have demonstrated that USP7 plays a crucial role in the development and progression of various tumors through its interactions with key proteins. In gastric cancer, overexpression of USP7 is a significant risk factor and significantly correlates with patient survival. However, to date, no USP7-targeting drugs have entered clinical trials, most of which have poor selectivity, and only a few have been evaluated in vivo. Therefore, the development of new, highly effective and specific USP7-targeted drugs has important potential value.

[0003] Phage display technology involves inserting DNA encoding a foreign protein or peptide into the gene sequence of a bacteriophage coat protein. This allows the foreign gene to be expressed along with the coat protein, displaying it on the phage surface with a specific spatial structure and biological activity. This technology allows for rapid screening and identification of various target ligands, offering advantages such as direct integration of genotype and phenotype, rapidity, efficiency, and high throughput. It has been widely applied to isolate various targets and natural ligands, study protein-protein interactions, validate targets, and elucidate their structure and function. However, there have been no reports of USP7-targeting peptides screened using phage display technology.

[0004] Proteolysis targeting chimeras (PROTAC) are a technology that uses the naturally occurring cleaning system in cells to efficiently remove specific proteins. Peptide PROTAC forms a ternary complex in vivo by connecting the target protein and the E3 ligase, promoting the transfer of ubiquitin to the target protein, causing the target protein to be degraded through the ubiquitin-proteasome pathway. Peptide PROTAC can bind to proteins with large interaction interfaces and shallow binding pockets, greatly expanding the range of druggable proteins, and only requires a catalytic amount to achieve high activity. At the same time, it can also reduce the occurrence of target protein resistance, with the advantages of high specificity and low toxicity. However, there is currently little research on peptide-PROTAC targeting USP7 in the existing technology.

[0005] Therefore, how to obtain a USP7-targeting peptide with good activity and use it for the construction of peptide-PROTAC is of great significance for the development of new cancer therapeutics and the treatment of related diseases. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the first object of the present invention is to provide a polypeptide targeting USP7, which is obtained by phage display technology and not only has good cell membrane permeability but also has excellent targeting to USP7 in cells.

[0007] The second object of the present invention is to provide the use of the above-mentioned polypeptide targeting USP7.

[0008] A third objective of the present invention is to provide a USP7-targeting polypeptide-protein hydrolysis targeting chimera, namely a polypeptide-PTOTAC, which consists of three parts: a cell membrane-penetrating USP7-targeting polypeptide screened through phage display technology as a recognition peptide sequence, a linker sequence, and an E3 ligase ligand sequence. This polypeptide-PTOTAC not only efficiently degrades highly expressed USP7 in tumor cells but also downregulates the immune checkpoint molecule PD-L1 on the tumor cell surface, reducing the binding of PD-L1 to PD-1, enhancing the sensitivity of immune cells to tumor cell killing, and promoting tumor immune responses. It also has the advantages of high efficiency, low toxicity, and high specificity.

[0009] The fourth object of the present invention is to provide applications of the aforementioned polypeptide-proteolysis targeting chimera targeting USP7.

[0010] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0011] A polypeptide targeting USP7, the amino acid sequence of which is shown in SEQ ID No. 1.

[0012] Use of the above-mentioned polypeptide targeting USP7 in the preparation of anti-tumor drugs.

[0013] As a preferred embodiment of the present invention, the anti-tumor drug is an anti-gastric cancer drug.

[0014] A polypeptide-protein hydrolysis targeting chimera targeting USP7, the amino acid sequence of which is shown in SEQ ID No.2.

[0015] Application of the above-mentioned polypeptide-protein hydrolysis targeting chimera targeting USP7 in the preparation of anti-tumor drugs.

