Polypeptide G9 specifically targeting human ovarian cancer cells and application thereof

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a polypeptide G9 specifically targeting human ovarian cancer cells and application of the polypeptide G9. Aiming at the problems that the existing polypeptide targeting ovarian cancer is insufficient in targeting in in-vivo tumor cells and polypeptide with better in-vivo targeting needs to be developed, the invention provides the polypeptide G9 specifically targeting human ovarian cancer cells, which is obtained by screening through a phage display technology, and the amino acid sequence is shown as SEQ ID NO: 1. The polypeptide G9 disclosed by the invention can realize a targeted affinity effect on SKOV3 human ovarian cancer cells, and in a SKOV3 human ovarian cancer tumor-bearing mouse body, the polypeptide disclosed by the invention can realize specific affinity on ovarian cancer tumor tissues so as to achieve a targeted delivery effect. The targeting polypeptide provided by the invention has strong specificity and high targeting property, and has good application potential in the fields of early diagnosis and treatment of tumors and targeted therapy.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a polypeptide G9 that specifically targets human ovarian cancer cells and its uses. Background Technology

[0002] Ovarian cancer is the leading cause of death among gynecological malignancies, constituting a significant public health burden on women worldwide. Its incidence ranks third among gynecological cancers, with over 70% of patients diagnosed at an advanced stage with metastasis. The deep anatomical location of the ovaries and the nonspecificity of early symptoms greatly increase the difficulty of treatment. Currently, surgical resection combined with chemotherapy has demonstrated broad clinical efficacy, while radiotherapy can be used to target residual lesions or high-risk areas. As novel molecular biology-based treatments, biotherapy, including immune checkpoint inhibitors and engineered immunotherapy, aims to restore the body's anti-tumor immune function. However, the efficacy and safety of these treatments still require further optimization, thus necessitating the development of innovative treatment strategies with stronger efficacy and lower toxicity.

[0003] Targeted drug delivery systems are a crucial direction in modern drug development. Their core lies in achieving specific enrichment of therapeutic drugs at lesion sites through active or passive targeting mechanisms, thereby improving efficacy and reducing systemic toxicity. Passive targeting primarily relies on the physicochemical properties of the carrier and its EPR effect in pathological tissues (such as tumors), but this method has limited specificity and inconsistent efficiency. In contrast, active targeting strategies represent higher precision. Based on the "key-lock" biomolecular recognition principle, it modifies drug carriers using specific ligands, enabling them to actively recognize and bind to specific biomarkers (such as receptors, antigens, or transporters) overexpressed at lesion sites. This precise recognition capability not only significantly improves delivery efficiency but also enhances drug internalization within cells. This advanced concept has been successfully translated into the design of cutting-edge drugs such as antibody-drug conjugates (ADCs), demonstrating significant clinical application value.

[0004] Among the many active targeting ligands explored, peptides have attracted considerable attention due to their unique comprehensive advantages. Peptides not only possess inherent advantages such as small molecular weight, strong tissue penetration, high targeting specificity, and low immunogenicity, but their molecular structures also offer high design flexibility and modifiability. They can be efficiently coupled to the surfaces of various gene delivery vectors (such as lipid nanoparticles and polymers) through chemical methods, thereby endowing these systems with excellent active targeting capabilities. This characteristic is particularly important in the field of ovarian cancer gene therapy, as designing targeting peptides that can specifically recognize ovarian cancer cell surface markers (such as folate receptors and mesothelin) is crucial for achieving precise gene drug delivery. Despite the enormous potential of peptide ligands, their development still faces significant challenges: on the one hand, the currently available libraries of peptide ligands with high affinity and specificity for ovarian cancer remain scarce, greatly limiting the development of novel delivery systems; on the other hand, existing research strategies largely rely on traditional, point-by-point optimization based on amino acid sequence structure-activity relationships, a time-consuming and inefficient method. Furthermore, ovarian cancer itself lacks highly specific and universal tumor surface targets, resulting in a high "off-target risk," which further exacerbates the difficulty of developing highly effective targeted peptides and constitutes a core bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0005] The technical problem to be solved by this invention is that existing peptides targeting ovarian cancer have insufficient targeting specificity to tumor cells in vivo, and there is a need to develop peptides with better in vivo targeting.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A polypeptide G9 that specifically targets human ovarian cancer cells is provided. The amino acid sequence of the polypeptide G9 that specifically targets human ovarian cancer cells described in this invention is shown in SEQ ID NO:1.

[0007] Amino acid sequence of a polypeptide that specifically targets human ovarian cancer cells (SEQ ID NO:1)

[0008] DPLKARHTSVWY.

