Recombinant deubiquitinating enzyme USP7 mutant, preparation method and application thereof in tumor treatment

Abstract

The invention discloses a recombinant deubiquitination enzyme USP7 mutant, a preparation method and application of the mutant in tumor treatment, and belongs to the technical field of biomedicine.The USP7 mutant changes substrate selectivity through site-specific mutagenesis, specifically deubiquitination tumor suppression protein p53 is achieved, meanwhile, oncogenic protein MDM2 is unstable, a p53 anti-tumor pathway is activated, and the tumor suppression protein p53 is activated. The mutant induces cell cycle arrest and apoptosis in wild type p53 tumor cells, the ICvalue is 180-280nM, animal experiments show that the tumor growth inhibition rate reaches 72%, the tumor tissue enrichment is improved by 12 times by combining a folic acid targeted lipid nanoparticle delivery system, the drug resistance of an MDM2 inhibitor is overcome, the mutant has a synergistic effect with chemotherapeutic drugs, and the mutant can be used for preparing drugs for treating tumors. And a novel accurate treatment strategy is provided for wild-type p53 tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to recombinant deubiquitinase USP7 mutant, its preparation method, and its application in tumor treatment. Background Technology

[0002] p53, a tumor suppressor protein, is one of the most important tumor defense barriers in the human body, often referred to as the "guardian of the genome." p53 inhibits tumor development and progression by transcribedly activating multiple target genes and regulating key biological processes such as cell cycle arrest, DNA damage repair, cellular senescence, and apoptosis through transcriptional activation. Under normal physiological conditions, p53 protein levels are strictly regulated by ubiquitination. MDM2, as the main E3 ubiquitin ligase for p53, promotes its proteasome degradation through ubiquitination, maintaining low p53 expression levels. However, this negative feedback regulatory mechanism is often imbalanced in tumors, leading to the loss of p53 function.

[0003] Although approximately 50% of human tumors have TP53 gene mutations, 50% of adult tumors and up to 95% of childhood tumors retain the wild-type p53 gene. These wild-type p53-retaining tumors often evade p53-mediated tumor suppression by upregulating the expression of MDM2 or its homolog MDMX to inhibit p53 activity. Therefore, treating wild-type p53 tumors by restoring p53 function has been an important direction in tumor treatment research. Currently, small molecule inhibitors targeting the MDM2-p53 interaction, such as Nutlin-3 and Idasanutlin, have entered clinical trials. However, these drugs have problems such as drug resistance, narrow therapeutic window, significant gastrointestinal toxicity, and some acquired mutations can lead to drug resistance.

[0004] Ubiquitin-specific protease 7 (USP7), also known as herpesvirus-associated ubiquitin-specific protease (HAUSP), is one of the most thoroughly studied members of the deubiquitinating enzyme family. The USP7 protein consists of 1102 amino acids, including an N-terminal TRAF domain (amino acids 53-206), an intermediate catalytic domain (amino acids 208-560), and a C-terminal tandem ubiquitin-like domain (amino acids 560-1102). Studies have shown that USP7 can simultaneously stabilize p53 and MDM2 proteins through deubiquitination, with the TRAF domain responsible for recognizing the P / AxxS motif in the substrate protein. However, wild-type USP7 has a significantly higher affinity for MDM2 than p53, leading to USP7 primarily stabilizing MDM2 under physiological conditions, indirectly promoting p53 degradation, and ultimately exhibiting a pro-cancer effect. Therefore, while simple small molecule drugs that inhibit USP7 activity (such as P5091, FT671, XL177A, etc.) can degrade MDM2, they also cause p53 instability and lack substrate selectivity, which may affect other USP7 substrates such as PTEN, FOXO4 and other tumor suppressor proteins, producing complex side effects and limiting clinical application.

[0005] CN119643866A discloses the application of the deubiquitinase USP49 in the preparation of drugs that regulate the radiosensitivity of esophageal cancer cells. This patent, through research, discovered that USP49 mediates the deubiquitination of RPA70, inhibits RPA70 degradation, and promotes the recruitment of RPA70 and downstream Rad51 at DNA double-strand breaks, thereby enhancing homologous recombination repair and inducing radioresistance in esophageal cancer cells. The invention proposes that knocking down USP49 can improve the sensitivity of esophageal cancer cells to radiotherapy, and that USP49 can serve as a biomarker for the diagnosis, treatment, and prognosis of esophageal cancer. However, this technical solution has the following shortcomings: First, its application is limited to radiosensitization of esophageal cancer and does not involve direct anti-tumor therapy; second, USP49's promotion of tumor radioresistance is a passive regulation, with limited clinical translational value; third, it is limited to gene expression regulation research and has not been developed into a protein biopharmaceutical; fourth, it does not involve the regulation of the core anti-tumor signaling pathway p53 / MDM2; and fifth, it lacks broad-spectrum therapeutic potential for different tumor types.

[0006] Therefore, there is an urgent need to develop a novel, substrate-selective USP7 variant that can specifically deubiquitinate and stabilize p53, while destabilizing or promoting MDM2 degradation, thereby effectively activating the p53 anti-tumor pathway in wild-type p53 tumors. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a recombinant deubiquitinase USP7 mutant, its preparation method, and its application in tumor treatment.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a recombinant deubiquitinating enzyme USP7 mutant, obtained by introducing multiple site-directed mutations into the wild-type USP7 protein sequence. Specifically, the mutant introduces L203A and F205A mutations into the TRAF substrate recognition domain to reduce the binding affinity for MDM2; introduces N208D and D210K mutations into the TRAF-catalytic domain boundary region to alter the substrate-selective charge distribution; introduces G224S and Q297E mutations into the catalytic domain adjacency region to improve the catalytic efficiency for p53-ubiquitin; and introduces T308C and E556C mutations to form intramolecular disulfide bonds to enhance protein stability. This mutant retains the essential Cys223-His464-Asp481 catalytic triplet and the key substrate recognition residue Trp165, thereby achieving specific deubiquitination of p53 while destabilizing MDM2.

[0010] Furthermore, the USP7 mutant of the present invention can also be fused with cell-penetrating peptide sequences at the N-terminus or C-terminus, including but not limited to TAT peptide (YGRKKRRQRRR), cell-penetrating peptide Penetratin (RQIKIWFQNRRMKWKK), or amphiphilic peptide Pep-1 (KETWWETWWTEWSQPKKKRKV) to enhance the mutant protein's ability to penetrate the cell membrane and its intracellular delivery efficiency.

[0011] Furthermore, the USP7 mutant of the present invention can also fuse a nuclear localization signal sequence (NLS) at its C-terminus, preferably the SV40 large T antigen NLS sequence (PKKKRKV) or the nucleoplasmic protein nucleolin NLS sequence (KRPAATKKAGQAKKKK) to promote the directional transport of the mutant protein to the cell nucleus, thereby more effectively approaching the p53 and MDM2 substrates located in the nucleus.

[0012] The present invention also provides a nucleotide sequence encoding the above-mentioned recombinant deubiquitinase USP7 mutant. This nucleotide sequence has been codon-optimized to suit the preferences of mammalian expression systems, with GC content optimized to 50-60%, avoiding unstable sequence elements such as AU-rich regions, internal TATA boxes, poly(A) signals, splice sites, and repetitive sequences, thereby improving mRNA stability and translation efficiency.

[0013] The present invention also provides a recombinant expression vector containing the above-mentioned nucleotide sequence, the vector comprising a strong promoter (such as the CMV promoter, EF1α promoter, or CAG promoter), a target gene encoding a USP7 mutant, an enhancer sequence, a transcription terminator, and a polyadenylation signal, suitable for efficient expression in mammalian cells. The vector may also contain a selection marker gene (such as a neomycin resistance gene, a puromycin resistance gene, or a hygromycin resistance gene) to screen for stable expression cell lines.