[0016] The polypeptide-protein hydrolysis-targeting chimera targeting USP7 provided by the present invention can be directly used to prepare anti-tumor drugs. In particular, all tumors associated with USP7 targeting are suitable for the anti-tumor drugs of the present invention. Therefore, the present invention does not specifically limit the types of tumors involved in the anti-tumor drugs. As a preferred embodiment of the present invention, the anti-tumor drug is an anti-gastric cancer drug. Furthermore, the anti-tumor drug of the present invention can be used alone or in the form of a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

[0017] The beneficial effects of the above technical solution of the present invention are:

[0018] The USP7-targeting polypeptide provided by the present invention is a novel bioactive peptide obtained through multiple rounds of screening using phage display technology and high-throughput sequencing. This polypeptide has good cell membrane penetrance and can effectively target USP7. Compared with the conventional method of solid-phase panning by coating the target protein on a culture dish, the present invention uses phage display technology for peptide screening. This avoids the nonspecific binding of bovine serum albumin (BSA) and uncoated polystyrene culture dishes to phages, which is very likely to occur during the screening process. This greatly improves the accuracy of the screening method and enhances the specificity and affinity of the target polypeptide obtained by screening.

[0019] The present invention further utilizes the polypeptide targeting USP7 obtained by the above screening as the target protein ligand of USP7, and then uses the polypeptide ligand of the VHL protein (ALAPYIP) as the E3 ligase ligand in the USP7 polypeptide-protein hydrolysis targeting chimera. Finally, the USP7 target protein ligand and the E3 ligase ligand are coupled through a linker (GSGS), and a polypeptide-protein hydrolysis targeting chimera targeting USP7 is successfully constructed. In preliminary experiments, the present invention found that the use of the polypeptide ligand of the VHL protein to couple with the USP7 target protein ligand is more conducive to the degradation of USP7 than the scheme of coupling the polypeptide ligands of the Keap1 protein, KLHDC2 protein, and MDM2 protein with the USP7 target protein ligand. Therefore, after verification, the present invention uses the polypeptide ligand of the VHL protein to couple with the USP7 target protein ligand, thereby achieving efficient degradation of USP7. In addition, the polypeptide-protein hydrolysis targeting chimera is composed of amino acids of natural origin, has a novel structure, high activity, is easy and simple to synthesize, and is low in cost, and has great application prospects.

[0020] Experiments have shown that the polypeptide-protein hydrolysis targeting chimera constructed by the present invention can not only efficiently degrade USP7, but also downregulate PD-L1 on the surface of tumor cells, reduce the binding of PD-L1 to PD-1, enhance the sensitivity of immune cells to killing tumor cells, promote the immune response of tumors, and comprehensively improve the therapeutic effect of tumors. In addition, the polypeptide-protein hydrolysis targeting chimera constructed by the present invention also has the advantages of high efficiency, low toxicity, and high specificity. Therefore, the present invention can provide a new treatment strategy and effective technical support for the development of new tumor-targeted drugs and the treatment of related cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the expression and purification result of USP7 catalytic domain protein in Experimental Example 1 of the present invention;

[0022] Figure 2 The blue-white color result of the phage titer determination in Test Example 1 of the present invention;

[0023] Figure 3 This is an agarose gel electrophoresis diagram of the phage DNA fragment amplified by PCR in Experimental Example 1 of the present invention;

[0024] Figure 4 This is a Venn diagram of the high-throughput sequencing results of the USP7 catalytic domain, BSA, and empty polystyrene culture dishes in Experimental Example 1 of the present invention;

[0025] Figure 5 The results of the kinetic analysis of the binding between UP01 polypeptide and USP7 in Experimental Example 2 of the present invention are as follows;

[0026] Figure 6This is a graph showing the results of flow cytometry detection of the transmembrane ability of UP01 polypeptide in Experimental Example 3 of the present invention;

[0027] Figure 7 This is a graph showing the targeting results of UP01 polypeptide to USP7 in Experimental Example 4 of the present invention;

[0028] Figure 8 The degradation results of USP7 by peptide-PROTAC at different concentrations in Experimental Example 5 of the present invention are as follows;

[0029] Figure 9 The degradation results of USP7 by peptide-PROTAC at different action times in Experimental Example 5 of the present invention are as follows;

[0030] Figure 10 This is the result of peptide-PROTAC downregulating PD-L1 on the surface of gastric cancer cells in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with specific embodiments and accompanying drawings, but the present invention is not limited to the scope of the embodiments. In the following examples, unless otherwise specified, the methods used are conventional methods in the art. The reagents used in the following examples, unless otherwise specified, are conventional reagents in the art and can be obtained from commercial sources.

[0032] Example 1

[0033] This embodiment provides a polypeptide targeting USP7, the amino acid sequence of which is shown in SEQ ID No. 1. The amino acid sequence shown in SEQ ID No. 1 is specifically FIGSAHSYGQGK.

