Polypeptide and application thereof in treating malignant solid tumors

Abstract

The invention discloses a polypeptide and application thereof in treating malignant solid tumors. The amino acid sequence of the polypeptide is FLFFASIISLIFYYSKKGRRSYK-NH2, and the polypeptide is named as the DRP. The compound can be prepared through chemical synthesis. In-vitro experiments show that the polypeptide shows remarkable broad-spectrum anti-tumor proliferation activity. In vivo experiments, the polypeptides exhibit potent growth inhibition against malignant solid tumors. In addition, cell experiments verify that the polypeptide DRP shows killing efficacy in cell lines such as CT26, OVCAR-8 and Panc-02. In animal experiments, mice dosed with the compound have no toxic reaction, and the compound has an anti-cancer effect and an anti-cancer application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide for the treatment or prevention of malignant solid tumors. Background Technology

[0002] Malignant solid tumors such as lung cancer, liver cancer, breast cancer, colorectal cancer, and pancreatic cancer are among the leading causes of death worldwide. Their clinical treatment primarily relies on a combination of methods including surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, existing therapies have significant limitations: surgery and radiotherapy, as local treatments, have limited effectiveness and are highly invasive for metastatic advanced tumors; chemotherapy lacks specificity, causing severe systemic side effects and multidrug resistance while killing tumor cells; the efficacy of targeted therapy is limited by specific biomarkers, and tumors are prone to acquired resistance; while immunotherapy (such as PD-1 / PD-L1 inhibitors), although groundbreaking, has a low overall response rate and is ineffective against immunologically "cold" tumors. Therefore, developing novel, highly effective, low-toxicity antitumor drugs that can overcome drug resistance remains an urgent clinical need. Peptide drugs, due to their high specificity, good tissue penetration, and low immunogenicity, are considered a highly promising therapeutic direction. Currently, therapeutic peptides mainly include targeted peptides, cytotoxic peptides, and immunomodulatory peptides. However, their clinical application still faces key bottlenecks such as poor in vivo stability, susceptibility to protease degradation, insufficient tumor accumulation efficiency, single mechanism of action, and difficulty in penetrating dense tumor stroma. Therefore, existing peptide drugs used to treat malignant solid tumors generally suffer from short in vivo half-life, limited targeting and permeability, and a tendency to develop drug resistance. The purpose of this invention is to provide a novel peptide to overcome or alleviate at least one of the above-mentioned defects, providing a new candidate drug with greater stability, targeting, and multiple anti-tumor activities for the treatment of malignant solid tumors.

[0003] Therefore, exploring novel protein peptides is of great significance in the treatment of malignant solid tumors. Summary of the Invention

[0004] The present invention aims to provide a novel polypeptide for the treatment or prevention of malignant solid tumors and its application, in order to overcome or alleviate at least one of the defects in the prior art.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a polypeptide having the amino acid sequence: FLFFASIISLIFYYSKKGRRSYK-NH2.

[0006] Secondly, the present invention provides the use of the polypeptide in the preparation of a medicament for the treatment or prevention of malignant solid tumors.

[0007] Thirdly, the present invention provides a method for treating or preventing malignant solid tumors, comprising administering a therapeutically effective amount of the polypeptide or a pharmaceutical composition comprising the polypeptide to a subject in need.

[0008] Furthermore, the malignant solid tumors include lung cancer, liver cancer, breast cancer, colorectal cancer, pancreatic cancer, etc.

[0009] Furthermore, the drug can be used in combination with at least one other therapeutic agent (such as chemotherapy drugs, targeted drugs, or immunomodulators).

[0010] Furthermore, the polypeptide may be a pharmaceutically acceptable salt, ester, amide, cyclized form, or an active sequence variant.

[0011] This invention investigates the therapeutic effect of DRP peptide on ovarian cancer. The amino acid sequence of the DRP peptide is: FLFFASIISLIFYYSKKGRRSYK-NH2. Through systematic in vitro experiments, it was discovered for the first time that this peptide exhibits broad-spectrum antitumor activity. Multiple experiments have demonstrated its significant inhibitory effect on the growth of malignant solid tumors, with particularly significant effects in cell lines such as CT26, OVCAR-8, and Panc-02.

[0012] Therefore, this invention proposes a novel polypeptide for the treatment or prevention of malignant solid tumors, wherein the polypeptide has the amino acid sequence FLFFASIISLIFYYSKKGRRSYK-NH2.

