A multi-target fusion protein for anti-tumor invasion and metastasis, its preparation method and application

Abstract

The present invention relates to a class of multi-target fusion proteins for anti-tumor invasion and metastasis, and a preparation method and application thereof. The fusion protein has the following amino acid sequence structure: RGD peptide - linker peptide - TRAIL functional peptide segment - linker peptide - NGR peptide; the fusion protein simultaneously has the anti-tumor cell proliferation effect of the TRAIL peptide segment and the tumor cell migration inhibitory effects of two targeting peptides, RGD and NGR; through comparison, it is found that the multi-target fusion protein has more significant anti-tumor activity, reverses the drug resistance of TRAIL to a certain extent, and can be used for preparing anti-tumor drugs.

Description

[0001] This application is a divisional application of application number 201610924303.1 filed on October 24, 2016, entitled "Preparation and Use of a Multi-Target Fusion Protein for Anti-tumor Invasion and Metastasis". Technical Field

[0002] This invention belongs to the field of bioengineering pharmaceutical protein technology, and relates to the preparation and use of a class of TRAIL protein-related multi-target anti-tumor fusion proteins. Background Technology

[0003] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as apoptin 2 ligand (Apo2L), is a cytokine synthesized by the body itself. It is expressed on the surface of a small number of lymphocytes and exists in a soluble, free form. Its main physiological functions are regulating the body's immune response, participating in immune surveillance, and inhibiting tumor development and metastasis. Studies have shown that human soluble TRAIL (sTRAIL), which contains amino acid sequences 114 to 281 of the TRAIL protein, possesses the activity of the complete TRAIL protein (Pitti RM, J. Biol. Chem, 1996). It can induce apoptosis in various tumor cells, and its target specificity makes it almost toxic to normal cells. However, its clinical trial results have been unsatisfactory, and the development of drug resistance has limited its clinical application. Based on TRAIL-related fusion proteins, it is possible to increase the sensitivity of tumor cells to TRAIL and, to some extent, reverse their drug resistance. Recombinant human soluble TRAIL (rhTRAIL) and agonist antibodies targeting the TRAIL receptor exhibit excellent antitumor activity and have made significant progress in clinical trials. However, the development of drug resistance and individual variability remain the biggest challenges to the clinical application of TRAIL and agonist antibodies targeting the TRAIL receptor. Studies have found that the antitumor activity of multi-target drugs recombined with sTRAIL can be significantly enhanced. For example, the recombinant protein resulting from the conjugation of an EGFR single-chain antibody with sTRAIL has a stronger apoptosis-inducing effect on tumor cells with high EGFR expression levels.

[0004] RGD peptides are short peptides containing the arginine-glycine-aspartic acid (Arg-Gly-Asp) sequence, serving as the recognition site for integrin-ligand protein interactions. Integrins ανβ3 and ανβ5 play crucial roles in tumor invasion and metastasis, and are typically highly expressed in tumor angiogenesis and certain tumor cells such as breast cancer, lung cancer, prostate cancer, and glioblastoma, thus serving as ideal targets for tumor therapy. RGD peptides have been widely used in tumor diagnosis and treatment research. Utilizing the specific binding of RGD peptides to integrins ανβ3 and ανβ5, antitumor drugs can be targeted and delivered to the tumor site, effectively reducing damage to normal tissues or organs. Studies have reported that the fusion protein resulting from the recombinant RGD and sTRAIL exhibits significantly superior activity compared to sTRAIL. The amino acid sequence of the cyclic RGD peptide used in this invention is ACDCRGDCFC (Koivunen E, Bio-Technology, 1995), also known as RGD4C.

[0005] NGR peptide is an antimicrobial peptide and a targeting peptide (Asn-Gly-Arg). It specifically binds to angiogenesis via aminopeptidase N (CD13). NGR modification allows cytotoxic drugs, cytokines, anti-angiogenic drugs, and contrast agents to target angiogenesis, improving efficacy and reducing damage to normal tissues. It is currently widely used in research on targeted delivery of therapeutic drugs, with NGR-hTNF already in Phase II and III clinical trials. Studies have reported that sTRAIL has a certain inhibitory effect on angiogenesis. Recombining NGR with sTRAIL yields a fusion protein that can induce apoptosis in tumor cells and can be targeted to tumor angiogenesis sites, inhibiting tumor angiogenesis. However, to date, there are no reports on the conjugation of NGR peptide with sTRAIL and its antitumor effects. The amino acid sequence of the NGR peptide used in this invention is CNGRCVSGCAGRC (Arap W, Science, 1998).