[0034] Example 2

[0035] This example provides the use of the USP7-targeting polypeptide of Example 1 in the preparation of an anti-tumor drug. The anti-tumor drug is an anti-gastric cancer drug.

[0036] Example 3

[0037] This example provides a polypeptide-proteolysis targeting chimera (referred to as polypeptide-PROTAC) targeting USP7, the amino acid sequence of which is shown in SEQ ID No. 2.

[0038] The amino acid sequence shown in SEQ ID No. 2 is specifically: FIGSAHSYGQGKGSGSALAPYIP.

[0039] The present invention uses the polypeptide provided in Example 1 as the target protein ligand of USP7 to design a polypeptide-PROTAC targeting USP7. At the same time, GSGS is selected as the intermediate linker and ALAPYIP is selected as the E3 ligase ligand. The synthesis operation is completed by Shanghai Chupeptide Biotechnology Co., Ltd., thereby successfully constructing the polypeptide-protein hydrolysis targeting chimera targeting USP7 in this example.

[0040] Example 4

[0041] This example provides the use of the USP7-targeting polypeptide-protein hydrolysis targeting chimera of Example 3 in the preparation of an anti-tumor drug. The anti-tumor drug is an anti-gastric cancer drug.

[0042] Experimental Example 1: Screening of USP7-targeting peptides

[0043] Step 1: Purification of USP7 catalytic domain protein

[0044] The catalytic domain of USP7 truncated protein (aa208-562) was used as the target protein (denoted as USP7 CD ), using pET-28b plasmid as expression vector, a recombinant expression vector (denoted as pET-28b-USP7) capable of expressing the target protein and carrying a His tag was constructed. CD Plasmid). The gene sequence number of USP7 is NM_003470.2, and the amino acid sequence of the catalytic domain of the USP7 truncated protein used is shown in SEQ ID No. 3.

[0045] The amino acid sequence shown in SEQ ID No.3 is specifically:

[0046] KKHTGYVGLKNQGATCYMNSLLQTLFFTNQLRKAVYMMPTEGDDSSKSVPLALQRVFYELQHSDKPVGTKKLTKSFGWETLDSFMQHDVQELCRVLLDNVENKMKGTCVEGTIPKLFRGKMVSYIQCKEVDYRSDRREDYYDIQLSIKGKKNIFESFVDYVAVEQLDGDNKYDAGEH GLQEAEKGVKFLTLPPVLHLQLMRFMYDPQTDQNIKINDRFEFPEQLPLDEFLQKTDPKDPANYILHAVLVHSGDNHGGHYVVYLNPKGDGKWCKFDDDVVSRCTKEEAIEHNYGGHDDDLSVRHCTNAYMLVYIRESKLSEVLQAVTDHDIPQQLVERLQEEKRIEAQKRKERQE.

[0047] pET-28b-USP7 CD The plasmid was transformed into BL21 (DE3) Escherichia coli and protein was purified using Ni-NTA affinity chromatography column to obtain high-purity USP7 catalytic domain protein. The expression and purification results of USP7 catalytic domain protein are shown in Figure 2. Figure 1 shown. Figure 1 In the figure, numbers 1-3 represent the purified USP7 catalytic domain protein bands. Figure 1 The results showed that the size of USP7 catalytic domain protein was approximately 40 kDa, which met the molecular weight requirement.

[0048] Step 2: Phage random twelve-peptide library selection

[0049] (1) Resuscitation of Escherichia coli ER2738

[0050] The steps are as follows: Take 5 μL of Ph.D. TM Add the E. coli ER2738 strain from the .-12 phage display peptide library kit to 0.5 mL of pre-sterilized liquid culture medium and shake thoroughly at 250 rpm at 37°C for 2 hours to activate the ER2738 strain. Pre-warm a tetracycline-resistant solid culture medium plate at 37°C in the dark. Use a sterile smear to evenly spread the activated ER2738 solution onto the pre-warmed tetracycline plate. Invert the plate and incubate at 37°C in the dark overnight.

