Polypeptide and application thereof in HPV (human papillomavirus) treatment

Abstract

The invention discloses an intelligent responsive fusion polypeptide (HPVP-02) of a targeted HPV (human papillomavirus) E6 / E7 protein and application of the intelligent responsive fusion polypeptide in treatment of cervical cancer. The amino acid sequence of the polypeptide is as shown in SEQ ID NO.1, the polypeptide is composed of a dual-targeting module (specifically bound with 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 all the modules are connected in series through flexible connecting peptides. The HPVP-02 is subjected to responsive disassembly in a tumor acidic microenvironment, efficiently penetrates through a cell membrane, degrades E6 / E7 protein through a ubiquitin-proteasome pathway, recovers the host cancer suppressor protein p53 / pRb function, blocks a PD-1 / PD-L1 pathway and activates anti-tumor immune response. The fusion polypeptide has the advantages of targeting accuracy, low toxicity and synergistic treatment, and provides 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 epithelial DNA virus that mainly infects human skin and mucosal epithelium. Among them, high-risk HPV (such as HPV-16, HPV-18) is closely related to the occurrence of cervical cancer. Epidemiological studies have shown that DNA sequences of high-risk HPV can be detected in more than 99% of cervical cancer cases. The carcinogenicity of HPV is mainly attributed to the two early proteins E6 and E7 encoded by it, which can synergistically inhibit the tumor suppressor signaling pathway of host cells, leading to unlimited cell proliferation and malignant transformation. E6 protein mediates the ubiquitination degradation of p53 protein by binding to E6AP (E6-associated ubiquitin ligase), causing cells to lose their ability to respond to DNA damage and regulate apoptosis. E7 protein can bind to and promote the degradation of pRb (retinoblastoma protein), thereby releasing E2F transcription factors, driving the abnormal advancement of the cell cycle from G1 phase to S phase, and ultimately leading to cell carcinogenesis.

[0003] Currently, the main treatments for cervical cancer include surgical resection, radiotherapy, and chemotherapy, but these methods have limited therapeutic effects in advanced patients and are accompanied by a high recurrence rate and serious toxic side effects. In addition, although HPV vaccines can effectively prevent infection, they cannot provide therapeutic effects for individuals who have already been infected or cells that have already become cancerous. Therefore, treatment strategies targeting the carcinogenic mechanism after HPV infection still face many challenges.

[0004] In recent years, the application of peptide and antibody therapy in antiviral and anti-tumor treatment has attracted widespread attention. However, there are relatively few studies on therapeutic peptides or antibodies targeting HPV E6 / E7 proteins. Most of the reported molecules have defects such as low affinity, poor stability, and weak cell membrane penetration, making it difficult to play an effective anti-HPV role in vivo. Therefore, the development of an efficient, low-toxic peptide with good cell membrane penetration ability to simultaneously target E6 and E7 proteins and block their functions is a scientific problem that needs to be solved urgently, and it is also the technical innovation point 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 was screened by molecular docking and binds to the zinc finger domain of HPV16 E6 (Kd = 3.2 nM), competitively inhibiting E6AP-mediated p53 degradation; the E7 targeting module blocks the binding of E7 to pRb in an α-helical conformation, restoring cell cycle regulation; experiments confirmed that HPVP-02 achieved E6 / E7 protein degradation rates of 94% and 86% respectively at pH 6.5.

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

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

[0010] 4. Long-term stability: The modification with D-amino acids enhances the anti-enzymatic ability of the polypeptide (retaining 85% activity after 24 h of trypsin treatment); the self-assembled nanoparticles (particle size 82.3 nm, PDI = 0.12) extend the blood circulation half-life to 12 h. Description of the Drawings

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

[0012] Figure 2 Western blot detection results of E6 and E7 after treatment under different conditions. Detailed Description of the Invention

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

[0014] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0015] Example 1: Design and construction of the fusion polypeptide HPVP-02

[0016] To achieve dual-targeted intervention against the HPV oncoproteins E6 and E7, and simultaneously enhance stability in vivo and the anti-tumor immune activation effect, a fusion polypeptide named HPVP-02 was designed. Its module composition and functions are as follows:

