VEGF / TIE-2 dual-targeting polypeptide and application thereof in preparation of antitumor drugs

Abstract

The invention discloses a VEGF / TIE-2 dual-targeting polypeptide and application thereof in preparation of antitumor drugs, and relates to the technical field of biological medicines. The VEGF / TIE-2 dual-targeting polypeptide (named as RTP) provided by the invention is formed by combining an amino acid sequence capable of identifying VEGF and TIE-2 in a targeting manner, an amino acid sequence capable of forming beta-sheet nanofibers through self-assembly, an amino acid sequence capable of identifying carbonic anhydrase IX in a targeting manner and an enzyme reactive amino acid sequence capable of realizing MMP-2 specific shearing. The RTP can be subjected to allosteric and self-assembly in tumor cells to form stable water-insoluble nanofibers, the formation and metastasis of tumor neovascularization are strongly inhibited through VEGF and TIE-2 double signal channels, and the tumor progress is inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a VEGF / TIE-2 dual-targeting polypeptide and an application thereof in the preparation of an anti-tumor drug. Background Art

[0002] Renal cell carcinoma (RCC), or kidney cancer for short, is the third most common malignant tumor of the urinary system worldwide. A key pathological feature of renal cancer is that the abnormal vasculature promotes metastasis and tumor growth. Tumor blood vessels not only provide the nutrients and oxygen required for the rapid development of cancer cells, but also the tumor neovascularization structure is immature, and the connection between endothelial cells is loose, which increases their permeability and promotes cancer metastasis. Metastatic renal cancer is also a highly vascularized solid malignancy. Conventional chemotherapy and radiotherapy are ineffective for RCC because it is highly vascularized. Treatment targeting vascular endothelial growth factor (VEGF) includes antibodies (bevacizumab) and other targeted therapeutic modalities, such as tyrosine kinase inhibitors (TKI), which also cause tumor cell death and are the first-line standard treatment for metastatic clear cell renal carcinoma (mRCC). In fact, although bevacizumab has been approved by the FDA for the treatment of RCC, bevacizumab has only shown marginal improvement in median progression-free survival (PFS) and no statistically significant advantage in overall survival. TKI-induced adverse reactions have been reported to be increasingly frequent. Although TKIs are known to affect multiple organs in the body, including the lungs, liver, gastrointestinal tract, kidneys, thyroid, blood, and skin, cardiac involvement is some of the most serious complications, and combination therapy (sunitinib plus bevacizumab) can cause severe cardiovascular toxicity, including hypertension and microangiopathic hemolytic anemia. In general, patients with locally advanced or metastatic RCC have a poor prognosis. Therefore, longer-term and safer combined inhibition strategies are needed to more effectively inhibit blood vessels in patients with renal cancer and improve the prognosis of patients with renal cancer. Summary of the invention

[0003] The purpose of the present invention is to provide a VEGF / TIE-2 dual-targeting polypeptide and its use in the preparation of anti-tumor drugs to solve the problems of the above-mentioned prior art. The VEGF / TIE-2 dual-targeting polypeptide strongly inhibits tumor angiogenesis and metastasis through the VEGF and TIE-2 dual signaling pathways, and inhibits the progression of renal cancer, and has important clinical application potential.

[0004] Clear cell renal cell carcinoma (ccRCC) has the highest expression of VEGFA among all epithelial cancers, which is one of the rationales for targeting VEGFA and its receptors, particularly 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 mechanism of resistance to VEGF targeting suggests that escape mechanisms induce or upregulate other proangiogenic 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, suggesting that ANG2 / TIE-2 signaling maintains resistance to VEGF / VEGFR2 inhibitors. It has been reported that when both ANG2 and VEGFA are blocked, normalization levels are increased by reducing the proportion of unstable vessels that initiate angiogenesis. In addition, studies have shown that antiangiogenic therapy targeting VEGF has Akt-dependent direct antitumor effects on human cancer cells, including renal cancer, by reducing tumor cell viability and proliferation. Therefore, dual inhibition of renal tumor angiogenic factors (VEGFA and TIE-2) is urgently needed to prevent the progression and metastasis of renal cancer.

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

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

[0007]

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

[0009] Furthermore, the anti-tumor drug exerts its anti-tumor effect by inhibiting tumor angiogenesis and metastasis.

[0010] Furthermore, the tumor is a tumor that highly expresses VEGF protein.

[0011] Furthermore, the tumor that highly expresses VEGF protein is renal cancer, prostate cancer, glioblastoma or head and neck squamous cell carcinoma.