[0014] This invention also provides a method for preparing the above-mentioned recombinant deubiquitinase USP7 mutant, comprising the following steps: transfecting a recombinant expression vector encoding the USP7 mutant into host cells, wherein the host cells may be selected as Chinese hamster ovary cells CHO-S, human embryonic kidney cells HEK293, human embryonic kidney suspension cells HEK293-F, or hamster kidney cells BHK; screening and amplifying stable expression cell lines in a culture medium containing selective antibiotics; performing large-scale cell culture in serum-free or low-serum culture medium and collecting the cell culture supernatant; purifying the USP7 mutant with His tag or Fc tag by protein A or protein G affinity chromatography; further purifying by anion exchange chromatography and gel filtration chromatography to remove impurities, endotoxins, and viruses; and finally performing quality tests including SDS-PAGE purity analysis, endotoxin detection, deubiquitinase activity assay, and substrate selectivity verification. A purity of 95% or higher and an endotoxin content of less than 0.5 EU / mg constitute a qualified USP7 mutant protein preparation.

[0015] The beneficial effects of this invention are as follows:

[0016] The USP7 mutant created through rational protein engineering design in this invention exhibits high substrate selectivity. Compared to wild-type USP7, its deubiquitination activity towards p53 is increased by approximately 8-15 times, while its binding affinity to MDM2 is reduced by approximately 80-95%, thus achieving specific activation of p53 without promoting MDM2 stability. In vitro biochemical experiments show that the USP7 mutant of this invention can extend the half-life of p53 protein from approximately 45 minutes under wild-type USP7 treatment to over 6 hours, while shortening the half-life of MDM2 protein from approximately 3 hours under wild-type USP7 treatment to approximately 30 minutes, demonstrating an excellent substrate selectivity reversal effect. Attached Figure Description

[0017] Figure 1 This is the dose-response curve of the USP7 mutant's inhibition of tumor cell proliferation.

[0018] Figure 2 Comparison of deubiquitination activity between USP7 mutant and wild type.

[0019] Figure 3This is due to the synergistic effect of the USP7 mutant combined with chemotherapy drugs. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0021] Unless otherwise specified, the experimental methods used in this invention are conventional methods. Unless otherwise specified, all materials and reagents used in this invention are commercially available.

[0022] Example 1: Construction of a recombinant deubiquitinase USP7 mutant expression vector

[0023] This embodiment constructs a mammalian expression vector encoding the USP7 mutant, laying the foundation for subsequent protein expression and functional verification.

[0024] 1. USP7 mutant gene design

[0025] Using the human USP7 wild-type gene sequence (GenBank accession number NM_003470) as a template, eight site-directed mutations were designed and introduced: L203A (CTG→GCG), F205A (TTC→GCC), N208D (AAC→GAC), D210K (GAC→AAG), G224S (GGC→AGC), Q297E (CAG→GAG), T308C (ACC→TGC), and E556C (GAG→TGC). Codon optimization was performed using DNAWorks software, favoring high-frequency codons from CHO-K1 cells of Chinese hamster ovaries, optimizing the GC content to 55%, and avoiding unstable sequence elements. The optimized USP7 mutant gene is 3306 bp in length, encoding 1102 amino acids. A Kozak sequence (GCCACC) was added to the 5' end of the gene to enhance translation initiation efficiency, and a His6 tag sequence (CATCATCATCATCATCAT) was added to the 3' end for protein purification. The designed gene sequence was entrusted to GenScript Biotech for whole-genome synthesis. The synthesized gene was cloned into the pUC57 vector, and the sequence was verified to be completely correct by DNA sequencing.

[0026] 2. Construction of mammalian expression vectors

[0027] The pcDNA3.4 vector was selected as the expression vector backbone. This vector contains the immediate early promoter of human cytomegalovirus (CMV), which can drive efficient expression of exogenous genes in various mammalian cells. The USP7 mutant gene was cloned into the multiple cloning site of the pcDNA3.4 vector using a double restriction endonuclease digestion method. The specific steps are as follows:

[0028] The pUC57-USP7 mutant plasmid was digested with EcoRI and XhoI at 37°C for 4 hours, separated by 1% agarose gel electrophoresis, and the approximately 3.3 kb USP7 mutant gene fragment was recovered using a gel recovery kit (Qiagen). Simultaneously, the pcDNA3.4 vector was digested with the same enzymes, dephosphorylated to prevent vector self-ligation, and the approximately 5.4 kb linearized vector fragment was recovered.

[0029] The USP7 mutant gene fragment was mixed with the linearized vector at a 3:1 molar ratio, and T4 DNA ligase (NEB) was added. Ligation was carried out overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Single clones were picked for colony PCR identification. Positive clones were cultured in a shaking culture, and plasmid DNA was extracted.

[0030] The correctness of the recombinant plasmid was verified by double enzyme digestion and sequencing. The recombinant plasmid was digested with EcoRI and XhoI, and agarose gel electrophoresis showed a vector band of approximately 5.4 kb and a target gene band of approximately 3.3 kb, consistent with the expected size. The recombinant plasmid was sent to Genewiz Biotechnology Co., Ltd. for full-sequence sequencing. The sequencing results showed that the USP7 mutant gene sequence was completely correct, all eight site-directed mutation sites were accurately introduced, the His6 tag was correctly fused, and it was correctly linked to the vector backbone, with no unexpected mutations or frameshift mutations.

[0031] The final recombinant expression vector was named pcDNA3.4-USP7-Mut-His, and plasmid DNA was prepared in large quantities and stored at -20℃ for later use. To improve protein expression levels, an enhanced expression vector pcDNA3.4-WPRE-USP7-Mut-His containing an enhanced CMV promoter and a WPRE post-transcriptional regulatory element was also constructed.

[0032] 3. Establishment of stable expression cell lines

[0033] The recombinant expression vector was transfected into Chinese hamster ovary cells CHO-K1 using liposome transfection. CHO-K1 cells were seeded in 6-well plates at a density of 2 × 10⁶ cells / well. 5Cells / well were cultured in F-12 medium containing 10% FBS until 70% confluence. Following the Lipofectamine 3000 (Thermo Fisher) instructions, 2.5 μg of pcDNA3.4-USP7-Mut-His plasmid was transfected into each well. Forty-eight hours after transfection, the original medium was replaced with selection medium containing 800 μg / mL G418, and this was repeated every 3 days. After 2-3 weeks of selection, resistant clones gradually emerged. Single clones were picked and seeded into 96-well plates for expansion. Western blot analysis was used to detect the expression level of the USP7 mutant protein, and high-expression clones CHO-USP7-Mut-14 and CHO-USP7-Mut-23 were selected. These high-expression clones were cryopreserved in liquid nitrogen.

[0034] Example 2: Large-scale expression and purification of recombinant deubiquitinase USP7 mutant protein

[0035] This embodiment establishes a large-scale expression and purification process for the USP7 mutant protein, resulting in a high-purity, high-activity recombinant protein formulation.

[0036] 1. Large-scale culture and protein expression of CHO cells

[0037] The stable expression cell line CHO-USP7-Mut-14 established in Example 1 was revived and expanded in F-12 medium containing 10% FBS and 400 μg / mL LG418. After passage to the 8th generation, the cells were gradually acclimatized in CD FortiCHO serum-free medium (Thermo Fisher) containing 5% FBS for approximately 2 weeks. After successful acclimatization, the cells doubled in serum-free medium in approximately 18 hours, maintaining a viability of over 95%.

[0038] The domesticated CHO-USP7-Mut-14 cells were seeded into 2L shake flasks at an initial density of 3×10⁶ cells / mL. 5 Cells / mL, culture volume 1.5L, placed in a shaker incubator, 37℃, 120rpm, 5% CO2. When cell density reaches 1.5×10⁶ cells / mL... 6 When the cell density reached 8 × 10⁶ cells / mL (approximately day 4 of culture), feed medium (containing concentrated glucose, amino acids, vitamins, and growth factors) was added to maintain high-density cell growth and sustained protein expression. Feed medium was added every 2 days, at a volume of 10% of the initial culture volume. After 14 days of continuous culture, the cell density reached a peak of 8 × 10⁶ cells / mL. 6 Cells / mL, at which point the viability begins to decrease to approximately 80%, and the culture is terminated.

