A VEGF / TIE-2 dual targeting polypeptide and its use in the preparation of anti-tumor drugs

Abstract

The application discloses a VEGF / TIE-2 double-targeting polypeptide and application thereof in preparation of an antitumor drug, and relates to the technical field of biological medicine.The VEGF / TIE-2 double-targeting polypeptide (named as RTP) provided by the application is composed of an amino acid sequence capable of target-recognizing VEGF and TIE-2, an amino acid sequence capable of self-assembling to form a beta-sheet nanofiber, an amino acid sequence capable of target-recognizing carbonic anhydrase IX and an enzyme-reactive amino acid sequence capable of being specifically cut by MMP-2.RTP can occur conformational change and self-assemble in tumor cells to form stable water-insoluble nanofibers, and can strongly inhibit tumor neovascularization and metastasis through the VEGF and TIE-2 double-signal pathways and inhibit the progress of the tumor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a VEGF / TIE-2 dual targeting polypeptide and application thereof in preparation of an anti-tumor drug. BACKGROUND

[0002] Renal cell carcinoma (RCC), also known as kidney cancer, is the third most common malignant tumor of the urinary system worldwide. A key pathological feature of renal cancer is the abnormal vasculature that promotes metastasis and tumor growth. Tumor blood vessels not only provide nutrients and oxygen required for rapid development of cancer cells, but also immature structure of tumor neovasculature, loose connection between endothelial cells, increased permeability of them, and promoted cancer metastasis. Metastatic renal cancer is also a highly vascularized solid malignancy, and conventional chemotherapy and radiotherapy are ineffective for RCC because of its high vascularization. Targeted therapies against vascular endothelial growth factor (VEGF) including antibodies (bevacizumab) and other targeted therapeutic modalities such as tyrosine kinase inhibitors (TKI) also cause tumor cell death and are the standard first-line treatment for metastatic clear cell renal carcinoma (mRCC). In fact, although bevacizumab has been approved by the FDA for the treatment of RCC, bevacizumab only shows marginal improvement in median progression-free survival (PFS) and has no statistically significant advantage in overall survival. The frequency of adverse reactions induced by TKI is reported to be increasing. Although TKI is known to involve multiple organs of the body, including the lungs, liver, gastrointestinal tract, kidneys, thyroid, blood, and skin, cardiac involvement is one of the most serious complications, and combination therapy (sunitinib plus bevacizumab) can cause severe cardiotoxicity, including hypertension and microvascular hemolytic anemia. In general, the prognosis of patients with locally advanced or metastatic RCC is poor. Therefore, there is a need for a longer-term, safer combination inhibition strategy to more effectively inhibit blood vessels in patients with renal cancer and improve the prognosis of patients with renal cancer. SUMMARY

[0003] The purpose of the present application is to provide a VEGF / TIE-2 dual targeting polypeptide and application thereof in preparation of an anti-tumor drug, in order to solve the problems existing in the prior art. The VEGF / TIE-2 dual targeting polypeptide strongly inhibits tumor neovascularization and metastasis through VEGF and TIE-2 dual signaling pathways, and inhibits the progression of renal cancer, which has important clinical application potential.

[0004] Among all epithelial cancers, clear cell renal cell carcinoma (ccRCC) has the highest expression of VEGFA, which is one of the reasons for targeting VEGFA and its receptors (especially VEGFR2) in this disease. Angiogenesis induced by VEGF (vascular endothelial growth factor) plays a key role in tumor growth and metastasis. Angiopoietin 2 (ANG2) is a cooperative driver of angiogenic factors and vascular instability. The ANG2 / TIE pathway controls vascular permeability under pathological conditions and is therefore a major regulator of vascular stability. One proposed VEGF-targeted resistance mechanism suggests that escape mechanisms induce or upregulate other pro-angiogenic factors at the transcriptional and translational levels, and studies have shown that both ANG2 and TIE-2 are upregulated in tumors resistant to VEGFR2-targeted drugs, indicating that ANG2 / TIE-2 signaling maintains resistance to VEGF / VEGFR2 inhibitors. It has been reported that when both ANG2 and VEGFA are blocked, the normalization level is improved by reducing the proportion of unstable blood vessels that initiate angiogenesis. In addition, studies have shown that anti-angiogenic therapy against VEGF has an Akt-dependent direct anti-tumor effect on human cancer cells, including renal cancer, by reducing the viability and proliferation of tumor cells. Therefore, there is an urgent need for dual inhibition of renal tumor angiogenic factors (VEGFA and TIE-2) to prevent the progression and metastasis of renal cancer.