[0006] Based on the above situation, this invention utilizes the selective apoptosis-inducing effect of TRAIL on tumor cells and the targeting effects of RGD peptide and NGR peptide. The above-mentioned RGD peptide and NGR peptide are introduced at both ends of sTRAIL to develop a TRAIL-related multi-target fusion protein that inhibits tumor cell metastasis and has a stronger tumor cell apoptosis-inducing effect and can effectively inhibit tumor cell metastasis in vivo and in vitro. Summary of the Invention

[0007] This invention provides a class of multi-target anti-tumor fusion proteins RGD-TRAIL-NGR and TRAIL-NGR.

[0008] This invention provides the encoding genes and amino acid sequences of the TRAIL-related fusion proteins RGD-TRAIL-NGR and TRAIL-NGR.

[0009] This invention provides a method for preparing TRAIL-related fusion proteins.

[0010] This invention provides the application of TRAIL-related fusion proteins in anti-tumor therapy.

[0011] The present invention also provides a composition comprising the fusion protein as the active ingredient and a pharmaceutically acceptable carrier, and its application in antitumor activity.

[0012] Existing reports on the preparation of TRAIL-related fusion proteins almost all employ prokaryotic expression systems. This invention utilizes genetic engineering technology to introduce RGD and NGR peptides at both ends of sTRAIL, constructing a recombinant vector for the TRAIL-related fusion protein RGD-TRAIL-NGR. The target protein was expressed using a Pichia pastoris secretory expression system, and after purification, a multifunctional pharmaceutical protein was obtained.

[0013] The present invention provides a multi-target fusion protein for anti-tumor invasion and metastasis, the structure of which is: RGD-linker peptide (G4S)-sTRAIL-linker peptide (G4S)-NGR, and the protein has the amino acid sequence of SEQ ID NO:1, which consists of 208 amino acids.

[0014] The present invention provides the encoding gene of the fusion protein, which has the nucleotide sequence shown in SEQ ID NO:2, totaling 624 bp.

[0015] The amino acid sequence of another anti-tumor invasion and metastasis multi-target fusion protein TRAIL-NGR provided by the present invention is SEQ ID NO:3, which contains 192 amino acids and has the nucleotide sequence shown in SEQ ID NO:4, which contains 576 bp.

[0016] This invention utilizes genetic engineering technology to provide a method for preparing novel multi-target fusion proteins related to TRAIL (Example 2). The advantage of this invention lies in the secretion and expression of three fusion proteins—RGD-TRAIL-NGR, RGD-TRAIL, and TRAIL-NGR—using a Pichia pastoris expression system, and comparing their antitumor effects. The three obtained fusion proteins can efficiently bind to tumor cells expressing related cell surface factors, significantly inhibiting tumor cell proliferation. The fusion proteins can also significantly inhibit the in vivo and in vitro metastasis of tumor cells. The addition of RGD and NGR peptides enhances the antitumor effect of TRAIL and partially reverses TRAIL resistance, demonstrating better application prospects. While RGD-TRAIL has been previously reported, comparisons show that the RGD-TRAIL-NGR fusion protein constructed in this invention exhibits superior antitumor activity. Attached Figure Description

[0017] Figure 1 Figure 1 shows the construction and identification results of TRAIL-related fusion protein expression vectors (M1: DNA marker DL 2000, 1: PCR product, 2: recombinant plasmid, 3: SnaB I and Not I double digestion, 4: Sal I digestion, M2: DNA marker DL15000). Among them, Figure A shows the construction and identification results of RGD-TRAIL expression vector, Figure B shows the construction and identification results of TRAIL-NGR expression vector, and Figure C shows the construction and identification results of RGD-TRAIL-NGR expression vector.