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

[0013] Furthermore, the anti-tumor drug also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the dosage form of the anti-tumor drug is tablets, granules, powders, capsules, pills or injections.

[0015] The present invention also provides a method for preparing the above-mentioned VEGF / TIE-2 dual-targeting polypeptide, comprising the step of preparing the VEGF / TIE-2 dual-targeting polypeptide by solid phase synthesis.

[0016] The present invention discloses the following technical effects:

[0017] The VEGF / TIE-2 dual-targeting polypeptide (named RTP) provided by the present invention is composed of an amino acid sequence that can target and recognize VEGF and TIE-2, an amino acid sequence that can self-assemble to form β-sheet nanofibers, an amino acid sequence that can target and recognize carbonic anhydrase IX, and an enzyme-reactive amino acid sequence that specifically cleaves MMP-2. RTP can undergo conformational changes and self-assemble in tumor cells to form stable water-insoluble nanofibers, strongly inhibit tumor angiogenesis and metastasis through the VEGF and TIE-2 dual signal pathways, and inhibit the progression of renal cancer.

[0018] In addition, the present invention focuses on practical issues of clinical transformation and application. The VEGF / TIE-2 dual-targeting peptide RTP is designed and constructed using a peptide with high biosafety as a material. Therefore, RTP has strong biosafety and great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 are the molecular structural formulas of polypeptide RTP and control polypeptide RTP-C; a is the molecular structural formula diagram of RTP, and b is the molecular structural formula diagram of RTP-C;

[0021] Figure 2 Transmission electron microscopy observations of the RTP and RTP-C polypeptides after co-incubation with the MMP-2 protein solution; scale: 200 nm;

[0022] Figure 3The results of thioflavin T (ThT) fluorescence detection after the peptides RTP and RTP-C were added to the 786-O cell culture medium and co-incubated; a is the result of ThT fluorescence detection 24 hours after RTP was added to the 786-O culture medium; b is the result of ThT fluorescence detection 24 hours after RTP was added to the MMP-2 protein; RTP is compared with RTP-C, *** P < 0.001;

[0023] Figure 4 The figure is the test result of the killing effect of PBS, RTP and RTP-C on 786-O cells;

[0024] Figure 5 The figure is the test result of the killing effect of PBS, RTP and RTP-C on HK-2 cells;

[0025] Figure 6 The figure is the test result of the killing effect of PBS, RTP and RTP-C on HUVEC cells;

[0026] Figure 7 The graph shows the test results of mouse liver function (ALT, AST, ALP) under the action of PBS, RTP and RTP-C;

[0027] Figure 8 The figure is the test result of mouse urea nitrogen (BUN) under the action of PBS, RTP and RTP-C;

[0028] Fig. 9 The figure shows the test results of mouse total protein (TP) and urine albumin (ALB) under the action of PBS, RTP and RTP-C;

[0029] Fig.10 The figure is the test result of mouse creatinine (CR) under the action of PBS, RTP and RTP-C;

[0030] Fig.11 H&E staining images of mouse organs under the action of PBS, RTP and RTP-C;

[0031] Fig.12 The results of the detection of the targeting and retention effects of RTP and RTP-C on 786-O and HUVEC cells; a is the detection result of the targeting and retention of HUVEC cell clusters by RTP and RTP-C; b is the detection result of the targeting and retention of 786-O cell clusters by RTP and RTP-C; the scale bars are all 50 μm;

[0032] Fig.13Figure 2 is the test result of the effect of PBS, RTP and RTP-C on the invasion ability of 786-O and HUVEC cells; a is the staining picture of HUVEC invaded cells; b is the quantitative analysis picture of a; c is the staining picture of 786-O invaded cells; d is the quantitative analysis picture of c; RTP is compared with RTP-C, *** P < 0.001;

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

[0034] Fig.15 Fluorescence imaging (a) and quantitative analysis (b) of mice under the action of PBS, RTP and RTP-C;

[0035] Fig.16 The graphs are the detection results of RTP and RTP-C inhibiting tumor progression in vivo; wherein, a is the statistical graph of mouse tumor volume; b is the statistical graph of mouse tumor weight; c is the observation graph of tumor lung metastasis; d is the statistical graph of c. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1 Preparation and Molecular Structure of Tubulin Targeting Polypeptide

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

[0043] The VEGF / TIE-2 dual-targeting peptide RTP (structural formula see Figure 1 a).

