A polypeptide and its application in HPV treatment

Abstract

The present invention discloses an intelligent responsive fusion polypeptide (HPVP-02) targeting HPV E6 / E7 protein and its application in the treatment of cervical cancer. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1. It is composed of a dual-targeting module (specifically binding to HPV E6 / E7 protein), a pH response module (H5E5 sequence), an immune activation module (PD-1 / PD-L1 inhibitory peptide) and a self-assembly module (amphiphilic nanostructure), and each module is connected in series through a flexible connecting peptide. HPVP-02 responsively disassembles in the acidic microenvironment of the tumor, efficiently penetrates the cell membrane, degrades E6 / E7 protein through the ubiquitin-proteasome pathway, restores the function of the host tumor suppressor protein p53 / pRb, and simultaneously blocks the PD-1 / PD-L1 pathway to activate the anti-tumor immune response. The fusion polypeptide of the present invention has the advantages of targeting precision, low toxicity and synergistic treatment, providing an innovative treatment strategy for HPV-related tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide and its application in HPV treatment. Background Art

[0002] HPV (human papillomavirus) is an epitheliotropic DNA virus that primarily infects the human skin and mucosal epithelium. High-risk HPV types (such as HPV-16 and HPV-18) are closely associated with the development of cervical cancer. Epidemiological studies have shown that DNA sequences of high-risk HPV types can be detected in over 99% of cervical cancer cases. The carcinogenicity of HPV is primarily attributed to the two early proteins it encodes, E6 and E7, which synergistically inhibit host cell tumor suppressor signaling pathways, leading to cell proliferation and malignant transformation. E6 binds to E6AP (E6-associated ubiquitin ligase) to mediate the ubiquitination and degradation of p53, resulting in cells losing their ability to respond to DNA damage and regulate apoptosis. E7, on the other hand, binds to and promotes the degradation of pRb (retinoblastoma protein), thereby releasing the E2F transcription factor, driving abnormal progression of the cell cycle from G1 to S phase, ultimately leading to cancerous transformation.

[0003] Currently, the main treatments for cervical cancer include surgical resection, radiotherapy, and chemotherapy. However, these approaches have limited efficacy in patients with advanced disease and are associated with high recurrence rates and severe side effects. Furthermore, while HPV vaccines can effectively prevent infection, they offer no therapeutic benefit for individuals already infected or for cells already cancerous. Therefore, therapeutic strategies targeting the carcinogenic mechanisms of HPV infection still face numerous challenges.

[0004] In recent years, the application of peptide and antibody therapies in antiviral and anti-tumor treatments has attracted widespread attention. However, relatively few therapeutic peptides or antibodies targeting HPV E6 / E7 proteins have been studied. Most reported molecules suffer from low affinity, poor stability, and weak cell membrane penetration, making them ineffective in vivo against HPV. Therefore, the development of a highly effective, low-toxic peptide with good cell membrane penetration that can simultaneously target both E6 and E7 proteins and block their function is a pressing scientific challenge and the technological innovation of this study. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide and its application in HPV treatment.

[0006] Therefore, the present invention designs a four-module fusion polypeptide HPVP-02, which mainly includes the following characteristics:

[0007] 1. Dual-targeted synergistic degradation: The E6-targeting module, screened through molecular docking, binds to the HPV16 E6 zinc finger domain (Kd = 3.2 nM), competitively inhibiting E6AP-mediated p53 degradation. The E7-targeting module blocks E7 binding to pRb via an α-helical conformation, restoring cell cycle regulation. Experiments confirmed that HPVP-02, at pH 6.5, degrades E6 and E7 proteins by 94% and 86%, respectively.

[0008] 2. Intelligent responsive delivery: The H5E5 module converts from an α-helix to a β-sheet in the acidic environment of the tumor (pH 6.5), triggering nanoparticle disassembly and increasing cellular uptake efficiency by 4 times. The EPR effect in vivo increases the drug concentration in tumor tissue to 8 times that of normal tissue.

[0009] 3. Immune synergistic activation: PD-1 inhibitory peptide blocks the binding of PD-L to T cells, increasing the CD8+ T cell activation rate to 68% (vs. 42% in the anti-PD-1 antibody group); synergistically with targeted degradation, IFN-γ secretion reached 1250 pg / mL in the co-culture model.