[0051] (2) Target protein solidification and first round of panning of phage random twelve-peptide library

[0052] The steps are as follows: (a) Use sterile pH 8.6, 0.1M NaHCO3 solution to prepare 70 μg / mL USP7 catalytic domain protein solution purified in step 1 and 5 mg / mL BSA protein solution, add them to two different sterile polystyrene culture dishes, add 1.5 mL of protein solution to each culture dish, and add 1.5 mL of NaHCO3 solution to another sterile polystyrene culture dish as a control. Place in a humidified box and incubate on a shaker at 80 rpm at 4°C overnight.

[0053] (b) Select a well-isolated ER2738 monoclonal strain and place it in a 250 mL sterile conical flask containing 20 mL of liquid culture medium. Carefully monitor its growth and do not exceed the early logarithmic growth phase (OD600: 0.01-0.05) for the first round of eluted phage amplification.

[0054] (c) Remove the dish containing the solidified protein from step (a), pour off the protein solution, and tap the dish on a clean paper towel to remove any residual solution. Add 2 mL of TBST and quickly rinse the dish six times, tapping the dish on a clean paper towel each time to remove any residual solution. Be quick to prevent the dish from drying out.

[0055] (d) Take 10 μL of Ph.D TM The original .-12 phage-displayed peptide library was diluted 100-fold with TBST to a final volume of 1 mL. This was added to the culture dish washed in step (c) and incubated at room temperature on a horizontal shaker at 80 rpm for 60 minutes. After incubation, unbound phage was poured out and the plate was tapped down on a clean paper towel. 2 mL of TBST was added to the culture dish and washed 10 times, using a clean paper towel each time the dish was tapped down to avoid cross-contamination. Then, 1 mL of acidic elution buffer was added and gently shaken for 10 minutes to elute the bound phage. The eluate was transferred to a new EP tube and immediately added with 150 μL of neutralization buffer.

[0056] (e) The eluted phage solution from step (d) was added to 20 mL of the ER2738 E. coli culture obtained in step (b) and amplified by vigorous shaking at 250 rpm at 37°C for 4.5-5 hours. The amplified solution was transferred to a sterile centrifuge tube and centrifuged at 12,000 g for 10 minutes in a refrigerated centrifuge at 4°C. The supernatant was transferred to a new EP tube and the centrifugation was repeated once. 80% of the supernatant was then transferred to a new EP tube, and 1 / 6 volume of PEG / NaCl solution was added. The tube was allowed to stand at 4°C overnight to precipitate the phage. The phage precipitate was centrifuged at 12,000 g for 15 minutes at 4°C, the supernatant discarded, and the tube was centrifuged again for 1 minute. The remaining supernatant was aspirated and the phage pellet was resuspended in 1 mL of TBS. The resulting phage suspension was then centrifuged at 14,000 rpm for 5 minutes at 4°C. The supernatant was transferred to a new EP tube. A 1 / 6 volume of PEG / NaCl solution was added and incubated on ice for 60 minutes to reprecipitate the phage. The suspension was then centrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant was discarded and centrifuged again for 1 minute. The remaining supernatant was removed with a pipette. The phage pellet was resuspended in 200 μL of TBS and briefly centrifuged again for 1 minute. The supernatant was transferred to a new EP tube to obtain the phage solution after the first round of panning amplification.

[0057] (3) Phage titer determination

[0058] The steps for phage titer determination are as follows: (a) Select a well-separated ER2738 monoclonal strain from a tetracycline-resistant solid culture medium, inoculate it into 10 mL of liquid culture medium containing 1‰ tetracycline, and culture it at 37°C and 200 rpm until the OD600 value is 0.5 (mid-logarithmic growth phase). (b) Melt the top agar in a microwave oven, and after melting, divide it into eight sterile 5 mL EP tubes, with 3 mL in each tube, and let it stand at 45°C for use. At the same time, preheat eight IPTG / X-gal plates at 37°C for at least one hour until they are ready for use. (c) Dilute the eluted phage solution after the first round of selection in step (2) and the amplified phage solution with LB culture medium. The dilution range for the eluted phage solution is 10 1 -10 4 The dilution range of the amplified phage solution is 10 8 -10 11 . (d) First add 200 μL of the ER2738 culture obtained in step (a) to the mid-logarithmic phase into each dilution concentration EP tube. (e) Add 10 μL of phage solution of different dilution concentrations to the tubes obtained in step (d), quickly pipette and evenly mix, and incubate at room temperature for 5 minutes for infection. (f) Pour the ER2738 infected in step (e) onto a 45°C preheated IPTG / X-gal plate, quickly tilt and rotate the plate to evenly distribute the top agar on it. (g) After the top agar on the plate in step (f) solidifies, turn it upside down and incubate it at 37°C overnight.