[0013] Animal experiments showed that the peptide exhibited significant anti-tumor activity in all mouse models, inhibiting tumor growth and reducing the endpoint tumor weight. Furthermore, the body weight of mice remained stable throughout the treatment period, and no significant systemic toxicity was observed.

[0014] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

[0015] Compared with the prior art, the present invention has the following significant advantages and beneficial effects: Broad-spectrum efficacy: This polypeptide DRP exhibited significant broad-spectrum anti-proliferative activity against 21 tumor cell lines from 8 tissue sources, confirming its broad-spectrum anti-tumor activity.

[0016] Clearly defined effect: This polypeptide has a high killing effect on solid tumors.

[0017] Low toxicity: No significant systemic toxicity was observed in animal experiments with this polypeptide. Attached Figure Description

[0018] Figure 1 This is a DRP mass spectrometry (MS) analysis report.

[0019] Figure 2 This is a DRP high-performance liquid chromatography (HPLC) analysis report.

[0020] Figure 3 This is a comparison of the dose-response curves of DRP and the positive control LL37 on CT26 cells.

[0021] Figure 4 This is a comparison of the dose-response curves of DRP and the positive control LL37 on OVCAR-8 cells.

[0022] Figure 5 This is a comparison of the dose-response curves of DRP and the positive control LL37 on Panc-02 cells.

[0023] Figure 6 For label-free quantitative proteomics of DRP.

[0024] Figure 7 This represents the change in mouse body weight.

[0025] Figure 8 Changes in tumor volume in mice.

[0026] Figure 9 Changes in tumor weight in mice. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods and reagents used in the present invention are conventional methods and techniques in the art.

[0028] The DRP polypeptide of this invention is a newly designed polypeptide with the amino acid sequence FLFFASIISLIFYYSKKGRRSYK-NH2.

[0029] Example 1: Chemical Synthesis and Purification of DRP Peptide The peptide compound DRP was chemically synthesized by Nanjing Peptide Biotechnology Co., Ltd. (Nanjing, China). Both DRP and its derivatives underwent C-terminal amidation modification to enhance stability. Molecular weight was confirmed by mass spectrometry (MS), and purity was analyzed by high-performance liquid chromatography (HPLC), confirming that the purity of DRP exceeded 90%.

[0030] Example 2: Detection of DRP peptide's in vitro anti-colon cancer activity (CCK-8 assay) Logarithmically growing mouse colon cancer cells CT26 were seeded at a density of 5 x 10^3 cells per well in 96-well plates. A blank control group (culture medium only), a negative control group (cells + culture medium), a positive control group (LL-37 peptide group, concentration gradient: 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.391, 0.195 μM), and a DRP experimental group (concentration gradient: 0.195, 0.391, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 μM) were set up, with three replicates per group. After 24 hours of culture, CCK-8 solution was added to each well, and incubation was continued for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability (%) was calculated as [(Experimental group OD - Blank group OD) / (Negative control group OD - Blank group OD)] x 100%. The dose-response curve was fitted using GraphPad Prism 8.0 software, and the half-maximal inhibitory concentration (IC50) was calculated.

[0031] The results are as follows Figure 3 As shown, the DRP peptide exhibited a dose-dependent inhibitory effect on the proliferation of CT26 cells. Under the experimental conditions, the inhibition curves of DRP and the positive control LL-37 on CT26 cells highly overlapped, indicating that both have comparable in vitro killing efficacy. These results demonstrate that the DRP peptide provided by this invention possesses significant inhibitory activity against colon cancer cells in vitro, proving its potential as an antitumor lead compound.

[0032] Example 3: Detection of DRP peptide's in vitro anti-ovarian cancer activity (CCK-8 assay) Log-phase human ovarian cancer cells (OVCAR-8) were seeded at a density of 5 x 10^3 cells per well in 96-well plates. The following groups were set up: a blank control group (culture medium only), a negative control group (cells + culture medium), a positive control group containing the endogenous peptide LL-37 (concentration gradients: 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.391, 0.195 μM), and a DRP experimental group (concentration gradients: 0.195, 0.391, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 μM), with three replicates per group. After 24 hours of culture, CCK-8 solution was added to each well, and incubation continued for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability (%) was calculated as [(Experimental group OD - Blank group OD) / (Negative control group OD - Blank group OD)] x 100%. The dose-response curve was fitted using GraphPad Prism 8.0 software, and the half-maximal inhibitory concentration (IC50) was calculated.