[0017] 1. Dual-targeting module: This module is used to specifically recognize and bind two major oncoproteins E6 and E7 of HPV, blocking their interaction with host tumor suppressor proteins, specifically including:

[0018] (1) E6 binding domain: Using molecular docking and virtual screening techniques, a highly affinity E6 binding peptide was screened from a polypeptide library. Its binding constant Kd with the zinc finger domain of the E6 protein is 3.2 nM, which can effectively compete with the interaction between E6 and E6AP and inhibit the degradation of p53. The specific amino acid sequence is: Cys-DArg-Pro-DGln-Lys-Gly-Ala-Trp-Arg-Cys (cyclized structure, forming a disulfide bond through Cys1 and Cys10).

[0019] (2) E7 binding domain: An artificially synthesized α-helix structure polypeptide was designed to specifically bind to the Rb binding site of the E7 protein, blocking its dissociation and degradation of pRb and restoring cell cycle regulation function. The specific amino acid sequence is: Ala-Thr-DArg-Ser-DGln-Val-Asp-Leu-Tyr-Lys (α-helix 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) was designed and inserted. This sequence can undergo a conformational change at around pH 6.5, exposing a hydrophobic transmembrane structure, which helps the polypeptide penetrate the tumor cell membrane and improve the cell uptake efficiency. The specific amino acid sequence is His-His-His-His-His-Glu-Glu-Glu-Glu-Glu.

[0021] 3. Immune activation module: To further activate the anti-tumor immune response, a verified inhibitory polypeptide of the PD-1 / PD-L1 pathway was fused into the polypeptide structure. This sequence can bind to PD-1 or PD-L1, blocking their interaction, thereby relieving the immunosuppressive state and enhancing the activity of tumor-specific T cells. The specific amino acid sequence is Arg-Met-Tyr-Phe-Asp-Asn-Trp-Val-Thr.

[0022] 4. Self-assembly module: A sequence of amphiphilic structure is fused to the C-terminus of the polypeptide, endowing it with self-assembly ability. Under physiological conditions, this module drives HPVP-02 to form stable nanoparticles with a particle size of approximately 80 nm, significantly enhancing its blood stability and in vivo circulation time, while facilitating tumor tissue enrichment mediated by the EPR effect. The specific amino acid sequence is Gly-Gly-Gly-Ser-(Leu-Ala-Val)3-Lys-Lys-Lys.

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

[0024] 6. Functions of the polypeptide. The polypeptide designed in this way has the following functions:

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

[0026] (2) Dual-targeted synergistic degradation: Inhibits HPV virus replication by inducing the ubiquitination and degradation of E6 / E7;

[0027] (3) Immunomicroenvironment remodeling: The 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 the polypeptide

[0030] 1.1 Solid-phase synthesis

[0031] (1) The solid-phase synthesis method using the Fmoc (9-fluorenylmethoxycarbonyl) strategy was adopted to synthesize the HPVP-02 fusion polypeptide on Rink Amide MBHA resin (0.6 mmol / g).

[0032] (2) Synthesis process: Pre-rinse with DMF solution three times. Remove the Fmoc group with 20% piperidine / DMF solution, and deprotect for 15 minutes at each step. Use HBTU / HOBt / DIEA (4:4:8 equiv.) for the coupling reaction, with a reaction time of 30 minutes. Double coupling was performed to improve efficiency. Arg of the E6 module 2 and Gln 4 , Arg of the E7 module 3 and Gln 5All use D-isomers (D-Arg, D-Gln) to improve protease stability and conformational rigidity. Cys in the E6 module 1 and Cys 10 are designed to form a stable disulfide bond ring. Use a mixture of TFA / TIS / H2O (95:2.5:2.5) to lyse at room temperature for 2 h, and filter to recover the polypeptide.

[0033] 1.2 Oxidative cyclization: Dissolve the crude product in 0.1 M NH4HCO3 buffer (pH 8.0) at a concentration of 1 mg / mL, and slowly shake (100 rpm) at room temperature for 24 h to allow Cys 1 and Cys 10 to form an endogenous disulfide bond.

[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 retention time of the main peak is 12.3 min, and the purity > 95%. Mass spectrometry analysis shows that the measured molecular weight is 5842.8 Da, which is 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 at a final concentration of 1 mg / mL. Let it stand in the dark at 37 °C for 24 h to complete self-assembly into a nanostructure.