[0009] Among them, the ovarian cancer cells targeted by the aforementioned peptide that specifically targets human ovarian cancer cells are the SKOV3 cell line.

[0010] The present invention also provides a nucleotide encoding method.

[0011] The encoding nucleotide can encode the aforementioned polypeptide that specifically targets human ovarian cancer cells.

[0012] Furthermore, the sequence of the encoded nucleotide is shown in SEQ ID NO:2.

[0013] The nucleotide sequence encoding a polypeptide that specifically targets human ovarian cancer cells (SEQ ID NO:2).

[0014] GACCCCCTGAAGGCCCGCCACACCAGCGTGTGGTAC.

[0015] The present invention also provides an expression vector.

[0016] Furthermore, the expression vector contains nucleotides as shown in SEQ ID NO:2.

[0017] The expression vector is either a prokaryotic vector or a eukaryotic vector.

[0018] The present invention also provides a host cell.

[0019] Furthermore, the host cell contains the aforementioned polypeptide, encoding nucleotide, or expression vector that specifically targets human ovarian cancer cells.

[0020] The present invention also provides the use of the above-mentioned polypeptide that specifically targets human ovarian cancer cells in targeting ovarian cancer cells.

[0021] Furthermore, the ovarian cancer cells mentioned are the SKOV3 cell line.

[0022] The present invention also provides the use of the above-mentioned polypeptide G9, which specifically targets human ovarian cancer cells, its encoded nucleotide, expression vector or host cell in the preparation of anti-ovarian cancer drugs or in the preparation of imaging agents for ovarian cancer diagnosis.

[0023] Furthermore, in the above-mentioned uses, the polypeptide is prepared as a solution with a concentration of 1 mg / mL.

[0024] Furthermore, in the above-mentioned applications, a buffer solution is also added to the polypeptide solution.

[0025] Furthermore, the buffer solution is a mixture of DMF buffer and HEPES buffer.

[0026] Furthermore, the volume ratio of the DMF buffer to the HEPES buffer is 1:19.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention provides a polypeptide that can specifically target the human ovarian cancer cell line SKOV3, and names it the G9 polypeptide. This invention is the first to discover that the G9 polypeptide has a strong affinity for SKOV3 ovarian cancer cells and can specifically target human ovarian cancer cells SKOV3, while it has no targeting ability against human LO2 cells and human peripheral blood cells. The targeting polypeptide of this invention has high specificity and high targeting ability, and can be used to develop drugs for the treatment of ovarian cancer, showing great application potential in the fields of early tumor diagnosis and targeted therapy. Attached Figure Description

[0029] Figure 1 The chemical structural formula of the polypeptide G9 obtained in this invention;

[0030] Figure 2 Evaluation of the specificity of phage binding to SKOV3, A549 and PMBC;

[0031] Figure 3 Assessment of the binding specificity of G9 peptide in different cell lines;

[0032] Figure 4 Systemic distribution of G9-FITC in mice after intravenous injection, as well as distribution of tumors and important organs;

[0033] Figure 5 Representative immunohistochemical (IHC) images of G9 phage binding in tissue microarrays (TMAs) from ovarian cancer patients (scale bar: 100 μm). Detailed Implementation

[0034] This invention utilizes phage display technology to screen for a highly specific targeting peptide against SKOV3 ovarian cancer cells, named G9 peptide. The amino acid sequence of the peptide is shown in SEQ ID NO:1, and its chemical structure is as follows: Figure 1 As shown.

[0035] The phage peptide library screened in this invention is randomly generated with a capacity greater than 10. 9 The dodecapeptide library underwent three rounds of screening before finally yielding the G9 peptide. During the screening process using SKOV3, this invention also selected adsorbent cells to remove peptides with affinity for other cells. This ensured that the final G9 peptide obtained could bind to SKOV3 cells while failing to recognize A549 and human peripheral blood cells.

[0036] Furthermore, the present invention also provides the encoding nucleotides, expression vectors, and host cells of the above-mentioned polypeptides that specifically target human ovarian cancer cells.

[0037] Furthermore, the present invention also provides the use of the above-mentioned peptides that specifically target human ovarian cancer cells in targeting ovarian cancer cells.

[0038] The present invention also provides the use of the above-mentioned polypeptide specifically targeting human ovarian cancer cells, its encoded nucleotide, expression vector or host cell in the preparation of drugs for the prevention or treatment of ovarian cancer.

[0039] The present invention also provides the use of the above-mentioned polypeptide specifically targeting human ovarian cancer cells, its encoded nucleotide, expression vector or host cell in the preparation of diagnostic reagents for ovarian cancer.