[0018] Figure 2 Figure A shows the isolation, purification, and identification results of TRAIL-related fusion proteins (M: protein marker, 1: Washing buffer I elution buffer, 2: Washing buffer II elution buffer, 3: Elution buffer elution buffer, 4: anti-His-tag immunoblotting results, 5: anti-TRAIL immunoblotting results). Among them, Figure A shows the isolation, purification, and identification results of RGD-TRAIL, Figure B shows the isolation, purification, and identification results of TRAIL-NGR, and Figure C shows the isolation, purification, and identification results of NGR-TRAIL-NGR.

[0019] Figure 3 The affinity activities of TRAIL-related fusion proteins with tumor cells are shown in Figure A, which shows the expression of various tumor cell surface receptors αν, CD13, DR4 and DR5, and Figure B shows the binding of TRAIL-related fusion proteins with tumor cells H460, A549, HT1080 and PANC-1 as detected by ELISA.

[0020] Figure 4The results of MTT assay were used to detect the inhibitory effect of TRAIL-related fusion proteins on the proliferation of tumor cells H460, A549, HT1080, and PANC-1.

[0021] Figure 5 The results of the scratch assay were used to detect the inhibitory effect of TRAIL-related fusion protein on the migration of A549 and HT1080 cells. Figure A shows A549 cells and Figure B shows HT1080 cells.

[0022] Figure 6 The Transwell assay was used to detect the inhibitory effect of TRAIL-related fusion proteins on the migration of A549 and HT1080 cells. Figure A shows A549 cells and Figure B shows HT1080 cells.

[0023] Figure 7 —The results of flow cytometry and Western blot analysis on the apoptosis-inducing effect of TRAIL-related fusion protein on tumor cells A549 and HT1080, where Figure A shows A549 cells and Figure B shows HT1080 cells.

[0024] Figure 8 The results show the inhibitory effect of TRAIL-related fusion protein on lung metastasis in a nude mouse model. Figure A shows the fluorescence intensity of each group, Figure B shows the number of lung metastatic nodules in each group, and Figure C shows the weight change curve of nude mice in each group. Detailed Implementation

[0025] The following examples are only intended to help those skilled in the art better understand the present invention, and are not intended to limit the present invention in any way.

[0026] Example 1

[0027] Construction of recombinant expression vectors pHBM-rgd-trail-ngr, pHBM-rgd-trail, and pHBM-trail-ngr

[0028] Recombinant plasmids pHBM-rgd-trail-ngr and pHBM-trail-ngr were constructed using enzyme digestion, PCR, and ligation molecular biology techniques with the pHBM expression vector.

[0029] The full gene sequence of the fusion protein RGD-linker peptide (G4S)-TRAIL-linker peptide (G4S)-NGR was optimized and synthesized by GenScript based on the preferred codons of Pichia pastoris. The gene sequences of the fusion proteins RGD-TRAIL and TRAIL-NGR were constructed using molecular biology techniques through primer design. The primers were synthesized by Invitrogen™. The expression vector was pHBM (CHKD Doctoral and Master's Thesis Full-text Database, 2014).

[0030] P1(SEQ ID NO:5): 5'-TCTG TACGTA GCTTGTGATTGTAGAGGAG-3'

[0031] (The underlined part is the SnaB I restriction site, followed by the start sequence of the RGD peptide encoding gene)

[0032] P2(SEQ ID NO:6): 5'-ATAAGAAT GCGGCCGC TTAGTGGTGGTGGTGGTGGTGACCAACCAAAAAGGC-3'

[0033] (The underlined part is the Not I restriction site, followed by the complementary sequence at the end of the His-tag encoding gene and the TRAIL protein encoding gene)

[0034] P3(SEQ ID NO:7): 5'-TCTG TACGTA GTTAGAGAAAGAGGACCTCAG-3'

[0035] (The underlined part is the SnaB I restriction site, followed by the TRAIL coding gene start sequence)

[0036] P4 (SEQ ID NO:8): 5'-ATAAGAAT GCGGCCGC TTAGTGGTGGTG-3'

[0037] (The underlined part is the NotI restriction site, followed by the complementary sequence at the end of the His-tag encoding gene)

[0038] 1) Extract the pUC57-rgd-trail-ngr plasmid provided by GenScript, digest it with SnaB I and Not I, and then ligate it with the pHBM plasmid vector that has been digested with the same enzymes to obtain the recombinant plasmid pHBM-rgd-trail-ngr.