[0005] Based on this, the present application provides the following solutions:

[0006] The present application provides a VEGF / TIE-2 dual targeting polypeptide, the structural formula of which is as follows:

[0007]

[0008] The present application also provides the use of the above-mentioned VEGF / TIE-2 dual targeting polypeptide in the preparation of an anti-tumor drug.

[0009] Further, the anti-tumor drug exerts an anti-tumor effect by inhibiting the formation of tumor neovasculature and metastasis.

[0010] Further, the tumor is a tumor with high expression of VEGF protein.

[0011] Further, the tumor with high expression of VEGF protein is renal cancer, prostate cancer, glioblastoma or head and neck squamous cell carcinoma.

[0012] The present application also provides an anti-tumor drug, the active ingredient of which comprises the above-mentioned VEGF / TIE-2 dual targeting polypeptide.

[0013] Further, the anti-tumor drug further comprises a pharmaceutically acceptable excipient.

[0014] Further, the dosage form of the antitumor drug is a tablet, granules, powder, capsule, pill or injection.

[0015] The application also provides a preparation method of the VEGF / TIE-2 dual-targeting polypeptide, comprising the step of preparing the VEGF / TIE-2 dual-targeting polypeptide by solid-phase synthesis.

[0016] The application discloses the following technical effects:

[0017] The VEGF / TIE-2 dual-targeting polypeptide (named RTP) provided by the application is composed of an amino acid sequence capable of recognizing VEGF and TIE-2, an amino acid sequence capable of self-assembling to form a beta-sheet nanofiber, an amino acid sequence capable of recognizing carbonic anhydrase IX and an enzyme-reactive amino acid sequence capable of being specifically cleaved by MMP-2. The RTP can undergo conformational change and self-assemble in tumor cells to form stable water-insoluble nanofibers, thereby strongly inhibiting tumor neovascularization and metastasis through the VEGF and TIE-2 dual signaling pathways and inhibiting the progression of renal cancer.

[0018] In addition, the application focuses on the practical problems of clinical transformation application, the VEGF / TIE-2 dual-targeting polypeptide RTP is designed and constructed by using a polypeptide with high biological safety as a material, and therefore, the RTP has high biological safety and great potential for clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0020] Figure 1 Fig. 1 is a molecular structure formula of the polypeptide RTP and a control polypeptide RTP-C; a is a molecular structure formula of the RTP, and b is a molecular structure formula of the RTP-C;

[0021] Figure 2 Fig. 2 is a transmission electron microscope observation diagram of the polypeptide RTP and the RTP-C polypeptide after being respectively incubated with an MMP-2 protein solution; the scale is 200 nm;

[0022] Figure 3Figures of thioflavin T (ThT) fluorescence detection results of polypeptides RTP and RTP-C after being added to 786-O cell culture solution for co-incubation; wherein, a is the ThT fluorescence detection result of RTP after being added to 786-O culture solution for 24 h; b is the ThT fluorescence detection result of RTP after being added with MMP-2 protein for 24 h; RTP and RTP-C are compared, *** P<0.001;

[0023] Figure 4 Figures of detection results of killing effects of PBS, RTP and RTP-C on 786-O cells;

[0024] Figure 5 Figures of detection results of killing effects of PBS, RTP and RTP-C on HK-2 cells;

[0025] Figure 6 Figures of detection results of killing effects of PBS, RTP and RTP-C on HUVEC cells;

[0026] Figure 7 Figures of detection results of liver functions (ALT, AST and ALP) of mice under the effects of PBS, RTP and RTP-C;

[0027] Figure 8 Figures of detection results of blood urea nitrogen (BUN) of mice under the effects of PBS, RTP and RTP-C;

[0028] Figure 9 Figures of detection results of total protein (TP) and urine albumin (ALB) of mice under the effects of PBS, RTP and RTP-C;

[0029] Figure 10 Figures of detection results of creatinine (CR) of mice under the effects of PBS, RTP and RTP-C;

[0030] Figure 11 Figures of H&E staining of organs of mice under the effects of PBS, RTP and RTP-C;

[0031] Figure 12 Figures of detection results of targeting and retention effects of RTP and RTP-C on 786-O and HUVEC cells; wherein, a is a figure of detection results of targeting and retention of RTP and RTP-C on HUVEC cell clusters; b is a figure of detection results of targeting and retention of RTP and RTP-C on 786-O cell clusters; the scales are all 50 μm;