[0040] The polypeptide of the present invention that specifically targets human ovarian cancer cells was obtained by screening using the following method:

[0041] Human ovarian cancer cells SKOV3 and A549 cells in good growth condition were seeded into 24-well plates. Blocking buffer (4% milk + PBS) was added, and the cells were blocked at 37°C for 1 h. The blocking buffer was removed, and the cells were washed three times with PBS. 10 μl of a phage library was mixed with 1 ml of PBS and incubated with A549 cells at 37°C for 1 h. The supernatant was then incubated with the blocked ovarian cancer SKOV3 cells at 37°C for 2 h. The phages bound to the cells were collected and amplified using ER2738 host bacteria. The amplified phages were used for the next screening of SKOV3 cells.

[0042] The above operation was repeated twice. The phages obtained in the third round of screening were titered and identified using LB / IPTG / X-Gal plates. The screened phage clones were identified using ELISA technology. Phage clones with OD values ​​greater than 0.8 in the ELISA test results were selected for purification and sequencing to obtain the specific polypeptide G9 of the present invention that can recognize SKOV3.

[0043] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0044] The preparation methods for each reagent in the following examples or test cases are as follows:

[0045] 0.2M Gly-HCl pH2.2: Weigh 1.5014 g g of glycine, dissolve it in water, adjust the pH to 2.2 with HCl, and then bring the volume to 100 mL with water.

[0046] 1M Tris-HCl pH9.1: Weigh 121.1 g Tris, dissolve it in water, adjust the pH to 9.1 with HCl, and then bring the volume to 100 mL with water.

[0047] 20% PEG / 2.5M NaCl: Dissolve 20 g of polyethylene glycol and 14.6 g of NaCl in water, then add water to a final volume of 100 mL.

[0048] All other reagents are commercially available products.

[0049] Example 1: Screening of phage polypeptide libraries for SKOV3 ovarian cancer cells

[0050] Human ovarian cancer cell line SKOV3 was used as the screening target cell, while human lung adenocarcinoma cell line A549, human cervical cancer cell line HeLa, and human breast cancer cell line MCF-7 were used as negative control cells.

[0051] All cell lines were cultured under optimal conditions and then seeded into 24-well plates at 90% confluence. After the cells reached confluence, they were washed twice with PBS, subjected to serum starvation for 1 h, and then blocked with PBS containing 4% skim milk for 1 h.

[0052] Dissolve 10 μL of a phage library in 1 mL of PBS and co-incubate with negative control cells for 1 h. After centrifugation, collect the supernatant and add it to blocked SKOV3 cells, incubating for another 2 h. Elute the bound phages with 1 mL of 0.2 mol / L glycine-HCl (pH 2.2), then neutralize with 150 μL of 1 mol / L Tris-HCl (pH 9.1). Perform 10³–10⁻⁶ cycles on the eluent. 5 After serial dilution, it was used to infect Escherichia coli ER2738 strain, with an infection time of 5 min.

[0053] Infected *E. coli* were inoculated onto LB / IPTG / X-gal agar and incubated overnight at 37 °C. Phage titers were determined by blue plaque counting. The eluted phages were amplified using a 20% PEG / 2.5 mol / L NaCl precipitation method. This enrichment cycle was repeated 3–4 times, and finally, phage clones that specifically bind to SKOV3 cells were screened.

[0054] Example 2: Identification of positive clones by enzyme-linked immunosorbent assay (ELISA)

[0055] Single phage plaques were picked from LB / IPTG / X-gal plates and transferred to 96-well plates, incubated at 37°C for 4.5 h to amplify the phage clones. SKOV3 cells were seeded into 96-well plates and incubated overnight at 37°C. Negative control cell lines (A549, HeLa, or MCF-7) were also seeded into plates under the same conditions.

[0056] After washing three times with PBS, the plates were blocked with 5% skim milk for 1 h. Then, 50 μL of monoclonal phage solution diluted with blocking buffer was added to the cells, and the plates were incubated for 1 h. After washing five times with PBS, the plates were incubated with anti-P8-HRP antibody at 37°C for 1 h. The plates were then washed five times with PBS, TMB substrate was added, and the reaction was allowed to proceed for 15 min. The reaction was terminated with 2 mol / L H2SO4, and the absorbance at 450 nm was measured using a BioTek Synergy H1 microplate reader. Phage clones with absorbance greater than 0.8 were selected for sequencing; the sequence was DPLKARHTSVW. The polypeptide of this sequence was named G9 polypeptide, and its structure is shown below. Figure 1 As shown.