[0039] 2) Using plasmid pHBM-rgd-trail-ngr as a template, PCR amplification reaction was performed with primers P1 and P2. The PCR product was recovered by gel, digested with SnaB I and NotI, and then ligated with the double-digested pHBM plasmid vector to obtain the recombinant plasmid pHBM-rgd-trail.

[0040] 3) Using plasmid pHBM-rgd-trail-ngr as a template, PCR amplification was performed with primers P3 and P4. The PCR product was recovered by gel extraction, digested with SnaB I and NotI, and then ligated with the double-digested pHBM plasmid vector to obtain the recombinant plasmid pHBM-trail-ngr.

[0041] The gene sequence of the fusion protein RGD-TRAIL-NGR expressed by Pichia pastoris is shown in SEQ ID NO:2, with a full length of 624 bp and encoding 208 amino acids. The RGD peptide gene and the TRAIL protein gene, as well as the TRAIL protein gene and the NGR peptide gene, are connected by GGGGS linker peptide genes (encoding 5 amino acids). The gene sequence of the TRAIL-NGR protein is shown in SEQ ID NO:4, with a full length of 576 bp and encoding 192 amino acids.

[0042] Example 2

[0043] Expression and purification of TRAIL-related fusion proteins in Pichia pastoris

[0044] The constructed expression plasmids pHBM-rgd-trail, pHBM-trail-ngr, and pHBM-rgd-trail-ngr were transformed into DH5α competent cells. After double enzyme digestion and sequencing identification, the plasmids were extracted and linearized by SalI digestion. Figure 1 Large molecular weight fragments were recovered via gel extraction, electroporated into Pichia pastoris GS115 competent cells, plated on MD plates, and single colonies were picked for colony PCR identification. His... + Colonies were induced to express the fusion protein, and strains with relatively high expression levels were selected as expression strains for large-scale fermentation. The strain was named GS115-RTN and deposited with the China General Microbiological Culture Collection Center (CGMCC) on September 19, 2016, with the accession number CGMCC No. 13016. The expression strain of the fusion protein was first inoculated onto BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 4 × 10⁻⁶). -5 Incubate in 1% biotin, 100mM pH 6.0 potassium phosphate buffer, 1% glycerol, at 30°C for 36 h. Collect bacterial cells by static incubation at room temperature or centrifugation, and transfer to BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB, 4×10⁻⁶ oz.). -5 1% biotin, 100mM pH 6.0 potassium phosphate buffer, 1% methanol) were added, and the mixture was cultured at 20℃ for 48-96 h (100% methanol was added every 24 h to a final concentration of 1% to induce fusion protein expression). The fusion protein was expressed in soluble form in Pichia pastoris in the culture medium. The supernatant was filtered through a 0.45 μm filter and then purified using GE Ni... 2+ Purification was performed using an affinity column, following the manufacturer's instructions. The expression and purification status of the fusion protein were detected by 12% SDS-PAGE. Western blot analysis confirmed that the purified protein contained both the TRAIL protein structure and the His-tag structure. Figure 2 ).

[0045] Example 3

[0046] Affinity activity analysis of fusion protein with tumor cells

[0047] 1) Western blot analysis to detect the expression levels of αν, CD13, DR4, and DR5 on the surface of different tumor cells.

[0048] Tumor cells HEK293, 3T3, A549, H460, PANC-1, BxPC-3, MIA, HCT-15, and HT1080 were all passaged and preserved in our laboratory. Tumor cells in logarithmic growth phase were washed three times with pre-chilled PBS, and then lysed on ice for 30 min with an appropriate amount of cell lysis buffer (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.2% SDS, 2% NP-40, 0.5% sodium deoxycholate, pH 8.0. 1% PMSF was added before use). The cells were centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was transferred to a new 1.5 ml EP tube. Protein quantification was performed on each cell lysate sample using a BCA kit. Each cell lysate sample was prepared with the same total protein content, mixed with an appropriate amount of 5× loading buffer, denatured in a boiling water bath for 10 min, cooled, and then loaded for SDS-PAGE electrophoresis analysis. After electrophoresis, the protein was transferred to a PVDF membrane. After blocking with 5% skim milk solution, the PVDF membrane was cut to size according to the molecular weight of the target protein. The membrane was incubated overnight at 4°C with the appropriate primary antibody, washed three times with TBST solution, and then incubated with secondary antibody and washed three more times with TBST solution. Finally, the membrane was developed and photographed. The primary antibody used was αν. Among the cells tested, H460 cells did not express CD13, but showed high levels of αν and DR4 / DR5 expression. A549, PANC-1, and HT1080 cells all expressed the aforementioned cell surface receptors. Therefore, the biological activity of the constructed TRAIL-related fusion protein was mainly compared using four tumor cell types: H460, A549, PANC-1, and HT1080.