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

[0045] 1) Targeted recognition of the amino acid sequence YNTNHVPLSPKY of carbonic anhydrase IX; micro carbonic anhydrase IX targeted recognition can be achieved, which acts as a target in RTP and specifically binds to the CAIX protein overexpressed in renal cancer cells;

[0046] 2) an enzyme-reactive peptide that can be specifically cleaved by MMP-2, whose amino acid sequence is PLGVR;

[0047] 3) The amino acid sequence KLVFF that can self-assemble into β-sheet nanofibers; the KLVFF peptide sequence derived from β-amyloid protein can self-assemble into water-insoluble nanofibers with β-sheet secondary structure due to hydrogen bond interactions;

[0048] 4) Targeted recognition of the amino acid sequences of VEGF and TIE-2, whose amino acid sequences are WHKPFRF and NLLMAAS, respectively, forms nanofibers by targeted recognition of VEGF or TIE-2 to be retained in renal cancer tissues for a long time.

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

[0050] The peptide RTP-C (structural formula see Figure 1 b), which includes the following four parts:

[0051] (1) Carbonic anhydrase IX (CAIX) binding motif, whose sequence is YNTNHVPLSPKY;

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

[0053] (3) The amino acid sequence that cannot undergo self-assembly deformation is KAAGG;

[0054] (4) Targeted recognition of the amino acid sequences of VEGF and TIE-2, whose sequences are WHKPFRF and NLLMAAS, respectively.

[0055] Example 2 After incubation with MMP-2 protein solution, polypeptide RTP undergoes conformational change and self-assembles into water-insoluble nanofibers. After polypeptides RTP and RTP-C were incubated with MMP-2 protein solution respectively, the solution samples were observed using a transmission electron microscope.

[0056] The results are as follows Figure 2 As shown, it can be seen from the results that the polypeptide RTP can be conformed and self-assembled to form hydrophobic nanofibers after co-incubation with the 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. The peptides RTP and RTP-C were dissolved in DMSO solvent respectively to prepare a peptide nanomaterial solution with a solution concentration of 20μM. Experimental cells in good logarithmic growth were randomly divided into RTP, RTP-C and PBS groups (where the PBS group was the solvent control group), and 100μL 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 the survival state of cells.

[0059] 1. Fluorescence detection of thioflavin T (ThT) after RTP polypeptide and RTP-C polypeptide were added to cell culture medium and incubated

[0060] 1×10 4 The cells were cultured in a 96-well plate for 24 h, and 100 μL of 20 μM RTP and RTP-C were added to the cell culture medium at 37°C and 5% CO 2 After 24 h of co-incubation, 50 μM ThT solution was added and incubated for another 30 min, and the fluorescence intensity was measured using a fluorescence microplate reader.

[0061] The results are as follows Figure 3As shown, it can be seen from the results that after co-incubation with MMP-2, RTP undergoes allosteric behavior and produces a β-sheet structure. The ThT reagent emits fluorescence after binding to the β-sheet structure, while RTP-C does not undergo allosteric and self-assembly behavior.

[0062] 2. Killing effect of peptide RTP on 786-O, HUVEC and HK-2 cells and biosafety detection

[0063] 786-O, HUVEC and HK-2 cells in good logarithmic growth were used at 1×10 4 Cells and a total volume of 100 μL were added to a 96-well plate and placed in a 37°C cell culture incubator. After 24 hours, they were randomly divided into RTP and RTP-C groups. 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 culture incubator. After 24 hours, the culture medium was discarded and the prepared CCK-8 solution was added. The plate was placed in a 37°C cell culture incubator for 4 hours, and the absorbance was measured to verify the effects of RTP and RTP-C on the survival status of cells.

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

[0065] The results are as follows Figure 4-Figure 11 As shown in the figure, it can be seen from the results that RTP has a killing effect on 786-O and HUVEC cells at a concentration of 20μM, but has no killing effect on HK-2 cells; RTP-C (20μM) has no killing effect on 786-O, HUVEC and HK-2 cells. It shows that the proliferation ability of 786-O and HUVEC cells is significantly inhibited by the peptide RTP (20μM). The results of liver function (ALT, AST, ALP), renal function (BUN, TP, ALB, CR) and H&E staining examinations showed that there was no significant difference between the three groups of mice, indicating that RTP and RTP-C are biosafe.