[0039] The cell culture supernatant was collected by centrifugation at 4000g for 15 minutes. Cell debris and particulate matter were removed by filtration through 0.45μm and 0.22μm filters to obtain approximately 6L of clear supernatant containing USP7 mutant protein.

[0040] 2. Nickel affinity chromatography purification

[0041] The USP7 mutant protein, fused with a His6 tag at its C-terminus, can be purified using nickel affinity chromatography as the first step. A HisTrap HP 5 mL nickel affinity chromatography column (Cytiva) was used, equilibrated with 5 column volumes of binding buffer (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole). 6 μL of cell culture supernatant was loaded onto the nickel column at a flow rate of 5 mL / min, taking approximately 20 hours to complete. After loading, the column was washed with 20 column volumes of binding buffer to remove non-specifically bound proteins until OD was reached. 280 Values ​​below 0.01. Perform gradient elution with elution buffer (20 mM sodium phosphate pH 7.4, 500 mM NaCl, 500 mM imidazole) and collect the elution peaks.

[0042] SDS-PAGE analysis of the elution peaks revealed a distinct band of USP7 mutant protein at approximately 125 kDa, with a purity of about 70%. The elution peaks were combined and concentrated to approximately 50 mL using a 10 kDa molecular weight cutoff ultrafiltration tube (Millipore), while simultaneously replacing the buffer with 20 mM Tris-HCl pH 8.0 and 50 mM NaCl.

[0043] 3. Anion exchange chromatography purification

[0044] The second purification step was performed using a HiTrap Q HP 5 mL anion exchange chromatography column (Cytiva) to remove impurities such as host cell proteins, nucleic acids, and endotoxins. The column was equilibrated with 5 column volumes of buffer A (20 mM Tris-HCl pH 8.0, 50 mM NaCl). The nickel affinity purified protein sample was loaded at a flow rate of 3 mL / min. After loading, the column was washed with 10 column volumes of buffer A. A linear NaCl gradient elution was performed, stepwise increasing from 50 mM to 500 mM, with a gradient volume of 30 column volumes. The USP7 mutant protein was eluted at approximately 200 mM NaCl, and the elution peak was collected.

[0045] SDS-PAGE analysis showed that the purity of the USP7 mutant protein increased to approximately 90% after anion exchange chromatography, with most of the host cell protein being removed. The elution peaks were combined and concentrated to approximately 20 mL.

[0046] 4. Gel filtration chromatography purification

[0047] Final purification was performed using a Superdex 200 Increase 10 / 300 GL gel filtration chromatography column (Cytiva) to remove aggregates, degradation products, and residual impurities, while also performing buffer replacement. The column was equilibrated with 2 column volumes of storage buffer (20 mM sodium phosphate, pH 7.2, 150 mM NaCl, 5% trehalose). 2 mL of sample was loaded per cycle (not exceeding 5% of the column bed volume) at a flow rate of 0.5 mL / min. The USP7 mutant protein elutes at approximately 13 mL of elution volume; the main peak was collected.

[0048] 5. Quality Inspection and Preparation

[0049] All batches of gel filtration chromatography products were combined and subjected to comprehensive quality testing.

[0050] SDS-PAGE purity analysis: 12% SDS-PAGE gel electrophoresis, Coomassie brilliant blue staining, ImageJ software scanning and quantitative analysis showed that the purity of the USP7 mutant protein was 97.5%, with a single band at approximately 125 kDa, and no obvious degradation products or aggregates.

[0051] Concentration determination: Protein concentration was determined using a BCA protein assay kit (Thermo Fisher), with bovine serum albumin as the standard. The final USP7 mutant protein concentration was 5.2 mg / mL. Approximately 62 mg of USP7 mutant protein was obtained from 6 L of cell culture supernatant, with a purification yield of approximately 52%.

[0052] Endotoxin detection: The endotoxin content was detected using the Lonza method with Limulus amebocyte lysate (LAL) reagent. The endotoxin content was 0.28 EU / mg protein, which is far below the requirement for injectable biological products (<0.5 EU / mg).

[0053] Activity assay: The deubiquitinating enzyme activity of the USP7 mutant was determined using the ubiquitin-AMC fluorescence substrate method. At 37℃ and pH 7.5, the Km value of the USP7 mutant to ubiquitin-AMC was 1.8 μM, the Vmax value was 85 nmol / min / mg, and the specific activity was approximately 472 U / mg, comparable to that of wild-type USP7 (approximately 420 U / mg), demonstrating that the mutation did not significantly affect the catalytic activity.

[0054] The purified USP7 mutant protein was aliquoted into sterile vials, 1 mL (5 mg) per vial, and stored at -80°C. Before use, the protein was slowly thawed at 4°C and gently mixed, avoiding repeated freeze-thaw cycles to maintain its activity.

[0055] Example 3: Validation of substrate selectivity of USP7 mutant for p53 and MDM2

[0056] like Figure 2 As shown, this embodiment systematically verified the substrate selectivity of the USP7 mutant for p53 and MDM2 through various in vitro biochemical experiments, confirming that it can specifically deubiquitinate p53 while destabilizing MDM2.

[0057] 1. Preparation of ubiquitinated p53 and MDM2 substrates

[0058] To evaluate the deubiquitination activity of the USP7 mutant on p53 and MDM2, it is first necessary to prepare ubiquitinated p53 and MDM2 substrate proteins.

[0059] Preparation of ubiquitinated p53: pcDNA3.1-p53-WT, pCMV-MDM2, pRK5-His-Ub(K48R) (retaining only the K63-linked polyubiquitin chain), and pCMV-E1 / E2 ubiquitinase plasmid were co-transfected into HEK293T cells. Forty-eight hours after transfection, cells were treated with 20 μM MG132 (a proteasome inhibitor) for six hours to accumulate ubiquitinated p53. Cells were collected and lysed with lysis buffer containing 6 M guanidine hydrochloride. His-labeled ubiquitinated p53 was purified by nickel affinity chromatography. After elution, the cells were dialyzed into physiological buffer. SDS-PAGE analysis showed that the p53 protein presented as ladder-like bands (65-200 kDa), indicating successful preparation of the polyubiquitinated p53 substrate. Western blot analysis using both anti-p53 and anti-ubiquitin antibodies confirmed the presence of ubiquitinated p53. The protein concentration is approximately 0.8 mg / mL. Aliquot and store at -80°C.

[0060] Preparation of ubiquitinated MDM2: Using a similar method, pcDNA3.1-MDM2, pRK5-His-Ub, and pCMV-E1 / E2 plasmids were co-transfected into HEK293T cells. After treatment with MG132, the ubiquitinated MDM2 was purified. SDS-PAGE analysis showed that the MDM2 protein presented as a ladder-like band (100-250 kDa), and Western blot confirmed it as ubiquitinated MDM2. The protein concentration was approximately 0.6 mg / mL.

[0061] 2. In vitro deubiquitination enzyme activity assay

[0062] The activity of the USP7 mutant against ubiquitinated p53 and MDM2 was evaluated using an in vitro deubiquitination reaction system.

[0063] Deubiquitination activity of p53: The reaction system consisted of 50 μL of deubiquitination buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, 0.1% Triton X-100), 1 μg of ubiquitinated p53 substrate, and 0, 50, 100, 200, and 400 nM USP7 mutant or wild-type USP7 (as a control). The reaction was carried out at 37 °C for 30 min, and the reaction was terminated by adding SDS-PAGE loading buffer and boiling at 95 °C for 5 min. SDS-PAGE and Western blot analysis were performed, and the degree of p53 deubiquitination was detected using anti-p53 antibody. Quantitative analysis using ImageJ software showed that the USP7 mutant at a concentration of 200 nM deubiquitinated approximately 75% of the ubiquitinated p53, while wild-type USP7 at the same concentration only deubiquitinated approximately 35% of the ubiquitinated p53. Calculations showed that the USP7 mutant exhibited approximately 11 times higher deubiquitination activity against p53 compared to the wild-type USP7 (according to EC). 50 The value is 85 nM for the USP7 mutant and 920 nM for the wild-type USP7.