[0057] Example 3 Synthesis and purification of G9 polypeptide

[0058] A certain amount of 2Cl resin was weighed and added to the reactor. Then, amino acids and alkali were added in a resin:Fmoc-L-Leu-OH:DIEA ratio of 1:3:10. The mixture was reacted with DCM (dichloromethane) as a solvent for 1 hour, then washed and dried to synthesize the resin. Resin of the appropriate specifications was selected and accurately weighed, and added to a clean reactor. DCM was added to swell the resin for 30 minutes. The DCM was removed, and 20% DMF was added for the reaction (5 minutes the first time, 15 minutes the second time). The DMF was then removed, and the mixture was washed 6 times alternately with DMF and DCM. A small amount of resin was placed in a detection tube, and one drop of ninhydrin solution was added to each tube. The tube was heated at 105–110 °C for 5 minutes. A deep blue color in the resin indicated a positive result, signifying that Fmoc had been removed. After removal, the resin was added to the reactor in the following order: 3 times amino acids: 3 times HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate): 10 times NMM (N-methylmorpholine). DMF was added to dissolve the residue, and the reaction was allowed to proceed for 30 minutes. Take a small amount of resin in a detection tube, add one drop of ninhydrin solution to each, and heat at 105–110 °C for 5 min. A negative result indicates a complete reaction; a positive result requires repeating the deprotection and condensation steps. After all peptide sequences are assembled, wash the resin alternately with DMF, methanol, and DCM, and then dry it. Add the dried resin to cleavage buffer (TFA (trifluoroacetic acid) 94.5%, water 2.5%, EDT (1,2-ethylenedithiol) 2.5%, TIS (triisopropylsilane) 1% at 10 ml per gram, and react on a shaker for 2 h. After the reaction is complete, filter to obtain the filtrate, dry it with nitrogen, add diethyl ether to precipitate the crude product, wash six times with diethyl ether, and then evaporate to dryness to obtain the crude polypeptide. Purify the crude polypeptide by HPLC: dissolve in 50% acetonitrile aqueous solution, filter, analyze by analytical HPLC, then purify by preparative HPLC, collect the fraction, identify the purity and molecular weight, and finally freeze-dry to obtain a white powder polypeptide, seal in a package, and store at -20 °C.

[0059] Experimental Example 1: Verification of G9 Peptide Cell Targeting Affinity

[0060] To investigate the affinity of ovarian cancer peptide G9 for cells, an assay was established for validation. First, well-grown SKOV3, A549, and PBMCs were collected, washed with PBS, centrifuged at 300 g for 3 min, and the supernatant was discarded. This process was repeated three times. Next, for blocking, cells were co-incubated with 10% goat serum for 1 h, centrifuged at 300 g for 3 min, and the supernatant was discarded. The corresponding phage diluted 1:10 with PBS was added to the cell pellet for incubation. Subsequently, anti-M13 phage antibody diluted 1:100 was added, followed immediately by DyLight 488-labeled secondary antibody, and the cells were incubated at room temperature in the dark for 30 min. Finally, the cells were washed with PBS and analyzed using a BD FACSCalibur flow cytometer. Results are as follows: Figure 2 As shown, G9 phage is clearly positive in SKOV3 cells. Compared with the control group, G9 phage showed a significantly stronger positive signal in SKOV3 cells, which confirms that G9 phage has a high degree of specific targeting ability for SKOV3 cells.

[0061] Experiment 2: In vitro verification of the binding ability of G9 peptide to different cell lines

[0062] The G9 peptide was synthesized by Genscript and FITC-labeled. SKOV3, 293T, and IOSE-80 (5 × 10⁻⁶) were then added. 4 Cells (cells / well) were seeded in culture plates. After washing the cells three times with PBS, 500 μL of PBS containing 2 μg / mL FITC-labeled G9 peptide (G9-FITC) was added, and the cells were incubated at 37°C for 5 min. After washing twice with PBS, the cells were stained with 1 μg / mL Hoechst 33342, and then analyzed by fluorescence microscopy and flow cytometry. The results are as follows: Figure 3 As shown, the results indicate that G9-FITC selectively binds to the cell membrane of SKOV3 cells, while its binding to 293T cells and IOSE-80 cells is negligible. This demonstrates that the G9 peptide screened in this invention exhibits specific targeting ability, specifically targeting SKOV3 cells, while showing no affinity for 293T cells and IOSE-80 cells.