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

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

[0041]

[0042] 3 pH-responsive verification experiment

[0043] 3.1 Experimental setup: Place equal amounts of HPVP-02 nanoparticles in buffers with pH 7.4 and pH 6.5 (adjusted with PBS) at a concentration of 1 mg / mL. Set 3 replicates for each group, and incubate in a shaker at 37 °C (100 rpm) for 72 h. Sample every 12 h to measure the particle size change and release rate.

[0044] 3.2 Particle size change analysis (DLS)

[0045] (1) At pH 7.4: The particle size remained stable (82 ± 2 nm), and there was no obvious depolymerization phenomenon.

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

[0047] 3.3 Polypeptide release experiment

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

[0049] (2) Cumulative release amount at 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 acidic microenvironment responsiveness.

[0051] 3.4 Circular dichroism analysis (CD Spectroscopy)

[0052] (1) At pH 7.4: The H5E5 module was mainly in the α-helix form (45% α-helix, 5% β-sheet).

[0053] (2) At pH 6.5: The structure changed significantly, with the α-helix decreasing to 10% and the β-sheet increasing to 55%.

[0054] (3) It shows that the structure of the H5E5 module can rearrange in response to pH changes, promoting the exposure of the membrane penetration region.

[0055] 4. Summary: In this example, the multifunctional fusion polypeptide HPVP-02 was successfully synthesized and characterized, and its good self-assembly ability, acidic responsiveness, and appropriate nanoparticle size were confirmed by physical and chemical means. These characteristics lay the foundation for subsequent cell uptake, pharmacodynamic evaluation, and in vivo delivery.

[0056] Example 3: In vitro functional verification

[0057] 1. Verification of the activity of targeting and degrading E6 / E7 proteins in the SiHa cell model

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

[0059] (2) Experimental grouping

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

[0061] The control groups were set as follows:

[0062] The DMSO treatment group (volume concentration 0.1%, pH 6.5);

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

[0064] The E7 siRNA group (50 nM).

[0065] (3) Treatment time: Cells were collected for subsequent analysis after incubation for 48 h in all groups.

[0066] (4) Western blot detection: Cells were lysed and total proteins were collected (RIPA buffer + protease inhibitor); After separation by SDS-PAGE, the membrane was transferred; Incubation with primary antibodies (E6, E7, p53, pRb, β-actin); Development was carried out using ECL and gray-scale quantitative analysis was performed.

[0067] (5) Co-immunoprecipitation analysis: Precipitation was carried out using anti-E6 and anti-E7 antibodies, and subsequent Western detection was carried out using anti-ubiquitin antibody; 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 the degradation of E6 / E7 under acidic conditions, and p53 / pRb was significantly restored, indicating that its "targeted degradation function" was superior to siRNA. Co-IP verified that HPVP-02 significantly promoted the ubiquitination of E6 / E7, indicating that its mechanism of action was mediated by the ubiquitin-proteasome pathway for degradation.

[0069] Table 2 Summary of data analysis of experimental results

[0070]

[0071] 2. Immunostimulatory effects in the PBMCs co-culture model

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

[0073] (2) Treatment groups:

[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) Cells and supernatants were collected after incubation for 48 h: The supernatant was used to detect IFN-γ and TNF-α (ELISA).

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

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

[0078] Table 3 Summary of data analysis of experimental results

[0079]

[0080] Example 4: Antitumor activity and immune memory in PDX model

[0081] 1. Establishment and treatment of PDX model

[0082] (1) Source: Tissues from HPV18+ cervical cancer patients, human-derived tumors were established by subcutaneous transplantation 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 combination group.

[0084] 2. Treatment effect (Table 4): HPVP-02 achieved a 60% complete remission rate in the PDX model, and the tumors were completely cleared, which was better than the cisplatin combined with anti-PD-1 regimen.

[0085] Table 4 Comparison of antitumor activity treatment results in PDX model

[0086]

[0087] 3 Immune memory assessment

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

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

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

[0091] Table 5 Results of memory T cell detection (flow cytometry)

[0092]

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope 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.

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

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