[0059] Count the number of blue spots on the plate with about 100, and then calculate the phage titer. Phage titer = number of phage blue spots × dilution factor / 10 (pfu / μL). The result is as follows Figure 2 shown. Figure 2 In the middle, from left to right are phage solution at 10 8 -10 11 Schematic diagram of blue spots at various concentrations when measuring titers within the dilution range, thereby achieving phage titer determination.

[0060] (4) Target protein solidification and second round of panning of phage random twelve-peptide library

[0061] The steps are as follows: (a) After determining the titer of the first round amplification eluate, re-solidify the protein in the same manner as the first round and add the same number of phage as the first round. Repeat the panning procedure above, but increase the concentration of Tween-20 in TBST to 0.5%. (b) Determine the titer of the second round amplification eluate on an IPTG / X-gal plate. (3) Solidify the protein culture plate used for the third round of panning.

[0062] (5) Target protein solidification and third round of panning of phage random twelve-peptide library

[0063] The steps are as follows: (a) Add the same amount of second-round phage amplification eluate as the first-round eluate and again use TBST buffer containing 0.5% Tween-20 for the wash step. (b) Titer the third-round amplification eluate on IPTG / X-gal plates; amplification of the third-round eluate is not required.

[0064] The results of phage random twelve-peptide library panning using USP7 catalytic domain protein, BSA, and empty polystyrene culture dishes are shown in Table 1 .

[0065] Table 1 Results of three rounds of phage panning using USP7 catalytic domain protein, BSA and empty polystyrene culture dishes

[0066]

[0067]

[0068] As shown in Table 1, after three rounds of panning with the phage twelve-peptide library, the phages binding to USP7, BSA and empty polystyrene culture dishes were significantly enriched.

[0069] Step 3: Phage DNA extraction

[0070] The steps are as follows: (a) Take the phage eluate after the third round of panning, add 400 μL of PEG / NaCl solution, invert several times to mix thoroughly, and let it stand at room temperature for 20 minutes. (b) Centrifuge at 4°C and 14,000 rpm for 10 minutes, discard the supernatant, centrifuge again for 1 minute, and remove any remaining solution with a pipette. (c) Add 200 μL of sodium iodide buffer to the precipitate obtained in step (b) and tap the bottom of the EP tube vigorously to completely resuspend the phage precipitate. (d) Add 500 μL of anhydrous ethanol and incubate at room temperature for 15 minutes to precipitate the phage single-stranded DNA and dissolve the phage protein in the anhydrous ethanol. (e) Centrifuge at 4°C and 14,000 rpm for 10 minutes, discard the supernatant, wash the precipitate with 1 mL of 70% ethanol solution stored at -20°C, re-centrifuge, discard the supernatant, and briefly dry the precipitate. (f) Resuspend the precipitate in 50 μL of ultrapure water and store at -20°C.

[0071] Step 4. Preparation and sequencing of high-throughput sequencing samples

[0072] According to the insertion position of the phage twelve peptide, the PCR primers were designed. The DNA fragment containing the twelve peptide in the third round of eluted phage was extracted and the target fragment was extended by the set PCR program. The size of the target fragment after PCR amplification was as follows: Figure 3 shown. Figure 3 The results showed that the size of the target fragment amplified by PCR was 231 bp.

[0073] PCR amplification conditions and parameters are as follows: Forward primer F: AAGCTGTTTTAAGAAATTCACCT; Back primer P: CAACAAATCGTTTTAGGGTATG; PCR mixture (25 μL) per sample: 1 μL each of primers F and R; 12.5 μL of TaKaRa PrimeSTAR HS (Prime) Mix; 1 μL of total DNA sample; 9.5 μL of ddH2O. PCR program: Initial denaturation: 98°C, 2 min; Denaturation: 98°C, 10 s; Annealing: 55°C, 10 s; Extension: 72°C, 30 s; Final extension: 72°C, 3 min. The denaturation, annealing, and extension steps were automatically cycled 50 times. After completion of the program, the samples were removed and stored at 4°C.