[0064] Deubiquitination activity against MDM2: Under the same reaction system and conditions, the substrate was replaced with ubiquitinated MDM2. Western blot results showed that the USP7 mutant exhibited almost no deubiquitination activity against ubiquitinated MDM2, with almost no change in band intensity even at a high concentration of 400 nM. In contrast, wild-type USP7 at a concentration of 200 nM could deubiquitinate approximately 65% ​​of ubiquitinated MDM2. This indicates that the deubiquitination activity against MDM2 in the USP7 mutant is significantly reduced or eliminated.

[0065] The substrate selectivity index (SI) was calculated by comparing the activity ratios of p53 and MDM2 for the USP7 mutant and wild-type USP7. The SI (p53 / MDM2) of the wild-type USP7 was approximately 0.54, indicating higher activity for MDM2. In contrast, the SI (p53 / MDM2) of the USP7 mutant was greater than 100, indicating extremely high selectivity for p53 and successful reversal of substrate selectivity.

[0066] 3. Protein binding affinity assay

[0067] The binding affinity of the USP7 mutant to p53 and MDM2 was determined using surface plasmon resonance (SPR) technology.

[0068] The experiment was performed on a Biacore T200 instrument (Cytiva). Purified p53 or MDM2 protein was immobilized on the surface of a CM5 sensor chip using amine coupling, with an immobilization volume of approximately 500 response units (RU). The mobile phase was HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant), with a flow rate of 30 μL / min and a temperature of 25 °C.

[0069] Different concentrations of USP7 mutant or wild-type USP7 (0, 10, 20, 50, 100, 200, 500 nM) were injected onto the chip surface, with binding for 180 seconds and dissociation for 300 seconds. The chip surface was regenerated with 10 mM glycine solution at pH 2.0. Kinetic analysis was performed using BiacoreT200 Evaluation Software, a 1:1 binding model was fitted, and the binding-dissociation constant (KD) was calculated.

[0070] The results showed that the binding affinity of the USP7 mutant to p53 (KD = 45 nM) was significantly higher than that of the wild-type USP7 to p53 (KD = 280 nM), an increase of approximately 6-fold. More importantly, the binding affinity of the USP7 mutant to MDM2 (KD > 5000 nM) was much lower than that of the wild-type USP7 to MDM2 (KD = 85 nM), a decrease of approximately 60-fold. This result is highly consistent with the in vitro deubiquitination activity assay results, confirming that the USP7 mutant achieves selectivity for p53 by altering substrate binding affinity.

[0071] 4. Protein stability experiment

[0072] The effect of the USP7 mutant on the stability of p53 and MDM2 proteins was evaluated using protein degradation assays.

[0073] HEK293T cells were transiently transfected with p53 and MDM2 expression plasmids. Twenty-four hours later, 100 μg / mL cyclohexylimide (CHX, a protein synthesis inhibitor) was added to block the synthesis of new proteins. Simultaneously, 500 nM USP7 mutant or wild-type recombinant USP7 protein (pre-coated with Bioporter protein transduction reagent to promote protein entry into cells) was added or not. Cells were collected at 0, 0.5, 1, 2, 4, and 6 hours, and p53 and MDM2 protein levels were detected by Western blot.

[0074] The results showed that in the control group without USP7, the half-life of p53 protein was approximately 30 minutes, and the half-life of MDM2 protein was approximately 2 hours. After adding wild-type USP7, the half-life of p53 protein increased to approximately 45 minutes, and the half-life of MDM2 protein increased to approximately 3 hours, indicating that wild-type USP7 stabilizes both proteins simultaneously, but has a stronger stabilizing effect on MDM2. After adding the USP7 mutant, the half-life of p53 protein significantly increased to over 6 hours, while the half-life of MDM2 protein shortened to approximately 30 minutes. This indicates that the USP7 mutant specifically stabilizes p53 while promoting MDM2 degradation, fully achieving the expected substrate selectivity.

[0075] Example 4: USP7 mutant activates p53 signaling pathway in tumor cells

[0076] This embodiment evaluated the activation of the p53 signaling pathway by the USP7 mutant in different tumor cell lines, verifying its biological effects at the cellular level.

[0077] 1. Tumor cell lines and culture conditions

[0078] The following tumor cell lines were selected: HCT116 (human colorectal cancer cells, p53 wild-type), A549 (human non-small cell lung cancer cells, p53 wild-type), MCF-7 (human breast cancer cells, p53 wild-type), MDA-MB-231 (human breast cancer cells, p53 mutant R280K), and H1299 (human non-small cell lung cancer cells, p53 deletion). All cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in their respective media containing 10% FBS at 37°C in a 5% CO2 incubator. HCT116, MDA-MB-231, and H1299 were cultured in McCoy's 5A medium, while A549 and MCF-7 were cultured in DMEM medium.

[0079] 2. USP7 mutant cell delivery and protein expression detection

[0080] Since the USP7 mutant is a recombinant protein, an effective cellular delivery method is required. Two methods were employed: protein transduction and liposome-mediated delivery.

[0081] Protein transduction method: Purified USP7 mutant protein was mixed with PULSI protein transduction reagent (Polyplus) at a mass ratio of 1:3 and incubated at room temperature for 15 minutes to form a protein-transduction reagent complex. The complex was then added to cultured tumor cells at final concentrations of 0, 100, 200, 500, and 1000 nM USP7 mutant. After incubation for 4-6 hours, the medium was replaced with fresh medium, and the cells were cultured until the specified time point.

[0082] Liposome-mediated method: The USP7 mutant protein is mixed with cationic lipid (Lipofectamine MessengerMAX, Thermo Fisher) to form a protein complex encapsulated by liposomes, which has higher delivery efficiency.

[0083] Western blot was used to detect the protein expression levels of USP7 mutant, p53, MDM2, and p53 downstream target genes p21 and PUMA in cells. HCT116 cells were treated with 500 nM USP7 mutant, and total protein was extracted from cells at 2, 6, 12, 24, and 48 hours. The results showed that USP7 mutant protein was detectable in cells 2 hours after treatment, peaked at 6-12 hours, and was still detectable at 48 hours. p53 protein levels began to increase 6 hours after treatment, peaked at 24 hours (approximately 12-fold higher than the control group), and remained at a high level at 48 hours. MDM2 protein levels began to decrease 12 hours after treatment, and decreased to approximately 20% of the control group at 24 hours. p53 downstream target proteins p21 and PUMA began to increase 12 hours after treatment, and increased approximately 15-fold and 20-fold, respectively, at 24 hours. These results confirm that the USP7 mutant successfully entered tumor cells and effectively activated the p53 signaling pathway.

[0084] In the comparative experiment, treatment of HCT116 cells with wild-type USP7 resulted in only a slight increase (approximately 2-fold) in p53 protein levels, a significant increase (approximately 5-fold) in MDM2 protein levels, and no significant changes in p21 and PUMA protein levels. This further confirms the functional differences between the USP7 mutant and wild-type USP7 at the cellular level.

[0085] 3. p53 transcriptional activity assay

[0086] The mRNA expression levels of p53 target genes were detected by real-time quantitative PCR (qRT-PCR) to assess the transcriptional activity of p53.

[0087] HCT116 cells were treated with 500 nM USP7 mutant for 24 hours, and total RNA was extracted. cDNA was synthesized using a reverse transcription kit (TaKaRa). Gene expression quantification was performed using SYBR Green qPCR Master Mix (Applied Biosystems) on an ABI 7500 real-time quantitative PCR instrument. The p53 target genes detected included: p21 (CDKN1A), PUMA (BBC3), BAX, MDM2, GADD45A, NOXA (PMAIP1), and DR5 (TNFRSF10B). GAPDH was used as an internal reference gene, and relative expression levels were calculated using the 2^-ΔΔCt method.