[0033] The results are as follows Figure 4 As shown, the DRP peptide has an effect on the IC50 of OVCAR-8 cells. 50 The M value was 3.90-5.94 μM. This result indicates that the DRP peptide provided by this invention exhibits significant inhibitory activity against the proliferation of OVCAR-8 ovarian cancer cells in vitro, demonstrating its potential as an anti-tumor lead compound.

[0034] Example 4: Detection of DRP peptide's in vitro anti-pancreatic cancer activity (CCK-8 assay) Human pancreatic cancer cells (Panc-02) in logarithmic growth phase were seeded at a density of 5 x 10^3 cells per well in 96-well plates. A blank control group (culture medium only), a negative control group (cells + culture medium), a positive control group containing the endogenous peptide LL-37 (concentration gradients: 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.391, 0.195 μM), and a DRP experimental group (concentration gradients: 0.195, 0.391, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 μM) were set up, with three replicates per group. After culturing for 24 hours, CCK-8 solution was added to each well, and incubation was continued for another 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability (%) was calculated as [(Experimental group OD - Blank group OD) / (Negative control group OD - Blank group OD)] x 100%. The dose-response curve was fitted using GraphPad Prism 8.0 software, and the half-maximal inhibitory concentration (IC50) was calculated.

[0035] The results are as follows Figure 5 As shown, the IC50 value of the DRP peptide against Panc-02 cells was 3.90-5.94 μM. This result indicates that the DRP peptide provided by this invention has significant inhibitory activity against the proliferation of pancreatic cancer Panc-02 cells in vitro, demonstrating its potential as an antitumor lead compound.

[0036] Example 5: Proteomics Analysis To elucidate the molecular mechanism underlying the regulatory effect of DRP peptides on CT26, OVCAR-8, and Panc-02 cells, and to identify potential protein targets or signaling pathways involved, this study employed liquid chromatography-tandem mass spectrometry combined with an independent data acquisition strategy for proteomics analysis, aiming to achieve high-depth and high-precision protein quantification and identification.

[0037] After harvesting, the cells were washed three times with phosphate-buffered saline. The cells were then lysed in lysis buffer to remove cell debris, and the supernatant was collected. Protein concentration was determined using a biuret protein assay kit.

[0038] During protein hydrolysis, 100 µg of extracted protein was taken, reduced with 8 µL of 100 mM dithiothreitol at 56 °C for 60 min, and then alkylated with 20 µL of 100 mM iodoacetamide at room temperature in the dark for 40 min. The protein solution was then loaded into a 10 kDa molecular weight cutoff filter membrane device and centrifuged.

[0039] Digestion was performed by adding trypsin and incubating overnight at 37°C. The resulting peptides were collected after centrifugation for 15 minutes; the membrane filter unit was further washed with ultrapure water and centrifuged again to maximize peptide recovery. The resulting peptide mixture was acidified with formic acid to terminate any potential residual enzyme activity and dried using a SpeedVac concentrator for subsequent mass spectrometry analysis.

[0040] Analysis was performed using an Orbitrap Eclipse Tribrid mass spectrometer via liquid chromatography-mass spectrometry (LC-MS / MS). After reconstitution of the dried peptides with loading buffer, they were loaded onto a chromatographic column for online desalting to eliminate interference from residual impurities. Chromatographic separation was performed using a 25 cm Acclaim PepMap column. TM A C18 analytical column (Thermo Fisher Scientific) was used with solvent B (80% acetonitrile, 0.1% formic acid) as the mobile phase, followed by linear gradient elution from 3% to 38% over 102 minutes, and then equilibration with 100% solvent B for 10 minutes to restore column performance. To improve chromatographic quality, the FAIMS compensation voltage (CV) was set to -45 V. Mass spectrometry data were acquired using Dynamic Ion Capture (DIA) mode, enabling comprehensive and unbiased detection of all peptides in the sample, achieving high-depth proteomic coverage. MS1 scans were performed in Orbitrap mode (resolution: 60,000; scan range: 350–1,250 m / z; automatic gain control target: standard; maximum IT: automatic; RF lens: 30%). MS2 scans were acquired in Orbitrap using a high-energy collisional dissociation (HCD) fragmentation mode (collision energy: 32%; isolation window: 9 m / z; AGC target: 2000%; maximum IT: auto; RF lens: 50%; data type: centroid).