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

[0067] To verify the interaction between nanofibers and VEGF protein, the present invention first used agar-liquid superposition method to construct multicellular spheroids (MCS). 2% agarose gel solution was placed on a 96-well plate to prevent cell adhesion, HK-2HUVEC or 786-O cells suspended in culture medium were seeded in 96-well plates, and multicellular spheroids (MCS) of 786-O, HUVEC and HK-2 cells were established to fully simulate the tumor microenvironment, and then RTP and RTP-C labeled with cyanine 7 dye (Cy7) were added to MCS respectively, and fluorescence images were captured using confocal laser scanning microscopy (CLSM) at 1, 24, 48, 72 and 96h.

[0068] The results are as follows Fig.12 As shown, the results show 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 assay, Matrigel (BD Biosciences, New Jersey, USA) was placed in 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 cell invasion ability. 786-O and HUVEC cells in good logarithmic growth were cultured at 10 cells / well. 5 The cells were inoculated at a density of 100 μL in the upper chamber of the Transwell containing 100 μL of 20 μM RTP, RTP-C and PBS solution, and 750 μL of 1640 complete culture medium was added to the lower chamber. After culturing in a cell culture incubator at 37°C for 48 h, the invaded cells in the lower layer were stained and counted.

[0071] The results are as follows Fig.13 As shown, 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 The effect of polypeptide RTP on the apoptosis of renal cancer 786-O cells

[0073] Renal cancer 786-O cells in good logarithmic growth were incubated with 20 μM RTP or RTP-C at 37°C and 5% CO 2 After 24 h of co-incubation, the cells were stained according to the instructions of the cell apoptosis detection kit (Biyuntian, China), and cell apoptosis was detected using flow cytometry.

[0074] The results are as follows Fig.14As shown, the results show that the apoptosis of renal cancer 786-O cells increased nearly 6 times, and RTP significantly promoted the apoptosis of renal cancer 786-O cells.

[0075] Example 7 Distribution of polypeptide RTP in mice

[0076] This experiment used Balb / c nude mice, and renal cancer cells were inoculated into the right buttocks of the mice. 3 , fluorescently labeled RTP and RTP-C (200×10 -6 M, 200 μL) solution. At 1, 4, 12, 24, 48, and 72 h after injection, the fluorescence imaging of mice was detected and quantitatively analyzed using a multi-spectral fluorescence small animal in vivo imaging system.

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

[0078] Example 8 Peptides RTP and RTP-C inhibit tumor progression in vivo

[0079] This experiment used Balb / c nude mice, and renal cancer cells were inoculated into the right buttocks of the mice. 3 Every 2 days, RTP or RTP-C (200×10 -6 M, 200 μL) solution was administered for 10 days. During the experiment, the tumor volume and mouse weight were measured regularly.

[0080] The results are as follows Fig.16 As shown, it can be seen from the results that the polypeptide RTP significantly inhibited the proliferation of tumors and inhibited lung metastasis in mice.

[0081] In summary, the polypeptide RTP provided by the present invention can specifically recognize CAIX overexpressed in renal cancer cells, and then be cleaved by MMP-2 in the tumor microenvironment, and then the probe residues form nanofibers by targeted recognition of VEGFA or TIE-2 to be retained in renal cancer tissue for a long time, strongly inhibit tumor angiogenesis and metastasis through the VEGF and TIE-2 dual signaling pathways, and inhibit the progression of renal cancer cells.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined 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. Use of the VEGF / TIE-2 dual-targeting polypeptide as claimed in claim 1 in the preparation of anti-tumor drugs.

3. The use according to claim 2, characterized in that: The tumor is a tumor that highly expresses VEGF protein.

4. The use according to claim 3, characterized in that: The tumor that highly expresses VEGF protein is renal cancer.

5. The use according to claim 3, characterized in that: The tumor that highly expresses VEGF protein is prostate cancer, glioblastoma or head and neck squamous cell carcinoma.

6. The use according to claim 2, characterized in that: The anti-tumor drug exerts its anti-tumor effect by inhibiting tumor angiogenesis and metastasis.

7. An anti-tumor drug, characterized in that: The active ingredient includes the VEGF / TIE-2 dual-targeting polypeptide according to claim 1.

8. The anti-tumor drug according to claim 7, characterized in that: The anti-tumor drug also includes pharmaceutically acceptable excipients.

9. The anti-tumor drug according to claim 7, characterized in that: The dosage form of the anti-tumor drug is tablets, granules, powders, capsules, pills or injections.

10. A method for preparing the VEGF / TIE-2 dual-targeting polypeptide according to claim 1, characterized in that: The method comprises the steps of preparing the VEGF / TIE-2 dual-targeting polypeptide by solid phase synthesis method.

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