[0032] Figure 13Figure for the detection results of the influence of PBS, RTP and RTP-C on the invasion ability of 786-O and HUVEC cells; wherein, a is the staining diagram of HUVEC cells invaded; b is the quantitative analysis diagram of a; c is the staining diagram of 786-O cells invaded; d is the quantitative analysis diagram of c; RTP and RTP-C are compared, *** P<0.001;

[0033] Figure 14 Figure for the detection results of the apoptosis of 786-O cells under the action of PBS, RTP and RTP-C; wherein, a is the flow cytometry detection diagram; b is the quantitative analysis diagram of a;

[0034] Figure 15 Figure for the detection results of the fluorescence imaging (a) and quantitative analysis diagram (b) of mice under the action of PBS, RTP and RTP-C;

[0035] Figure 16 Figure for the detection results of the tumor progression inhibition of RTP and RTP-C in vivo; wherein, a is the statistical diagram of the tumor volume of mice; b is the statistical diagram of the tumor weight of mice; c is the observation diagram of tumor lung metastasis; d is the statistical diagram of c. DETAILED DESCRIPTION

[0036] The various illustrative embodiments of the present application will now be described in detail in connection with the following figures. This description is made for the purpose of demonstrating and describing particular embodiments of the present application and is not intended to limit the scope of the present application in any way. It is understood that the present application is not limited to the particular embodiments described and illustrated herein, and that the scope of the present application includes any modifications and variations within the scope of the present application.

[0037] It is understood that the terms used herein are merely for the purpose of describing particular embodiments of the present application and are not intended to limit the present application in any way. In addition, for numerical ranges in the present application, it is understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value between any stated value or stated range, and any other stated value or intermediate value in the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described herein. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0039] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the teachings of the description of the application, which are within the scope of the present application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples of the application are exemplary only.

[0040] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0041] Preparation and molecular structure of tubulin targeting polypeptide

[0042] 1. Preparation of VEGF / TIE-2 dual targeting polypeptide RTP [WHKPFRF-K(SAAMLLN)LVFF-PEG4-PLGVR-YNTNHVPLSPKY]

[0043] The VEGF / TIE-2 dual targeting polypeptide RTP (structural formula see Figure 1 a) in the specification is synthesized by solid phase synthesis.

[0044] The RTP polypeptide can recognize and bind to VEGF protein, which is composed of the following four parts:

[0045] 1) Amino acid sequence YNTNHVPLSPKY for targeting recognition of carbonic anhydrase IX; it can achieve micro carbonic anhydrase IX targeting recognition, which acts as a target head in RTP and specifically binds to the overexpressed CAIX protein in renal cancer cells;

[0046] 2) Enzyme reactive peptide that can be specifically cleaved by MMP-2, with an amino acid sequence of PLGVR;

[0047] 3) Amino acid sequence KLVFF that can self-assemble into β-sheet nanofibers; it is derived from the KLVFF peptide sequence in β-amyloid protein, and can self-assemble into water-insoluble nanofibers with β-sheet secondary structure due to hydrogen bond interaction;

[0048] 4) Amino acid sequence for targeting recognition of VEGF and TIE-2, with amino acid sequences of WHKPFRF and NLLMAAS, respectively, which form nanofibers by targeting recognition of VEGF or TIE-2 to remain in renal cancer tissue for a long time.

[0049] 2. Preparation of polypeptide RTP-C [WHKPFRF-K(SAAMLLN)AAGG-PEG4-PLGVR-YNTNHVPLSPKY]

[0050] The polypeptide RTP-C (structural formula see Figure 1 b) in the specification is synthesized by solid phase synthesis, which includes the following four parts:

[0051] (1) carbonic anhydrase IX (CAIX) binding motif, the sequence of which is YNTNHVPLSPKY;

[0052] (2) enzyme reactive peptide that can be specifically cleaved by MMP-2, the sequence of which is PLGVR;

[0053] (3) amino acid sequence that cannot undergo self-assembly deformation, the sequence of which is KAAGG;

[0054] (4) amino acid sequences that target VEGF and TIE-2, the sequences of which are WHKPFRF and NLLMAAS, respectively.