[0074] The samples were sent to Beijing Qingke Biotechnology for high-throughput sequencing. The analysis results are shown in the Venn diagram. Figure 4 As shown. Figure 4 It can be seen that a total of 29,819 USP7-specific binding dodecapeptide sequences, 356,730 BSA-nonspecific binding dodecapeptide sequences, and 1,048,575 nonspecific binding dodecapeptide sequences that were not completely coated on the culture dish wall were obtained. After screening and removing these two types of nonspecific binding dodecapeptide sequences and amino acid sequences containing unknown or sequenced abnormalities, a total of eight USP7-specific binding dodecapeptides were identified, which were named UP01 to UP08, and their sequences are shown in Table 2.

[0075] Table 2 Amino acid sequences of USP7-specifically binding phage dodecapeptides

[0076]

[0077]

[0078] As shown in Table 2, UP01 is the most frequently occurring peptide among the USP7-binding twelve peptides, indicating that it has the strongest binding affinity. Therefore, the UP01 peptide was selected as the sequence to be verified, and only the UP01 peptide was further studied.

[0079] Experimental Example 2: Analysis of the Binding Kinetics of UP01 and USP7 Using Biofilm Interferometry

[0080] The steps are as follows: (1) Place the SA sensor in a pre-wetted plate for 5 minutes. Dilute the biotin-tagged UP01 peptide synthesized by Shanghai Chupeptide Biotechnology Co., Ltd. to 100 nM with 0.1 M NaHCO3, pH 8.6 solution. Add 200 μL of the diluted peptide to a black 96-well plate. Set up a blank SA probe and a negative control group without peptide buffer. Set the operating program (equilibration 60 seconds, curing 3600 seconds, equilibrium 60 seconds) on the BLI molecular interaction instrument to allow the biotin-tagged UP01 peptide to be cured on the SA sensor.

[0081] (2) 0.02% Tween-20 was added to a 0.1 M NaHCO3, pH 8.6 solution. The purified USP7 catalytic domain protein was diluted 10-fold from 50 μM to five concentration gradients using this solution. The diluted proteins were added to a new black 96-well plate in descending order of concentration. A negative control group without peptide buffer was set up. The BLI molecular interaction instrument was set to the operating program (equilibrium 60 s, association 180 s, dissociation 180 s), and each concentration was cycled once.

[0082] (3) Data processing: export the results to Graphpad to calculate the KD value and draw the binding and dissociation curve. The results are as follows Figure 5 shown.

[0083] Figure 5 middle, Figure 5 A is the binding and dissociation curve between USP7 and UP01, Figure 5 B is the steady-state analysis of the average response signal obtained at different concentrations. Figure 5 It can be seen that UP01 polypeptide has excellent binding affinity to USP7.

[0084] Experimental Example 3: Evaluation of the Cell Entry Ability of UP01 Peptide

[0085] The steps are as follows: (1) Four types of gastric cancer cells (AGS, BGC-803, MGC-823, MKN-45) were plated in a six-well plate. After the cells adhered to the wall, 10 μM UP01 peptide carrying a FITC fluorescent label synthesized by Shanghai Chupeptide Biotechnology Co., Ltd. was added and incubated for 12 hours. (2) After the incubation, the culture medium was discarded, and the cells were washed three times with PBS and centrifuged. PBS was added to wash the cell pellet once, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. (3) The cell pellet was resuspended in 300 μL PBS, filtered, and analyzed on a flow cytometer. The results are shown in Figure 2. Figure 6 shown.

[0086] Figure 6 The flow cytometry results showed that in AGS ( Figure 6 A), BGC-803( Figure 6 B)MGC-823( Figure 6 C) MKN-45( Figure 6 D) In ​​four different gastric cancer cell lines, UP01 polypeptide has a certain ability to penetrate the cell membrane.

[0087] Test Example 4: UP01 Targeted Detection

[0088] In this experiment, protein in vitro binding assay (Pull down), immunoprecipitation assay (IP), and cell thermal shift assay (CETSA) were performed to investigate the targeting of UP01.