[0088] The results showed that after treatment with the USP7 mutant, p21 mRNA expression increased by approximately 18-fold, PUMA by approximately 25-fold, BAX by approximately 8-fold, MDM2 by approximately 12-fold (due to feedback upregulation of MDM2 transcription after p53 activation), GADD45A by approximately 14-fold, NOXA by approximately 10-fold, and DR5 by approximately 7-fold. These genes cover multiple functions regulated by p53, including cell cycle arrest (p21, GADD45A), apoptosis (PUMA, BAX, NOXA, DR5), and negative feedback (MDM2), confirming that the USP7 mutant comprehensively activates the transcriptional activity of p53.

[0089] In p53 mutant cells MDA-MB-231 and p53-deficient cells H1299, the expression of the above target genes did not change significantly after treatment with the USP7 mutant, confirming that the effect of the USP7 mutant is entirely dependent on wild-type p53.

[0090] 4. p53-DNA binding activity assay

[0091] The binding of p53 to the promoter of the target gene was detected by chromatin immunoprecipitation (ChIP) assay.

[0092] HCT116 cells were treated with 500 nM USP7 mutant for 24 hours, fixed with 1% formaldehyde for 10 minutes, and the fixation was terminated with 0.125 M glycine. Chromatin was sonicated to fragments of 200-500 bp. The p53-DNA complex was immunoprecipitated with anti-p53 antibody (Santa Cruz, sc-126), and the immune complex was captured with protein A / G magnetic beads. After washing and decrosslinking, DNA was extracted, and the enrichment of p53 target gene promoter regions was detected by qPCR.

[0093] The results showed that after treatment with the USP7 mutant, p53 enrichment increased approximately 16-fold in the p21 promoter (containing two p53 binding sites), approximately 22-fold in the PUMA promoter, and approximately 10-fold in the BAX promoter. No significant enrichment changes were observed in the control region (GAPDH promoter). This confirms that the USP7 mutant not only increases p53 protein levels but also enhances p53 binding to target gene promoters, thus enabling it to perform its full transcription factor function.

[0094] 5. Comparison of p53 activation in different tumor cell lines

[0095] In HCT116, A549, MCF-7 (all p53 wild type) and MDA-MB-231, H1299 (p53 mutant or deleted) cells, the p53 and p21 protein levels were detected by Western blot after treatment with 500 nM USP7 mutant for 24 hours.

[0096] The results showed that p53 protein levels were significantly increased in all three wild-type p53 cell lines: approximately 12-fold in HCT116, approximately 9-fold in A549, and approximately 10-fold in MCF-7. p21 protein levels were correspondingly increased: approximately 15-fold in HCT116, approximately 11-fold in A549, and approximately 13-fold in MCF-7. In MDA-MB-231 and H1299 cells, treatment with the USP7 mutant showed no significant changes in p53 mutant protein or p53 deficiency, and p21 protein levels did not increase significantly. This indicates that the USP7 mutant can effectively activate the p53 pathway in different wild-type p53 tumor cells, and its effect is p53-dependent, having no effect on p53-mutant or deficient tumor cells, demonstrating good tumor selectivity.

[0097] Example 5: Inhibitory effect of USP7 mutant on tumor cell proliferation

[0098] like Figure 1 As shown, this embodiment evaluated the inhibitory effect of the USP7 mutant on the proliferation of different tumor cells and compared its effect with that of the MDM2 inhibitor Nutlin-3.

[0099] 1. MTT cell proliferation assay

[0100] The inhibitory effect of the USP7 mutant on tumor cell proliferation was detected using the MTT assay. HCT116, A549, MCF-7, MDA-MB-231, and H1299 cells were seeded in 96-well plates at a density of 5 × 10³ cells / well and cultured for 24 hours. Different concentrations of the USP7 mutant (0, 10, 20, 50, 100, 200, 500, 1000, 2000 nM) were added, with six replicates for each concentration. After 72 hours of further culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37°C for 4 hours. The culture medium was then removed, and 150 μL of DMSO was added, followed by shaking for 10 minutes to fully dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader.

[0101] Cell viability (%) = (Experimental group OD) 490 - Blank group OD 490 ) / (control group OD 490 - Blank group OD 490 )×100%

[0102] The dose-response curve was fitted using GraphPad Prism 9.0 software, and the IC50 was calculated. 50 Value (drug concentration that inhibits 50% of cell proliferation).

[0103] The results showed that the USP7 mutant had an IC50 effect on wild-type p53 cells. 50The values ​​were: HCT116 180±25 nM, A549 220±32 nM, and MCF-7 195±28 nM. IC50 values ​​for p53 mutant or deletion cells... 50 The values ​​were: MDA-MB-231 2150±320 nM, H1299 2480±410 nM. IC50 values ​​were: wild-type p53 cells vs. p53 mutant / deletion cells. 50 The values ​​differ by about 10-13 times, indicating that the USP7 mutant is highly selective for wild-type p53 tumor cells.

[0104] In the comparative experiment, the same cells were treated with the MDM2 inhibitor Nutlin-3, and the IC50 value was [not specified]. 50 The values ​​were: HCT116 450±65 nM, A549 520±78 nM, and MCF-7 480±72 nM. The antitumor activity of the USP7 mutant was approximately 2-2.5 times higher than that of Nutlin-3.

[0105] 2. Cloning experiment

[0106] The effect of the USP7 mutant on the long-term proliferation and clonogenic ability of tumor cells was evaluated using a clonogenic assay. HCT116 and A549 cells were seeded in 6-well plates at a density of 500 cells / well and cultured for 24 hours. Different concentrations of the USP7 mutant (0, 50, 100, and 200 nM) were then added. The medium containing the mutant was changed every 3 days, and the cells were cultured continuously for 10–14 days until control clones were clearly visible. Cells were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet for 30 minutes, washed with PBS, and air-dried. The number of cell clones was counted (>50 cells were defined as one clone).

[0107] Clonal formation rate (%) = (Number of clones in the experimental group / Number of clones in the control group) × 100%

[0108] The results showed that in HCT116 cells, treatment with 50, 100, and 200 nM USP7 mutants reduced the colony formation rate to 65%, 32%, and 8% of the control group, respectively. In A549 cells, the colony formation rate decreased to 62%, 35%, and 12%, respectively. This indicates that the USP7 mutant not only inhibits the short-term proliferation of tumor cells but also significantly inhibits their long-term colony formation ability, exhibiting a durable anti-tumor effect.

[0109] 3. EdU cell proliferation labeling assay

[0110] The effect of the USP7 mutant on tumor cell DNA synthesis was evaluated using an EdU (5-ethynyl-2'-deoxyuridine) incorporation assay. HCT116 cells were seeded in 24-well plates at a density of 3 × 10⁶ cells / well. 4Cells were cultured for 24 hours, then 500 nM MUSP7 mutant was added, and the cells were cultured for another 24 hours. 10 μM EdU was added and incubated for 2 hours. Cells were fixed with 4% paraformaldehyde and stained with EdU according to the Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher) instructions. Cell nuclei were counterstained with DAPI. The proportion of EdU-positive cells (green fluorescence) was observed and counted under a fluorescence microscope.

[0111] The results showed that the proportion of EdU-positive cells in the control group was about 55% (indicating that 55% of the cells were in the S phase), while the proportion of EdU-positive cells in the USP7 mutant treatment group decreased to about 12%, indicating that the USP7 mutant significantly inhibited the DNA synthesis of tumor cells, causing the cells to exit the proliferation cycle.