[0055] Example 2 Conformational change and self-assembly of polypeptide RTP into water-insoluble nanofibers after incubation with MMP-2 protein solution

[0056] Results are shown in Table 1. Figure 2 From the results, it can be seen that polypeptide RTP can undergo conformational change and self-assembly to form hydrophobic nanofibers after incubation with MMP-2 protein solution.

[0057] Example 3

[0058] RCC cell line (786-O), vascular endothelial cell line (HUVEC) and normal control human renal tubular epithelial cells (HK-2) were selected for the experiment. Polypeptides RTP and RTP-C were dissolved in DMSO solvent respectively to prepare polypeptide nanomaterial solution with a solution concentration of 20 μM. The experimental cells in good condition in logarithmic growth were randomly divided into RTP, RTP-C and PBS groups (among them, the PBS group is the solvent control group), and 100 μL of RTP solution, RTP-C solution and solvent were added to the culture medium to verify the effects of RTP, RTP-C and solvent on cell survival state, respectively.

[0059] 1. Thioflavin T (ThT) fluorescence detection of polypeptide RTP and RTP-C after being added to cell culture solution and incubated

[0060] 1 × 10 4 cells were cultured in a 96-well plate for 24 h, 100 μL of RTP and RTP-C with a concentration of 20 μM were added to the cell culture solution, and after incubation at 37°C, 5% CO2 for 24 h, 50 μM ThT solution was added for further incubation for 30 min, and the fluorescence intensity was measured using a fluorescence microplate reader.

[0061] Results are shown in Table 1. Figure 3As shown in the results, RTP and MMP-2 were co-incubated, and RTP occurred conformational change and produced β-sheet structure, and ThT reagent combined with β-sheet structure to emit fluorescence, while RTP-C did not occur conformational change and self-assembly behavior.

[0062] 2. Killing effect of polypeptide RTP on 786-O, HUVEC and HK-2 cells and biological safety detection

[0063] The 786-O, HUVEC and HK-2 cells in a good state of logarithmic growth were added to a 96-well plate at 1×10 4 cells per well, a total volume of 100 μL, and placed in a 37°C cell incubator. After 24 h, they were randomly divided into RTP and RTP-C groups, and the RTP and RTP-C solutions were added to the culture medium at concentrations of 5, 10, 20, 50, 100 and 200 μM, respectively, and placed in a 37°C cell incubator. After 24 h, the culture medium was discarded, and the prepared CCK-8 solution was added, and placed in a 37°C cell incubator for 4 h. The absorbance was measured to verify the effect of RTP and RTP-C on the survival state of the cells.

[0064] Three groups of mice were given RTP, RTP-C and PBS (200×10 -6 M, 200 μL) respectively, once every 48 h, intravenously administered for 5 times. On the 28th day after the first administration, the blood of the mice was drawn for liver and kidney function tests, and the organs were taken for H&E examination.

[0065] The results are shown in Figures 4-11 From the results, it can be seen that RTP had a killing effect on 786-O and HUVEC cells at a concentration of 20 μM, but had no killing effect on HK-2 cells. RTP-C (20 μM) had no killing effect on 786-O, HUVEC and HK-2 cells. It was indicated that the proliferation ability of 786-O and HUVEC cells was significantly inhibited by polypeptide RTP (20 μM). The liver function (ALT, AST, ALP), kidney function (BUN, TP, ALB, CR) and H&E staining examination results showed that there was no significant difference among the three groups of mice, indicating that RTP and RTP-C had biological safety.

[0066] Example 4 Targeting effect and retention of polypeptide RTP on 786-O and HUVEC cells

[0067] To verify the interaction between the nanofiber and the VEGF protein, the present application first constructs a multi-cell spheroid (MCS) using an agar-liquid overlay method. A 2% agarose gel solution is placed on a 96-well plate to prevent cell adhesion, and HK-2 HUVEC or 786-O cells suspended in culture medium are seeded in the 96-well plate to establish multi-cell spheroids (MCS) of 786-O, HUVEC, and HK-2 cells to fully simulate the tumor microenvironment, and then RTP and RTP-C labeled with cyanine 7 dye (Cy7) are added to the MCS, respectively, and fluorescence images are captured at 1, 24, 48, 72, and 96 h using a confocal laser scanning microscope (CLSM).

[0068] The results are shown in Figure 12 From the results, it can be seen that RTP can be retained in 786-O and HUVEC cells for a long time.