[0089] The in vitro protein pull-down assay (Pull Down) procedure is as follows: 50 μL of streptavidin magnetic beads are washed three times with PBS, and the beads are then magnetically separated using a magnetic stand to remove the protective solution. 10 μM biotin-tagged UP01 peptide is then added to the streptavidin beads and incubated on a rotary shaker at room temperature for two hours to allow the UP01 peptide to bind to the streptavidin beads, allowing subsequent target protein capture. After incubation, the supernatant is discarded, and the beads are washed three times with PBS to remove unbound UP01 peptide. The purified USP7 catalytic domain protein solution described above is then added, and the beads are incubated on a rotary shaker at room temperature for two hours. The supernatant is discarded, and the beads are washed three times with PBS to remove unbound protein. SDS-PAGE protein loading buffer is then added for denaturation, and the results are analyzed by Western blot. The results are shown in 7A.

[0090] The immunoprecipitation (IP) assay procedure involved washing 50 μL of streptavidin magnetic beads three times with PBS, then performing magnetic separation on a magnetic rack to remove the protective buffer from the beads. MKN-45 gastric cancer cell lysate, treated with 10 μM biotin-tagged UP01 peptide, was then added to the streptavidin beads. The beads were incubated on a rotary shaker at room temperature for two hours. The beads were then washed and denatured with SDS-PAGE protein loading buffer. The results were analyzed by Western blot. The results are shown in 7B.

[0091] The CETSA assay was performed as follows: MKN-45 gastric cancer cells were plated in two 10 cm cell culture dishes. When cells reached mid-logarithmic phase, 10 μM UP01 peptide and an equal volume of DMSO were added, respectively, for 8 hours. The culture medium was discarded, and the cells were washed once with PBS. The cells were harvested using a cell scraper and transferred to a 1.5 mL EP tube. The suspension was centrifuged at 3000 rpm for 5 minutes. The cell pellet was resuspended in 300 μL of PBS and divided equally into six aliquots. Each aliquot was incubated in a water bath at 42, 44, 46, 48, 50, and 52°C for 3 minutes, followed by recovery on ice for 3 minutes. The cell suspension was repeatedly frozen and thawed three times in liquid nitrogen to disrupt the cells. The supernatant was then collected by centrifugation at 10,000 rpm for 15 minutes at 4°C. The suspension was denatured with SDS-PAGE protein loading buffer, and USP7 protein levels were analyzed by Western blot. The results are shown in Figure 7C.

[0092] In this experiment, in order to prove the targeting of UP01 peptide to USP7, UP01 peptide with biotin tag was bound to streptavidin magnetic beads, making it act as a "bait" to bind to USP7. Figure 7 As shown in A, UP01 peptide successfully binds to USP7 catalytic domain protein, proving that UP01 peptide has targeting effect on USP7 at the molecular level. Furthermore, the gastric cancer cell lysate treated with UP01 peptide was incubated with streptavidin magnetic beads, proving that UP01 peptide can also target USP7 in cells ( Figure 7 B). Further, Figure 7 The cell thermal migration test of C showed that when the temperature rose from 42°C to 52°C, the degradation trend of USP7 protein after treatment with UP01 peptide was slower than that in the DMSO group, indicating that UP01 peptide can enhance the thermal stability of USP7, once again proving the targeting of UP01 peptide to USP7 in cells.

[0093] Experimental Example 5: Peptide PROTAC induces degradation of USP7 in gastric cancer cells

[0094] The steps are as follows: (1) MKN-45 gastric cancer cells were cultured at a rate of 3×10 5 Cells were seeded into 6-well plates at 100 μg / well. After the cells adhered, different concentration gradients (0 μM, 1 μM, 10 μM, 100 μM) of the polypeptide-PROTAC provided in Example 3 were added. The cells were collected to extract total protein, denatured with protein loading buffer, and then loaded onto SDS-PAGE gel for electrophoresis.

[0095] (2) Transfer: Soak the nitrocellulose (NC) membrane in electrotransfer solution for activation. Cut the gel block according to the protein marker indication. Transfer the NC membrane and gel block into the transfer rack according to the "black gel and white membrane" pattern. Finally, place the membrane into the transfer tank filled with electrotransfer solution. Transfer the membrane at a constant current of 300 mA for two hours. Keep the membrane in an ice bath during the entire process.