[0112] 4. Comparison with Nutlin-3

[0113] In HCT116 cells, the inhibitory effects of equivalent doses of USP7 mutant (200 nM) and Nutlin-3 (500 nM, producing similar p53 activation levels) on cell proliferation were compared. MTT assays showed that after 72 hours, the cell viability in the USP7 mutant treatment group was 28 ± 5%, while in the Nutlin-3 treatment group it was 42 ± 6%, indicating a stronger inhibitory effect from the USP7 mutant. In clonogenic assays, the clonogenic rate in the USP7 mutant treatment group was 8 ± 2%, while in the Nutlin-3 treatment group it was 18 ± 4%. This suggests that, under conditions producing similar p53 activation levels, the USP7 mutant exhibits a stronger antitumor effect than Nutlin-3. This may be because the USP7 mutant simultaneously degrades MDM2, eliminating the sustained inhibition of p53 by MDM2, while Nutlin-3 only blocks the MDM2-p53 interaction; the MDM2 protein remains and may exert a pro-cancer effect through other mechanisms.

[0114] Example 6: USP7 mutant induces tumor cell cycle arrest and apoptosis

[0115] This embodiment evaluated the effect of the USP7 mutant in inducing tumor cell cycle arrest and apoptosis, and elucidated its anti-tumor cell biological mechanism.

[0116] 1. Cell cycle analysis

[0117] Flow cytometry was used to analyze the effect of the USP7 mutant on tumor cell cycle distribution. HCT116 and A549 cells were seeded in 6-well plates at a density of 2 × 10⁶ cells / well. 5Cells were cultured in wells for 24 hours, then different concentrations of USP7 mutant (0, 200, 500 nM) were added, and the cells were cultured for another 48 hours. Cells (including adherent and suspension cells) were collected, washed with PBS, fixed with 70% cold ethanol, and incubated overnight at 4°C. After washing, cells were stained with a staining solution containing 50 μg / mL propidium iodide (PI) and 100 μg / mL RNase A at room temperature in the dark for 30 minutes. Cell DNA content was detected by flow cytometry (BD FACSCanto II), and cell cycle distribution was analyzed using ModFit LT software.

[0118] The results showed that in HCT116 cells, the cell cycle distribution in the control group was: G1 phase 45%, S phase 42%, and G2 / M phase 13%. After treatment with 200 nM USP7 mutant, the number of cells in G1 phase increased to 68%, S phase decreased to 22%, and G2 / M phase decreased to 10%. After treatment with 500 nM USP7 mutant, the number of cells in G1 phase further increased to 85%, S phase decreased to 9%, and G2 / M phase decreased to 6%. A similar G1 phase arrest effect was also observed in A549 cells. This indicates that the USP7 mutant induces G1 phase arrest in tumor cells by activating p53 and upregulating the cell cycle repressor protein p21, thus preventing cells from entering the S phase for DNA replication.

[0119] In the control experiment, no significant changes were observed in cell cycle distribution after treatment with p53 mutant cells MDA-MB-231 and USP7 mutant, confirming that the G1 phase arrest effect depends on wild-type p53.

[0120] 2. Apoptosis detection

[0121] USP7 mutant-induced tumor cell apoptosis was detected using the Annexin V / PI double staining method. HCT116 and A549 cells were seeded in 6-well plates and cultured for 24 hours. Different concentrations of USP7 mutant (0, 200, and 500 nM) were added, and the cells were cultured for another 48 hours. Cells were collected and stained using the Annexin V-FITC / PI apoptosis detection kit (BD Biosciences). Cells were resuspended in 100 μL binding buffer, and 5 μL of Annexin V-FITC and 5 μL of PI were added. The cells were incubated at room temperature in the dark for 15 minutes. 400 μL of binding buffer was added, and the cells were immediately analyzed by flow cytometry. Annexin V⁺ / PI⁻ represented early apoptotic cells, Annexin V⁺ / PI⁺ represented late apoptotic cells, and the total apoptosis rate was the sum of the two.

[0122] The results showed that in HCT116 cells, the total apoptosis rate in the control group was approximately 8%. Treatment with the 200 nM USP7 mutant increased the total apoptosis rate to approximately 38% (25% early apoptosis and 13% late apoptosis). Treatment with the 500 nM USP7 mutant further increased the total apoptosis rate to approximately 60% (32% early apoptosis and 28% late apoptosis). In A549 cells, treatment with the 500 nM USP7 mutant increased the total apoptosis rate to approximately 45%. This indicates that the USP7 mutant induces programmed cell death in tumor cells by activating p53 and upregulating pro-apoptotic proteins such as PUMA, BAX, and NOXA.

[0123] In the control experiment, the apoptosis rate of p53 mutant cells MDA-MB-231 and p53-deficient cells H1299 did not increase significantly after treatment with the same concentration of USP7 mutant, confirming that the apoptosis effect depends on wild-type p53.

[0124] 3. Caspase activity assay

[0125] Caspase activation induced by the USP7 mutant was quantitatively analyzed using a Caspase-3 / 7 activity assay kit (Promega). HCT116 cells were treated with 500 nM USP7 mutant for different time periods (0, 12, 24, and 48 hours), and caspase-3 / 7 activity was measured according to the kit instructions. In short, cell lysis buffer was mixed with caspase-3 / 7 substrate (Ac-DEVD-AMC), incubated at 37°C for 1 hour, and the AMC fluorescence signal was detected using a fluorescence microplate reader (excitation wavelength 360 nm, emission wavelength 460 nm).

[0126] The results showed that caspase-3 / 7 activity in the control group was approximately at baseline. Caspase activity began to increase after 12 hours of treatment with the USP7 mutant (approximately 3 times that of the control group), increased to approximately 8 times after 24 hours, and to approximately 12 times after 48 hours. Pretreatment with the caspase-3 specific inhibitor Z-DEVD-FMK (50 μM) completely blocked USP7 mutant-induced caspase activation and apoptosis, confirming that the USP7 mutant induces tumor cell death through a caspase-dependent intrinsic apoptotic pathway.

[0127] 4. Mitochondrial membrane potential detection

[0128] The effect of the USP7 mutant on the mitochondrial membrane potential (ΔΨm) of tumor cells was detected using JC-1 staining. HCT116 cells were treated with 500 nM USP7 mutant for 24 hours, stained with JC-1 staining solution (Beyotime), and incubated at 37°C for 20 minutes. After washing with PBS, flow cytometry was used to detect red fluorescence (590 nm, representing normal mitochondrial membrane potential, JC-1 aggregates) and green fluorescence (529 nm, representing loss of membrane potential, JC-1 monomers). Cells with loss of membrane potential showed a decreased red / green fluorescence ratio.

[0129] The results showed that approximately 12% of cells in the control group exhibited loss of mitochondrial membrane potential. After treatment with the USP7 mutant, the number of cells with loss of membrane potential increased to approximately 55%, indicating that the USP7 mutant induces mitochondrial dysfunction and triggers the mitochondrial-mediated intrinsic apoptosis pathway. Western blot analysis of mitochondrial cytochrome c release showed that the level of cytochrome c in the cytoplasm was significantly increased after treatment with the USP7 mutant, further confirming the activation of the mitochondrial apoptosis pathway.

[0130] 5. Cell senescence detection

[0131] SA-β-galactosidase staining was used to detect USP7 mutant-induced cellular senescence. HCT116 cells were treated with 200 nMUSP7 mutant (subapoptotic dose) for 7 days and stained with a Senescence β-Galactosidase Staining Kit (CellSignaling). After cell fixation, the cells were incubated overnight at 37°C with staining solution containing X-Gal, and the proportion of blue SA-β-gal positive cells was observed and counted under a microscope.

[0132] The results showed that approximately 5% of cells in the control group were SA-β-gal positive, while approximately 40% of cells in the USP7 mutant treatment group were SA-β-gal positive, indicating that the USP7 mutant, in addition to inducing cell cycle arrest and apoptosis, can also induce irreversible cellular senescence in tumor cells. Western blot analysis of senescence-related markers p16, p21, and p53 showed that these proteins were significantly elevated in the USP7 mutant treatment group, confirming the senescence phenotype. Cellular senescence is one of the important mechanisms by which p53 exerts its tumor-suppressive effect, and these results demonstrate that the USP7 mutant can comprehensively activate multiple anti-tumor functions of p53.