[0010] 4. Long-term stability: D-amino acid modification enhances the peptide's resistance to enzymatic degradation (85% activity retained after 24 hours of trypsin treatment); self-assembled nanoparticles (particle size 82.3 nm, PDI = 0.12) extend the blood circulation half-life to 12 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 TEM image of the fusion polypeptide shows that HPVP-02 forms uniform, compact spherical nanoparticles with a diameter of approximately 80 nm after self-assembly.

[0012] Figure 2 Western blot detection results of E6 and E7 after treatment with different conditions. DETAILED DESCRIPTION

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0014] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0015] Example 1: Design and Construction of Fusion Peptide HPVP-02

[0016] To achieve dual targeted intervention against HPV oncogenic proteins E6 and E7, while simultaneously enhancing in vivo stability and anti-tumor immune activation, a fusion peptide, named HPVP-02, was designed. Its modular composition and functions are as follows:

[0017] 1. Dual-targeting module: This module is designed to specifically recognize and bind to HPV's two major oncogenic proteins, E6 and E7, blocking their interaction with host tumor suppressor proteins. Specifically, it includes:

[0018] (1) E6 binding domain: Molecular docking and simulation screening techniques were used to screen a high-affinity E6-binding peptide from a peptide library. Its binding constant, Kd, ​​for the zinc finger domain of the E6 protein is 3.2 nM, effectively competing for the interaction between E6 and E6AP, inhibiting the degradation of p53. The specific amino acid sequence is: Cys-DArg-Pro-DGln-Lys-Gly-Ala-Trp-Arg-Cys (cyclized structure, with a disulfide bond formed by Cys1 and Cys10).

[0019] (2) E7 binding domain: A synthetic α-helical peptide was designed to specifically bind to the Rb binding site of the E7 protein, blocking its dissociation and degradation of pRb, thereby restoring its cell cycle regulatory function. The specific amino acid sequence is: Ala-Thr-DArg-Ser-DGln-Val-Asp-Leu-Tyr-Lys (α-helical design, D-amino acids enhance stability).

[0020] 2. pH-responsive module: To achieve targeted release and membrane penetration in the tumor microenvironment, a histidine-glutamic acid repeat sequence (H5E5) is designed and inserted. This sequence undergoes a conformational change at around pH 6.5, exposing a hydrophobic transmembrane structure that helps the peptide penetrate tumor cell membranes and improve cellular uptake. The specific amino acid sequence is His-His-His-His-His-Glu-Glu-Glu-Glu-Glu.

[0021] 3. Immune Activation Module: To further stimulate anti-tumor immune responses, a validated PD-1 / PD-L1 pathway inhibitory peptide is fused to the peptide structure. This sequence binds to PD-1 or PD-L1, blocking their interaction, thereby relieving immunosuppression and enhancing tumor-specific T cell activity. The specific amino acid sequence is Arg-Met-Tyr-Phe-Asp-Asn-Trp-Val-Thr.

[0022] 4. Self-assembly module: An amphiphilic structural sequence is fused to the peptide's C-terminus, enabling self-assembly. Under physiological conditions, this module drives HPVP-02 to form stable nanoparticles approximately 80 nm in size, significantly improving its blood stability and in vivo circulation time while also facilitating EPR-mediated tumor tissue accumulation. The specific amino acid sequence is Gly-Gly-Gly-Ser-(Leu-Ala-Val)3-Lys-Lys-Lys.

[0023] 5. Polypeptide structure arrangement and amino acid sequence: HPVP-02 = [E6 binding domain] - [pH response module] - [E7 binding domain] - [immune activation module] - [self-assembly module]. Each module is separated by a flexible linker peptide GGGS. The amino acid sequence of HPVP-02 after this design is shown in SEQ ID NO. 1.

[0024] 6. The functions of the polypeptide. The designed polypeptide has the following functions:

[0025] (1) Intelligent responsive delivery: Remains inert in normal tissue (pH 7.4), activates transmembrane function in the tumor microenvironment, and reduces systemic toxicity;

[0026] (2) Dual-targeted synergistic degradation: inhibiting HPV viral replication by inducing E6 / E7 ubiquitination and degradation;

[0027] (3) Immune microenvironment remodeling: PD-1 inhibitory peptide reverses T cell exhaustion and produces a synergistic effect with targeted therapy.

[0028] Example 2: Synthesis, Assembly and Characterization of HPVP-02 Polypeptide

[0029] 1. Synthesis and purification of peptides

[0030] 1.1 Solid-phase synthesis

[0031] (1) HPVP-02 fusion peptide was synthesized on Rink Amide MBHA resin (0.6 mmol / g) using the solid-phase synthesis method of Fmoc (9-fluorenylmethoxycarbonyl) strategy.