[0049] 2) ELISA method was used to analyze the affinity activity of the fusion protein for different tumor cells.

[0050] Tumor cells H460, A549, HT1080, and PANC-1 were seeded into 96-well plates after reaching confluence within 24 hours. The seeding density of each tumor cell type was adjusted according to cell size and growth rate, approximately 1 × 10⁻⁶ cells / well. 4Cells / well were cultured at 37°C for 24 h, then washed twice with PBS (3 min / well). 50 μl / well of pre-chilled 0.05% glutaraldehyde (4°C) was added, and the cells were fixed at 4°C for 20 min. After fixation, the cells were washed three times with PBS (3 min / well), and after shaking off residual liquid, 200 μl / well of 5% skim milk solution was added and gently shaken on a shaker at room temperature for 2 h. The cells were then washed three times with PBST buffer (PBS containing 0.05% Tween-20) (3 min / well). The fusion protein was serially diluted with PBS and added to 96-well plates, with three parallel wells per concentration, 50 μl / well, and incubated at 37°C for 2 h. After washing three times with PBST (3 min / well), the fusion protein was added... Anti-His-tag monoclonal antibody (Abmart, 1:2000 dilution), 50 μl / well, incubated at 37℃ for 2 h; after washing 3 times with PBST (3 min / wash), add HRP-labeled goat anti-mouse IgG antibody (1:2500 dilution), 50 μl / well, incubated at 37℃ for 2 h; after washing 5 times with PBST (5 min / wash), add 100 μl of horseradish peroxidase substrate-soluble single-component TMB solution (Beijing Tiangen Biotech Co., Ltd.) to each well, react at room temperature in the dark for 10-20 min. When the liquid changes from colorless to blue, add 100 μl of 2 mol / L sulfuric acid to each well to stop the reaction, and immediately measure the absorbance at 450 nm on a microplate reader. Results are as follows. Figure 3 As shown, the three proteins all exhibited strong affinity activities for H460, A549, PANC-1, and HT1080 cells. The figure shows that the affinity activities of the four fusion proteins with the above-mentioned tumor cells are not significantly different, indicating that the introduction of the targeting peptide has little effect on the binding activity of the TRAIL protein, and also does not significantly improve the affinity of the fusion protein with tumor cells.

[0051] Example 4

[0052] MTT assay for the inhibitory effect of fusion proteins on the proliferation of various tumor cells

[0053] Tumor cells H460, A549, HT1080, and PANC-1 in logarithmic growth phase were digested with trypsin and counted. Based on cell growth rate, 2500-5000 cells were seeded per well and cultured at 37°C for 24 hours to allow cell adhesion. The fusion protein was serially diluted with PBS, and 100 μl was added to each well. Three replicates were set up for each fusion protein concentration. A control group and a blank group were also established. Cells were cultured at 37°C for another 48 hours. 20 μl of 5 mg / ml MTT was added to each well, and the cells were cultured at 37°C for another 4 hours. The liquid in the wells was carefully aspirated, and 150 μl of DMSO was added to each well. The cells were shaken slowly at room temperature for 10 minutes, and the absorbance at 570 nm was measured using a microplate reader. The average absorbance of parallel wells was taken, and cell viability was calculated using the formula: Viability = (AT-AB) / (AC-AB) × 100%, where AB, AC, and AT represent the average absorbance values ​​of the blank group, control group, and recombinant protein group, respectively. A concentration-response curve was plotted with cell viability on the ordinate and fusion protein concentration on the abscissa, and the IC50 value was calculated using SPSS software.