[0063] Example 3: In vivo targeting analysis of G9 peptide

[0064] To construct a subcutaneous xenograft model of SKOV3 cells, female BALB / c nude mice (4–6 weeks old, purchased from Beijing Huafukang Biotechnology Co., Ltd.) were housed in a specific pathogen-free (SPF) environment. The tumor was diagnosed by subcutaneous injection of 5 × 10⁵ cells into the lateral dorsal flank of the mice. 6 SKOV3 cells were used to induce tumor formation. When the tumor volume reached 500 mm³, six mice were intravenously injected with 100 μL of PBS solution containing 10 μg FITC-labeled G9 peptide (10 μg / mL). Whole-body fluorescence imaging of the mice was performed at 15 min, 2 h, 4 h, 8 h, 24 h, and 48 h post-injection using a PerkinElmer IVISSpectrum in vivo imaging system. One mouse was sacrificed at each time point, and in vitro fluorescence imaging of the tumor and major organs was performed. The results are shown below. Figure 4 As shown in the figure, in vivo fluorescence imaging results showed that the FITC-labeled G9 peptide rapidly accumulated at the tumor site within 15 minutes after injection, reaching a peak fluorescence intensity at 2 hours. Significant tumor fluorescence signals could still be detected thereafter, up to 48 hours, indicating that the G9 peptide has the characteristics of rapid tumor accumulation and long-term retention, and can be used for the development of targeted drugs.

[0065] Experimental Example 4: Immunohistochemical staining verification test of G9 peptide on tissue sections from ovarian cancer patients

[0066] To verify whether the G9 peptide might have a targeted effect on ovarian cancer in patients, immunohistochemistry was used to stain tissue sections from ovarian cancer patients. After processing according to standard histopathological procedures, the sections were dewaxed and rehydrated; subsequently, antigen retrieval was performed at 95 °C for 20 min using citrate buffer (pH 6.0). Antigen retrieval was then performed using 5% goat serum and 3% hydrogen peroxide. After blocking the section for 15 min, the section was mixed with G9 phage (concentration of 2×10⁻⁶). 9 Incubate overnight at 4 °C with PFU / mL (dissolved in PBS).

[0067] After washing the sections three times with PBS (5 min each time), they were incubated with anti-M13 enzyme-labeled antibody (1:500 dilution) at 37 °C for 1 h. DAB chromogenic reagent was added for 10 min, followed by hematoxylin counterstaining of the cell nuclei. After dehydration and mounting, representative images were acquired using a pathological section scanning system. Results are as follows: Figure 5 As shown, tissue microarray analysis of patient-derived tissues revealed that, compared to adjacent non-tumor tissues, strong positive signals were specifically localized in the ovarian cancer tumor region, indicating that G9 phage has the ability to specifically recognize human ovarian cancer tissues.

[0068] In summary, this invention has screened and obtained a polypeptide that can specifically target ovarian cancer cells. This polypeptide has a good targeting effect on ovarian cancer SKOV3 cells and can be further developed for the preparation of drugs for the treatment of ovarian cancer or for the preparation of imaging agents for the diagnosis of ovarian cancer, showing great application prospects.

Claims

1. A polypeptide G9 that specifically targets human ovarian cancer cells, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. The polypeptide G9 specifically targeting human ovarian cancer cells according to claim 1, characterized in that: The ovarian cancer cells mentioned are the SKOV3 cell line.

3. The nucleotide encoding the polypeptide G9 that specifically targets human ovarian cancer cells as described in claim 1.

4. The encoded nucleotide according to claim 3, characterized in that: The sequence of the encoded nucleotide is shown in SEQ ID NO:

2.

5. An expression vector, characterized in that: Contains the polypeptide G9 that specifically targets human ovarian cancer cells as described in claim 1 or 2, or the encoded nucleotide as described in claim 3 or 4.

6. The expression vector according to claim 5, characterized in that: The expression vector is a prokaryotic vector or a eukaryotic vector.

7. A host cell containing the polypeptide G9 that specifically targets human ovarian cancer cells as described in claim 1 or 2, the encoding nucleotide as described in claim 3 or 4, and the expression vector as described in claim 5 or 6.

8. Use of the polypeptide G9 specifically targeting human ovarian cancer cells as described in claim 1, the encoding nucleotide as described in claim 3 or 4, the expression vector as described in claim 5 or 6, and the host cell as described in claim 7 in the preparation of a medicament for the prevention or treatment of ovarian cancer.

9. Use of the polypeptide G9 specifically targeting human ovarian cancer cells according to claim 1, the encoded nucleotide according to claim 3 or 4, the expression vector according to claim 5 or 6, and the host cell according to claim 7 in the preparation of an imaging agent for ovarian cancer diagnosis.

10. The use according to claim 8 or 9, characterized in that: The aforementioned polypeptide G9 is prepared as a solution with a concentration of 1 mg / mL.

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