[0032] (2) Synthesis process: Pre-wash with DMF solution 3 times. Remove the Fmoc group with 20% piperidine / DMF solution, and deprotect for 15 min per step. Use HBTU / HOBt / DIEA (4:4:8 equiv.) for coupling reaction, reaction time 30 min, double coupling to improve efficiency. Arg 2 With Gln 4 , Arg of E7 module 3 With Gln 5D-isomers (D-Arg, D-Gln) are used to improve protease stability and conformational rigidity. 1 With Cys 10 A stable disulfide bond ring was designed between them. A mixture of TFA / TIS / H2O (95:2.5:2.5) was used for lysis at room temperature for 2 hours, and the peptide was recovered by filtration.

[0033] 1.2 Oxidative cyclization: The crude product was dissolved in 0.1 M NH4HCO3 buffer (pH 8.0) at a concentration of 1 mg / mL and slowly shaken (100 rpm) at room temperature for 24 h to allow Cys 1 With Cys 10 Forms endogenous disulfide bonds.

[0034] 1.3 Purification and identification

[0035] (1) Purification: Reverse-phase high performance liquid chromatography (RP-HPLC), C18 column (250 mm × 10 mm), flow rate 1 mL / min, elution gradient 10%-90% acetonitrile (containing 0.1% TFA), completed in 40 min.

[0036] (2) Purity and identification results: The main peak retention time was 12.3 min, and the purity was >95%. Mass spectrometry analysis showed that the measured molecular weight was 5842.8 Da, which was consistent with the theoretical value of 5843.2 Da.

[0037] 2 Self-assembly and physical characterization of nanoparticles

[0038] 2.1 Self-assembly conditions: Dissolve the purified HPVP-02 polypeptide in PBS buffer to a final concentration of 1 mg / mL. Incubate at 37°C in the dark for 24 hours to allow for self-assembly into nanostructures.

[0039] 2.2 Characterization results are shown in Table 1.

[0040] Table 1 Statistical table of characterization results after synthesis of fusion peptides

[0041]

[0042] 3pH responsiveness verification experiment

[0043] 3.1 Experimental Setup: Equal amounts of HPVP-02 nanoparticles were placed in pH 7.4 and pH 6.5 buffers (pH adjusted with PBS) at a concentration of 1 mg / mL. Three replicates were set up in each group and incubated at 37°C in a shaker (100 rpm) for 72 hours. Samples were collected every 12 hours to measure particle size change and release rate.

[0044] 3.2 Particle size change analysis (DLS)

[0045] (1) pH 7.4: The particle size remains stable (82±2 nm) and there is no obvious disaggregation.

[0046] (2) pH 6.5: The particle size decreased to about 30 nm after 24 h and to 15 nm after 48 h, indicating that the nanoparticles gradually disassembled.

[0047] 3.3 Peptide release experiment

[0048] (1) FITC-labeled HPVP-02 (FITC-HVP02) was used for release experiments.

[0049] (2) Cumulative release after 72 h: At pH 6.5, the release reached 90%; at pH 7.4, the release was only 10%.

[0050] (3) This indicates that HPVP-02 has significant responsiveness to acidic microenvironment.

[0051] 3.4 Circular Dichroism Analysis (CD Spectroscopy)

[0052] (1) pH 7.4: The H5E5 module is mainly composed of α-helices (45% α-helices, 5% β-sheets).

[0053] (2) pH 6.5: The structure changes significantly, with α-helix reduced to 10% and β-sheet increased to 55%.

[0054] (3) showed that the H5E5 module structure can rearrange in response to pH changes, leading to the exposure of the membrane-penetrating region.

[0055] 4. Summary: This example successfully synthesized and characterized the HPVP-02 multifunctional fusion polypeptide. Physicochemical analysis confirmed its excellent self-assembly ability, acidic responsiveness, and suitable nanoparticle size. These properties lay the foundation for subsequent cellular uptake, efficacy evaluation, and in vivo delivery.

[0056] Example 3: In vitro functional verification

[0057] 1. Validation of the activity of targeted degradation of E6 / E7 proteins in SiHa cell models

[0058] (1) Cell treatment: SiHa cells were seeded in 6-well plates (2 × 10 5 cells) and cultured overnight; the next day the culture medium was replaced with pH 6.5 buffer.