[0041] Raw mass spectrometry data were processed using DIA-NN software (version 1.8.1). Library retrieval and label-free quantitative analysis were performed by comparing the data with the UniProt human proteome database. Search parameters included: N-terminal methionine cleavage as a variable modification and cysteine ​​carbamylation as a fixed modification, consistent with the previous reduction and alkylation steps. Peptide lengths were allowed to be set between 7 and 30 amino acids, and trypsin digestion allowed a maximum of one cleavage site deletion to balance identification coverage and confidence. False discovery rates for both precursor ions and protein levels were set to 1% to ensure high-quality identification results; other parameters remained at their default values.

[0042] The results are shown in the attached diagram in the instruction manual. Figure 6 As shown, DRP downregulated the key cytokine GTF2A2 and upregulated NAB2 in the three cell types CT26, OVCAR-8, and Panc-02, which helped to prevent cell division.

[0043] Example 6: Animal Model Validation To evaluate the in vivo antitumor efficacy and systemic safety of DRP, and to explore its efficacy in ovarian cancer, this study established a subcutaneous xenograft tumor model using the OVCAR-8 cancer cell line, providing direct evidence for the therapeutic potential of peptide compounds. Based on the characteristics of the OVCAR-8 cell line, the optimal mouse strain for tumor formation was selected: female BALB / c nude mice were used for the OVCAR-8 (ovarian cancer) model.

[0044] Cancer cells in the logarithmic growth phase were collected, counted, and resuspended in sterile PBS. During tumor inoculation, cells containing 1-5 × 10⁻⁵ cells were... 6 100 μL of cell suspension was subcutaneously injected into the right dorsal and ventral region of each mouse to establish a xenograft model. When the tumor volume reached 30-140 mm³ (a size that ensures stable tumor growth without excessive burden), the mice were randomly divided into two groups based on tumor volume and body weight to ensure homogeneous baseline characteristics: a solvent control group and a DRP treatment group. The sample size for each model was as follows: OVCAR-8 (n=5).

[0045] Mice in the treatment group received intravenous DRP (10 mg / kg), while the solvent control group received an equal volume of sterile PBS. Treatment was administered every other day for six consecutive days. Throughout the experiment, tumor size and mouse weight were monitored every other day. Tumor volume was measured using electronic calipers and calculated using the formula V = 0.5 × L × W² (where L is the tumor length (longest diameter) and W is the tumor width (shortest diameter perpendicular to the length)). Systemic toxicity of the peptide was assessed by recording changes in body weight. Mice were sacrificed at the end of the treatment period, tumors were excised, and mice were weighed.

[0046] The results are shown in the attached diagram in the instruction manual. Figure 7, Figure 8 , Figure 9 As shown, after administration, DRP exhibited significant anti-solid tumor activity in a mouse model, inhibiting the growth of solid tumor volume and reducing the endpoint tumor weight. Furthermore, throughout the entire treatment period, as... Figure 7 As shown, the weight change curves of mice in the treatment group and the solvent control group basically overlapped, with no statistically significant difference (p>0.05), indicating that DRP did not cause significant systemic toxicity at effective doses and had good safety. In summary, DRP has broad-spectrum antitumor activity.

[0047] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A polypeptide for the treatment or prevention of malignant solid tumors, characterized in that, The amino acid sequence of the polypeptide is FLFFASIISLIFYYSKKGRRSYK-NH2.

2. Use of the polypeptide of claim 1 in the preparation of a medicament for the treatment or prevention of malignant solid tumors.

3. The use according to claim 2, characterized in that, The solid tumors include, but are not limited to, lung cancer, liver cancer, breast cancer, colorectal cancer, and pancreatic cancer.

4. The use according to claim 2, characterized in that, The drug comprises a pharmaceutically acceptable salt, ester, amide, or cyclized form of the polypeptide.

5. The use according to claim 2 or 3, characterized in that, The drug is intended for use in combination with at least one other therapeutic agent selected from chemotherapy drugs, targeted therapies, immunomodulators, glucocorticoids, or any combination thereof.

Images