[0054] From MTT results ( Figure 4 It can be seen that these three proteins did not show a particularly significant difference in their inhibitory effects on the proliferation of H460 and A549 cells. In fact, H460 and A549 cells are relatively sensitive to TRAIL, while HT1080 and PANC-1 cells are relatively insensitive to TRAIL. Therefore, the activity differences of fusion proteins modified with targeting peptides were not significant in H460 and A549 cells, with RGD-TRAIL-NGR showing slightly stronger activity, while the activity of fusion proteins modified with a single targeting peptide was slightly weaker. In PANC-1 cells, the activity differences between RGD-TRAIL and the other two fusion proteins were more significant, indicating that the NGR peptide can enhance the activity of fusion proteins. In HT1080 cells, the fusion protein TRAIL-NGR showed the strongest proliferation inhibition effect, followed by RGD-TRAIL-NGR. This may be related to the high expression level of CD13 on the surface of HT1080 cells, and its role is particularly important for HT1080 cells. The RGD-TRAIL-NGR fusion protein may have a certain spatial limitation on the full effect of the NGR peptide due to the presence of the RGD peptide, thus its activity is slightly weaker than that of TRAIL-NGR (Table 1).

[0055] Table 1. IC50 values ​​of fusion proteins RGD-TRAIL, TRAIL-NGR and RGD-TRAIL-NGR for various cell types.

[0056]

[0057] Example 5

[0058] Inhibitory effect of fusion protein on tumor cell migration

[0059] 1) Scratch assay to detect the effect of fusion protein on cell migration

[0060] Passaged tumor cells were seeded into 6-well plates and cultured at 37°C for 24 hours to allow cell adhesion. Using a sterile 200 μl yellow pipette tip, cells were streaked along the diameter of each well, close to the bottom of the plate. The cells were washed three times with PBS to remove floating cells, and fresh cell culture medium and the appropriate concentration of fusion protein were added. Observations and photographs were taken under an inverted microscope at 0 h and 24 h after streaking. Experimental results are as follows: Figure 5 As shown, since HT1080 and A549 cells have different sensitivities to the fusion protein, the concentration of the fusion protein was set at 0.12 μmol·L⁻¹. -1 and 0.016 μmol·L -1 Compared with the control group, the fusion protein significantly inhibited the migration of tumor cells HT1080 and A549. The inhibitory effects, from weakest to strongest, were RGD-TRAIL, TRAIL-NGR, and RGD-TRAIL-NGR, indicating that the introduction of the dual-targeting peptides can inhibit tumor cell migration, with the NGR peptide showing a more significant effect in migration inhibition.

[0061] 2) Transwell assay to detect the effect of fusion proteins on cell migration

[0062] Add 100 μl and 600 μl of serum-free culture medium to the inner and outer chambers of the Transwell chamber, respectively, and incubate at 37°C for 1-2 hours to equilibrate and improve cell adhesion efficiency. After digesting the passaged tumor cells, wash twice with serum-free culture medium, count the cells, and adjust the cell density according to the migration ability of different cell types. The A549 cell concentration was 8 × 10⁻⁶. 5 HT1080 cells / ml, cell density was 5×10⁶ / ml. 5Cells / ml; Discard the liquid in the inner and outer chambers, add cell suspension and corresponding concentration of fusion protein to each chamber, total volume 100 μl; add 600 μl of serum-containing culture medium to the outer chamber, using 20% ​​serum for A549 cells and 10% serum for HT1080 cells; after incubation at 37℃ for 24 h, remove the chambers, discard the culture medium in both chambers, wash the nested membrane once with PBS, fix the cells with pre-cooled methanol for 10 min; rinse three times with PBS, place in 0.1% crystal violet staining solution, stain at room temperature for 30 min; rinse three times with PBS, wipe away the liquid and non-migrated cells from the inner side of the nested membrane chamber with a cotton swab, retaining the cells on the outer side of the nested membrane, dry and observe and photograph under a microscope; to more objectively evaluate the migration of tumor cells, cut the nested membrane along the edge with a blade, place it in a 96-well plate, add 150 μl of... Dissolve crystal violet in 33% acetic acid, shake slowly for 10 minutes, carefully remove the membrane, and measure the absorbance at OD570 on an ELISA reader.