[0059] (2) Experimental groups

[0060] Drug treatment group: HPVP-02 (final concentration 10 μM) was added and incubated for 24 h;

[0061] The control group was set up as follows:

[0062] DMSO-treated group (volume concentration 0.1%, pH 6.5);

[0063] E6 siRNA group (50 nM, liposome transfection);

[0064] E7 siRNA group (50 nM).

[0065] (3) Treatment time: All groups were incubated for 48 h before cell collection for subsequent analysis.

[0066] (4) Western blot assay: cells were lysed and total proteins were collected (RIPA buffer + protease inhibitors); SDS-PAGE separation and transfer to membrane; primary antibody incubation (E6, E7, p53, pRb, β-actin); ECL development and grayscale quantitative analysis were performed.

[0067] (5) Immunoprecipitation analysis: Anti-E6 and anti-E7 antibodies were used for precipitation, followed by Western blotting with anti-ubiquitin antibodies; the ubiquitination levels of E6 / E7 in different groups were compared to evaluate their degradation pathways.

[0068] (6) Experimental results (Table 2, Figure 2 HPVP-02 potently induced E6 / E7 degradation under acidic conditions and significantly restored p53 / pRb, demonstrating its superior targeted degradation compared to siRNA. Co-IP assays confirmed that HPVP-02 significantly promoted E6 / E7 ubiquitination, suggesting a ubiquitin-proteasome pathway-mediated degradation mechanism.

[0069] Table 2 Experimental results data analysis summary

[0070]

[0071] 2. Immune Activation in PBMCs Co-culture Model

[0072] (1) Establishment of co-culture system: SiHa cells were seeded in 24-well plates (2 × 10 cells per well). 5 ), and PBMCs were added after adherence.

[0073] (2) Treatment group:

[0074] Group 1: no treatment; Group 2: anti-PD-1 antibody (10 μg / mL); Group 3: HPVP-02 (10 μM); Group 4: HPVP-02 (10 μM) + anti-PD-1 antibody (10 μg / mL).

[0075] (3) After 48 h of incubation, cells and supernatant were collected. The supernatant was used to detect IFN-γ and TNF-α (ELISA).

[0076] (4) Cells were used for flow cytometric analysis: CD8+ T cells (activation markers CD69, CD107a), PD-L1 expression (tumor cells).

[0077] (5) Results (Table 3) showed that HPVP-02 performed better than anti-PD-1 antibody alone in activating CD8+ T cells and inhibiting PD-L1 expression, showing synergistic immune activation characteristics.

[0078] Table 3 Experimental results data analysis summary

[0079]

[0080] Example 4: Anti-tumor activity and immune memory in PDX models

[0081] 1. PDX model establishment and processing

[0082] (1) Source: Humanized tumors were established by subcutaneous transplantation of HPV18+ cervical cancer patient tissues in SCID mice.

[0083] (2) Grouping (n=10 / group): control group (normal saline); cisplatin + anti-PD-1 group (positive control); HPVP-02 group (10 mg / kg, iv, twice a week); HPVP-02 + anti-PD-1 combined group.

[0084] 2. Therapeutic effect (Table 4): HPVP-02 achieved a 60% complete response rate in the PDX model, and the tumor was completely cleared, which was superior to the cisplatin combined with anti-PD-1 regimen.

[0085] Table 4 Comparison of anti-tumor activity treatment results in PDX models

[0086]

[0087] 3. Immune memory assessment

[0088] (1) Secondary challenge experiment: On day 60 after treatment, mice with complete tumor remission were inoculated with SiHa cells (2×10 6 ).

[0089] (2) Experimental results: In the HPVP-02 group and the combination group, 100% of the patients had no tumor formation; in the cisplatin + PD-1 group, 80% of the patients had tumor formation; and in the PBS group, 100% of the patients had tumor formation.

[0090] (3) Memory T cell detection (flow cytometry) As shown in Table 5, the HPVP-02 group and the combined group had better effects.

[0091] Table 5 Memory T cell detection (flow cytometry) results

[0092]

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fusion polypeptide targeting HPV E6 / E7 protein, characterized in that: The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.

1. The fusion polypeptide further includes a cyclization structure, which forms a disulfide bond through the first Cys and the tenth Cys.

2. Use of the fusion polypeptide according to claim 1 in preparing a drug for treating HPV-related cervical cancer.

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