[0133] Example 7: USP7 mutant overcomes MDM2 inhibitor resistance

[0134] This embodiment evaluated the killing effect of the USP7 mutant on tumor cells resistant to the MDM2 inhibitor Nutlin-3, demonstrating that it can overcome some MDM2 inhibitor resistance.

[0135] 1. Establishment of Nutlin-3 resistant cell lines

[0136] HCT116 cells were treated with progressively increasing concentrations of Nutlin-3 (starting at 0.5 μM, increasing by 0.5 μM every 2 weeks, ending at 5 μM) for 6 months to establish the Nutlin-3 resistant cell line HCT116-NutR. The IC50 of the resistant cells to Nutlin-3 was determined. 50 The MDM2 protein level increased from 450 nM in parental cells to approximately 2800 nM, representing a resistance index of approximately 6.2-fold. Western blot analysis showed that the MDM2 protein level in HCT116-NutR cells was significantly elevated (approximately 4 times that in parental cells). Whole-exome sequencing revealed a C305F point mutation in the MDM2 gene, a mutation that has been reported to reduce the binding affinity of Nutlin-3 to MDM2, leading to resistance.

[0137] 2. Sensitivity determination of USP7 mutant to drug-resistant cells

[0138] The MTT assay was used to detect the inhibitory effect of the USP7 mutant on the proliferation of HCT116-NutR resistant cells. The results showed that the USP7 mutant had an IC50 inhibitory effect on HCT116-NutR cells. 50 The value was 280±35 nM, only lower than the IC50 of parental HCT116 cells. 50 The value (180 nM) increased by approximately 1.6 times, which is far lower than the resistance fold to Nutlin-3 (6.2 times). This indicates that the USP7 mutant can largely overcome Nutlin-3 resistance caused by the MDM2-C305F mutation.

[0139] 3. Comparison of p53 pathway activation in drug-resistant cells

[0140] Western blot compared the activation of the p53 pathway by Nutlin-3 and USP7 mutants in HCT116-NutR resistant cells. 5 μM Nutlin-3 (IC50 for resistant cells) was used. 50 (concentration) or 500 nM USP7 mutant (exceeding the IC50 concentration of resistant cells) 50 HCT116-NutR cells were treated with the concentration of [specific concentration] for 24 hours.

[0141] The results showed that Nutlin-3 treatment only slightly increased p53 protein levels (approximately 1.8-fold), while MDM2 protein levels increased (approximately 1.5-fold), and p21 protein levels increased approximately 2-fold. Conversely, treatment with the USP7 mutant significantly increased p53 protein levels (approximately 9-fold), significantly decreased MDM2 protein levels (approximately 70%), and increased p21 protein levels approximately 12-fold. This indicates that although the MDM2-C305F mutation prevents Nutlin-3 from effectively blocking the MDM2-p53 interaction, the USP7 mutant can still effectively stabilize p53 and degrade mutated MDM2 through ubiquitination regulation, thereby overcoming drug resistance.

[0142] 4. Analysis of drug resistance mechanisms

[0143] To gain a deeper understanding of the mechanism by which the USP7 mutant overcomes Nutlin-3 resistance, the following experiments were conducted:

[0144] SPR experiments were conducted to examine the binding affinity of the MDM2-C305F mutant with p53, Nutlin-3, and USP7 mutants. The results showed that the binding affinity of MDM2-C305F with p53 (KD=520 nM) was slightly lower than that with wild-type MDM2 (KD=350 nM), and the binding affinity with Nutlin-3 was significantly reduced (KD increased from 90 nM in wild-type to >5000 nM). However, the binding affinity with the USP7 mutant remained very low (KD>5000 nM, similar to wild-type MDM2). This explains why the USP7 mutant remains effective against the MDM2-C305F mutant, as the USP7 mutant itself does not rely on a high affinity binding to MDM2.

[0145] The ubiquitination assay was used to investigate the effect of the USP7 mutant on MDM2-C305F. His-labeled ubiquitin, MDM2-C305F, E1 / E2 enzyme, and either the USP7 mutant or wild-type USP7 were mixed in an in vitro ubiquitination reaction system, and the ubiquitination level of MDM2-C305F was measured. The results showed that wild-type USP7 significantly reduced the ubiquitination of MDM2-C305F, while the USP7 mutant had almost no effect or slightly promoted the ubiquitination of MDM2-C305F, indicating that the USP7 mutant destabilizes MDM2-C305F and promotes its degradation.

[0146] These results collectively demonstrate that the USP7 mutant can effectively overcome Nutlin-3 resistance caused by MDM2 point mutations through a ubiquitination regulation mechanism independent of the MDM2-p53 binding interface, providing a new treatment option for patients with drug-resistant tumors.

[0147] Example 8: Synergistic antitumor effect of USP7 mutant and chemotherapy drugs

[0148] like Figure 3 As shown, this embodiment evaluates the combined therapeutic effect of USP7 mutant with commonly used chemotherapy drugs, verifying its potential for sensitizing chemotherapy.

[0149] 1. Design of combination drug regimens

[0150] The following chemotherapy drugs were selected for combination therapy experiments: 5-fluorouracil (5-FU, a pyrimidine antimetabolite), cisplatin (DDP, a platinum alkylating agent), doxorubicin (DOX, an anthracycline topoisomerase II inhibitor), and paclitaxel (PTX, a microtubule stabilizer). The combination therapy regimen was as follows: pretreatment with a subtherapeutic dose of the USP7 mutant (100 nM, which did not cause significant cytotoxicity) for 24 hours, followed by chemotherapy drugs for another 48 hours.

[0151] 2. Cell proliferation inhibition experiment of combined therapy

[0152] The inhibitory effect of USP7 mutant combined with chemotherapy drugs on the proliferation of HCT116 cells was detected by the MTT assay. The following experimental groups were set up: control group, USP7 mutant alone (100 nM) group, chemotherapy drug alone group (5-FU 5 μM, DDP 2 μM, DOX 0.5 μM, PTX 5 nM), and combination therapy group.

[0153] The results showed that the cell viability of 100 nM USP7 mutant alone was approximately 70% (slightly inhibiting proliferation). The cell viability of 5-FU alone was approximately 55%, decreasing to approximately 15% after combination with the USP7 mutant. The cell viability of DDP alone was approximately 48%, decreasing to approximately 12% after combination. The cell viability of DOX alone was approximately 42%, decreasing to approximately 10% after combination. The cell viability of PTX alone was approximately 52%, decreasing to approximately 18% after combination. All combination therapy groups showed significantly stronger inhibitory effects than the single-drug groups (p<0.001).

[0154] 3. Analysis of drug synergistic effects

[0155] The Chou-Talalay method was used to calculate the Combination Index (CI) to quantitatively assess the interaction between the USP7 mutant and chemotherapeutic drugs. CI < 0.9 indicated a synergistic effect, CI = 0.9–1.1 indicated an additive effect, and CI > 1.1 indicated an antagonistic effect. CompuSyn software was used to calculate the CI values.

[0156] The results showed that the CI values ​​for the USP7 mutant with 5-FU were 0.42 (strong synergism), with DDP were 0.38 (strong synergism), with DOX were 0.35 (strong synergism), and with PTX were 0.48 (strong synergism). All combinations exhibited significant synergistic antitumor effects.

[0157] 4. Apoptosis-enhancing effect of combined therapy

[0158] Annexin V / PI flow cytometry was used to detect apoptosis induced by combined therapy. HCT116 cells were pretreated with 100 nMUSP7 mutant for 24 hours, followed by treatment with 5-FU (5 μM) for another 48 hours.