[0069] Example 5 Inhibitory effect of polypeptide RTP on the invasion ability of 786-O and HUVEC cells

[0070] In the invasion experiment, Matrigel (BD Biosciences, New Jersey, USA) was laid on the upper chamber of a Transwell chamber (pore size 8 μm, polycarbonate filter, diameter 6.5 mm; Corning) to verify the effect of RTP on the invasion ability of cells. Well-grown 786-O and HUVEC cells in the logarithmic phase were seeded in the upper chamber of the Transwell at a cell density of 10 5 cells per well in 100 μL of a solution containing 20 μM of RTP, RTP-C, and PBS, and 750 μL of 1640 complete medium was added to the lower chamber, and after incubation at 37°C in a cell incubator for 48 h, the invaded cells in the lower layer were stained and counted.

[0071] The results are shown in Figure 13 The invasion ability of 786-O and HUVEC cells was inhibited to 17.22% ± 0.86% and 23.52 ± 1.76%, respectively, indicating that RTP significantly inhibited the invasion ability of 786-O and HUVEC cells.

[0072] Example 6 Promoting effect of polypeptide RTP on apoptosis of renal cancer 786-O cells

[0073] Well-grown renal cancer 786-O cells in the logarithmic phase were incubated with 20 μM of RTP or RTP-C at 37°C and 5% CO2 for 24 h, and then the cells were stained according to the instructions of the cell apoptosis detection kit (Bi Yun Tian, China), and the flow cytometer was used to detect the apoptosis of the cells.

[0074] The results are shown in Figure 14As shown in the results, apoptosis in renal cell carcinoma 786-O cells increased nearly 6-fold, and RTP significantly promoted apoptosis in renal cell carcinoma 786-O cells.

[0075] Example 7: Distribution of peptide RTP in mice

[0076] This experiment used Balb / c nude mice, and renal cell carcinoma cells were inoculated into the right buttock of the mice. When the tumor volume reached 50 mm... 3 Fluorescently labeled RTP and RTP-C (200×10⁻⁶) were administered intravenously. -6 M (200 μL) solution. At 1, 4, 12, 24, 48, and 72 h after injection, fluorescence imaging in mice was detected and quantitatively analyzed using a multispectral fluorescence small animal in vivo imaging system.

[0077] The results are as follows Figure 15 As shown in the results, RTP is mainly distributed in the tumor site, indicating that it has good targeting properties.

[0078] Example 8: In vivo inhibition of tumor progression by peptides RTP and RTP-C

[0079] This experiment used Balb / c nude mice, and renal cell carcinoma cells were inoculated into the right buttock of the mice. When the tumor volume reached 50 mm... 3 RTP or RTP-C (200×10) is administered intravenously every 2 days. -6 M (200 μL) solution was administered for 10 days. During the experiment, tumor volume and mouse weight were measured periodically.

[0080] The results are as follows Figure 16 As shown in the results, the peptide RTP significantly inhibited tumor proliferation and suppressed lung metastasis in mice.

[0081] In summary, the peptide RTP provided by this invention can specifically recognize CAIX overexpressed in renal cell carcinoma cells, and then be cleaved by MMP-2 in the tumor microenvironment. The probe residues then form nanofibers by targeting and recognizing VEGFA or TIE-2, which can be retained in renal cell carcinoma tissue for a long time. It strongly inhibits tumor angiogenesis and metastasis through the dual signaling pathways of VEGF and TIE-2, and inhibits the progression of renal cell carcinoma cells.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A VEGF / TIE-2 dual-targeting polypeptide, characterized in that, The structural formula is as follows: 。 2. The application of the VEGF / TIE-2 dual-targeting polypeptide as described in claim 1 in the preparation of antitumor drugs, characterized in that, The tumor is a tumor that highly expresses VEGF protein; The tumors that highly express VEGF protein are renal cell carcinoma, prostate cancer, glioblastoma, or head and neck squamous cell carcinoma.

3. An antitumor drug, characterized in that, The active ingredient includes the VEGF / TIE-2 dual-targeting polypeptide as described in claim 1.

4. The antitumor drug according to claim 3, characterized in that, The antitumor drugs also include pharmaceutically acceptable excipients.

5. The antitumor drug according to claim 3, characterized in that, The dosage form of the antitumor drug is tablets, granules, powders, capsules, pills, or injections.

6. A method for preparing the VEGF / TIE-2 dual-targeting polypeptide as described in claim 1, characterized in that, The process includes the step of preparing the VEGF / TIE-2 dual-targeting polypeptide using a solid-phase synthesis method.

Images