[0096] (3) Ponceau staining: After the transfer is completed, place the transferred NC membrane in Ponceau staining solution to observe whether the protein lane is clear, which can be used to preliminarily judge the transfer effect;

[0097] (4) Milk blocking: Transfer the NC membrane to 5% skim milk prepared in advance and block at room temperature for one hour;

[0098] (5) Primary antibody blocking: After milk blocking, the strips were removed, washed in PBS, and transferred to a wet chamber. They were then blocked with USP7 primary antibody solution prepared in PBST at 4°C overnight.

[0099] (6) Secondary antibody blocking: After the primary antibody blocking is completed, the strips are washed with PBST solution on a shaker for 5 minutes × 4 times; then the strips are transferred to the horseradish peroxidase secondary antibody solution prepared in milk and blocked at room temperature on a shaker for one hour. After the secondary antibody blocking is completed, the strips are washed again with PBST solution for 5 minutes × 4 times;

[0100] (7) Exposure: Prepare the luminescent solution according to ECL A solution: ECL B solution = 1:1, and analyze the image on a fully automatic chemiluminescence image analyzer. The results are as follows: Figure 8 、 9 shown.

[0101] in, Figure 8 The degradation results of USP7 by peptide-PROTAC at different concentrations. Figure 9 The degradation results of USP7 by peptide-PROTAC with different action times. Figure 8 and Figure 9 It shows that the degradation of USP7 by peptide PROTAC is concentration-dependent and time-dependent.

[0102] Experimental Example 6: Peptide PROTAC Induces Downregulation of PD-L1 on the Surface of Gastric Cancer Cells

[0103] The steps are as follows: (1) Collect MKN-45 gastric cancer cells after incubation with 10 μmol / L peptide-PROTAC in Example 3 for 24 hours, wash once with PBS, centrifuge at 500g for 5 minutes, and discard the supernatant. (2) Add diluted PE-coupled PD-L1 antibody and incubate on ice in the dark for 40 minutes. After incubation, centrifuge and discard the supernatant, and wash three times with PBS. (3) Resuspend the cell pellet in PBS, filter through a filter, and analyze on a flow cytometer. The results are as follows Figure 10 shown.

[0104] Figure 10 The results showed that after PROTAC treatment, PD-L1 on the surface of gastric cancer cell membranes was significantly downregulated. This suggests that the peptide-PROTAC can further reduce the binding of tumor cells to PD-1 on the surface of T cells, enhance the sensitivity of T cells to tumor cells, reduce the immune escape of tumor cells, and improve the therapeutic effect on tumors.

[0105] In summary, the polypeptide targeting USP7 provided by the present invention is a bioactive peptide obtained through multiple rounds of screening using phage display technology and high-throughput sequencing. The polypeptide has good cell membrane permeability and can target USP7. The present invention utilizes the polypeptide obtained by the above screening as the target protein ligand of USP7, and couples the USP7 target protein ligand with the E3 ligase ligand (the polypeptide ligand of the VHL protein) through a linker to construct a polypeptide-protein hydrolysis targeting chimera targeting USP7. The polypeptide-protein hydrolysis targeting chimera can not only efficiently degrade USP7, but also downregulate PD-L1 on the surface of tumor cells, thereby improving the effect of tumor treatment. Therefore, the present invention can provide effective technical support for the research and development of tumor-targeted drugs and the treatment of related cancers.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A polypeptide targeting USP7, characterized in that The amino acid sequence of the polypeptide targeting USP7 is shown in SEQ ID No.

1.

2. Use of the polypeptide targeting USP7 as claimed in claim 1 in the preparation of anti-tumor drugs.

3. Use of the polypeptide targeting USP7 according to claim 2 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is an anti-gastric cancer drug.

4. A polypeptide-protein hydrolysis targeting chimera targeting USP7, characterized in that The amino acid sequence of the polypeptide-proteolysis targeting chimera targeting USP7 is shown in SEQ ID No.

2.

5. Use of the USP7-targeting polypeptide-protein hydrolysis targeting chimera according to claim 4 in the preparation of anti-tumor drugs.

6. Use of the polypeptide-protein hydrolysis targeting chimera targeting USP7 according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is an anti-gastric cancer drug.

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