[0159] The results showed that the apoptosis rate induced by USP7 mutant alone was approximately 18%, that of 5-FU alone was approximately 32%, and the apoptosis rate induced by the combination therapy reached approximately 78%, far exceeding the expected value (50%) of the sum of the two, confirming a synergistic pro-apoptotic effect. Western blot analysis showed that the expression levels of p53, p21, PUMA, and cleaved caspase-3 in the combination therapy group were significantly higher than those in the single-drug group, indicating that the USP7 mutant enhanced the apoptosis signal induced by chemotherapy drugs by activating p53.

[0160] 5. The impact of combination therapy on chemotherapy tolerance

[0161] To assess whether the USP7 mutant can reduce the toxicity of chemotherapy drugs to normal cells. Using normal human colonic epithelial cells NCM460 as a control, the selectivity of 5-FU alone (5 μM) and the combination of USP7 mutant (100 nM) + low-dose 5-FU (2.5 μM) on normal cells and tumor cells HCT116 was compared.

[0162] The results showed that 5-FU (5 μM) alone resulted in approximately 55% survival of HCT116 cells and approximately 62% survival of NCM460 cells, with a selectivity index (SI = NCM460 IC50). 50 / HCT116 IC 50 The selectivity index was approximately 1.1, indicating poor selectivity. The combination therapy (USP7 mutant 100 nM + 5-FU 2.5 μM) resulted in approximately 22% survival of HCT116 cells and approximately 75% survival of NCM460 cells, with a selectivity index of approximately 3.4, showing a significantly improved selectivity. This indicates that by combining the USP7 mutant with chemotherapy, the anti-tumor effect can be maintained while reducing the dosage of chemotherapy drugs, decreasing toxicity to normal cells, and broadening the therapeutic window.

[0163] This phenomenon may be because tumor cells often have p53 pathway dysregulation (such as MDM2 overexpression), making them more sensitive to p53 activation, while normal cells have normal p53 pathway function and are less responsive to exogenous USP7 mutants, thus achieving tumor selectivity.

Claims

1. A recombinant deubiquitinase USP7 mutant, characterized in that, The USP7 mutant introduces the following site-directed mutations into the human USP7 wild-type protein sequence: L203A, F205A, N208D, D210K, G224S, Q297E, T308C, and E556C. The L203A and F205A mutations are located in the TRAF substrate recognition domain to reduce binding affinity to the MDM2 protein; the N208D and D210K mutations are located at the TRAF domain-catalytic domain interface to alter substrate selectivity; and the G208D and D210K mutations are located in the TRAF domain-catalytic domain interface to alter substrate selectivity. The 4S and Q297E mutations are located in the catalytic domain adjacency region to improve the catalytic efficiency for p53-ubiquitin, while the T308C and E556C mutations form intramolecular disulfide bonds to enhance protein stability. The USP7 mutant retains the Cys223-His464-Asp481 catalytic triplet and the Trp165 substrate recognition key residues, and can specifically deubiquitinate the tumor suppressor protein p53 while destabilizing the oncogenic protein MDM2, thereby activating the p53-dependent antitumor signaling pathway.

2. The recombinant deubiquitinase USP7 mutant according to claim 1, characterized in that, The USP7 mutant is fused with a cell-penetrating peptide sequence at its N-terminus or C-terminus. The cell-penetrating peptide sequence is selected from TAT peptide, Penetratin peptide, or Pep-1 peptide to enhance the cell membrane penetration ability and intracellular delivery efficiency of the mutant protein. The sequence of the TAT peptide is YGRKKRRQRRR, the sequence of the Penetratin peptide is RQIKIWFQNRRMKWKK, and the sequence of the Pep-1 peptide is KETWWETWWTEWSQPKKKRKV.

3. The recombinant deubiquitinase USP7 mutant according to claim 1 or 2, characterized in that, The USP7 mutant is fused with a nuclear localization signal sequence at its C-terminus. The nuclear localization signal sequence is selected from the SV40 large T antigen NLS sequence or the nucleoplasmic protein nucleolin NLS sequence to promote the directional transport of the mutant protein to the cell nucleus. The SV40 large T antigen NLS sequence is PKKKRKV, and the nucleoplasmic protein nucleolin NLS sequence is KRPAATKKAGQAKKKK.

4. The recombinant deubiquitinase USP7 mutant according to claim 1, characterized in that, The USP7 mutant exhibits 8-15 times higher deubiquitination activity for p53 compared to wild-type USP7, 80-95% lower binding affinity for MDM2 compared to wild-type USP7, and a substrate selectivity index greater than 100. The USP7 mutant can extend the half-life of p53 protein from about 45 minutes to more than 6 hours and shorten the half-life of MDM2 protein from about 3 hours to about 30 minutes.

5. The method for preparing the recombinant deubiquitinase USP7 mutant according to any one of claims 1-4, characterized in that, The procedure includes the following steps: transfecting a recombinant expression vector encoding the USP7 mutant into host cells, wherein the host cells are selected from Chinese hamster ovary cells (CHO-S), human embryonic kidney cells (HEK293), human embryonic kidney suspension cells (HEK293-F), or hamster kidney cells (BHK); screening and amplifying stable expression cell lines in a culture medium containing selective antibiotics; performing large-scale cell culture in serum-free or low-serum medium and collecting the cell culture supernatant; purifying the USP7 mutant with His or Fc tags by protein A or protein G affinity chromatography; further purifying by anion exchange chromatography and gel filtration chromatography to remove impurities, endotoxins, and viruses; and performing quality tests including SDS-PAGE purity analysis, endotoxin detection, deubiquitinating enzyme activity assay, and substrate selectivity verification, achieving a purity of over 95% and an endotoxin content of less than 0.5 EU / mg.

6. The preparation method according to claim 5, characterized in that, The recombinant expression vector includes a strong promoter, a target gene encoding the USP7 mutant, an enhancer sequence, a transcription terminator, and a polyadenylation signal. The strong promoter is selected from the CMV promoter, the EF1α promoter, or the CAG promoter. The recombinant expression vector also contains a selection marker gene to screen for stable expression cell lines. The selection marker gene is selected from neomycin resistance genes, puromycin resistance genes, or hygromycin resistance genes.

7. The preparation method according to claim 5, characterized in that, The nucleotide sequence encoding the USP7 mutant has been codon-optimized to suit mammalian expression systems, with GC content optimized to 50-60%, avoiding unstable sequence elements including AU-enriched regions, internal TATA boxes, poly(A) signals, splice sites, and repetitive sequences, thereby improving mRNA stability and translation efficiency.

8. The use of the recombinant deubiquitinase USP7 mutant according to any one of claims 1-4 in the preparation of a tumor-treating drug, characterized in that, The tumor is a solid tumor or a hematologic malignancy that retains the wild-type p53 gene. The solid tumor is selected from non-small cell lung cancer, colorectal cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, or neuroblastoma. The hematologic malignancy is selected from acute lymphoblastic leukemia, chronic lymphoblastic leukemia, or multiple myeloma.

9. The application according to claim 8, characterized in that, The USP7 mutant is delivered to tumor tissues and tumor cells via a tumor-targeted delivery system. The tumor-targeted delivery system includes lipid nanoparticles, polymer nanoparticles, liposomes, or exosomes as carriers. The carrier surface is modified with a tumor-targeting ligand, which is selected from folic acid, RGD peptide, tumor-penetrating peptide iRGD, or NGR peptide targeting tumor blood vessels. The delivery system introduces pH-sensitive lipids or polymers to utilize the acidity of the tumor microenvironment to achieve tumor-specific drug release. The pH-sensitive lipids or polymers are selected from DOPE or polyhistidine.

10. The application according to claim 8 or 9, characterized in that, The USP7 mutant can be used alone or in combination with chemotherapy drugs, radiotherapy or immune checkpoint inhibitors. The chemotherapy drugs are selected from 5-fluorouracil, cisplatin, doxorubicin, paclitaxel, gemcitabine or irinotecan. The immune checkpoint inhibitors are selected from anti-PD-1 antibody, anti-PD-L1 antibody or anti-CTLA-4 antibody. The combination therapy achieves a synergistic anti-tumor effect with a combination index of less than 0.9.

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