[0063] In the Transwell assay, the concentration of the fusion protein was consistent with that in the scratch assay, and the results were as follows: Figure 6 As shown, cell migration was significantly inhibited after treatment with the fusion protein, and the experimental results were consistent with the scratch assay, further indicating that the fusion protein can target the peptides RGD and NGR.

[0064] Example 6

[0065] The apoptosis-inducing effect of fusion proteins on tumor cells A549 and HT1080

[0066] 1) Flow cytometry detection of apoptosis induced by fusion protein in A549 and HT1080 cells

[0067] A549 and HT1080 cells were seeded in 6-well plates and cultured overnight at 37°C to allow them to adhere. The appropriate concentration of fusion protein was added, and the cells were cultured for another 24 hours. Cells were collected, washed twice with PBS, and resuspended in 100 μl of 1×Annexin V Binding Solution. 5 μl of Annexin V and FITC conjugate, followed by 5 μl of PI, were added to the cell suspension, and the cells were cultured at room temperature in the dark for 15 minutes. 400 μl of 1×Annexin V Binding Solution was added, and the cells were analyzed by flow cytometry.

[0068] Based on the MTT assay results, concentrations of 0.05 and 1 μmol·L⁻¹ were selected respectively. -1 The fusion protein acts on A549 and HT1080 cells. For example... Figure 7As shown, in A549 cells, which are relatively sensitive to TRAIL, the fusion protein RGD-TRAIL-NGR had the strongest apoptosis-inducing effect, followed by TRAIL-NGR, which is consistent with the MTT results. However, in HT1080 cells, although the apoptosis rate showed the same trend as the MTT results, the apoptosis rate was lower and the difference was smaller, indicating that although the fusion protein could induce apoptosis in HT1080 cells, which are relatively insensitive to TRAIL, the effect was not significant. This also indicates that RGD peptide and NGR peptide can play a role.

[0069] 2) Western blot analysis of the induction of apoptosis-related proteins in A549 and HT1080 by the fusion protein.

[0070] A549 and HT1080 cells were seeded in 6-well plates and cultured at 37°C for 24 h to allow cell adhesion. Appropriate concentrations of fusion protein were added, and the cells were cultured for another 24 h. Cells were then collected, and proteins were lysed on ice. After quantification using a BCA kit, the proteins were electrophoresed and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h, and the primary antibody was diluted with blocking buffer and incubated overnight. The membrane was washed three times with TBST, and a secondary antibody diluted with TBST was added. The membrane was incubated for another 2 h, and then washed five times with TBST. Millipore luminescent detection solutions A and B were mixed in a 1:1 ratio and added to the PVDF membrane for color development. Images were taken using a gel imaging system.

[0071] Based on the MTT assay results, concentrations of 0.025 and 0.05 μmol·L⁻¹ were selected respectively. -1 The RGD-TRAIL, TRAIL-NGR, and RGD-TRAIL-NGR proteins were used to treat A549 cells at concentrations of 0.5 and 1 μmol·L⁻¹. -1 The above three proteins were applied to HT1080 cells. After 24 hours, total protein was extracted and Western blot was used to detect the expression of apoptosis-related proteins PARP and caspase-3 (e.g., ...). Figure 7 In A549 cells, low concentrations of all four proteins induced apoptosis in tumor cells, indicating that the fusion protein could function as a part of the TRAIL protein. The RGD-TRAIL-NGR fusion protein showed a more pronounced effect, consistent with the MTT assay results, suggesting that both the RGD and NGR peptides played their respective roles. In HT1080 cells, expression of apoptosis-related proteins was also detected, but caspase-3 spliceosome expression was not prominent. This is related to the relatively low sensitivity of HT1080 cells to TRAIL. Furthermore, the three proteins showed similar effects, and the MTT results showed that the fusion protein containing the NGR peptide was more effective in inhibiting the proliferation of HT1080 cells, suggesting that the NGR peptide may have exerted a stronger inhibitory effect.

[0072] Example 7

[0073] Inhibitory effect of fusion protein on lung metastasis in nude mouse model

[0074] Example 5: In vitro transfer inhibition results showed that TRAIL-NGR and RGD-TRAIL-NGR significantly inhibited cell migration, while RGD-TRAIL had a weaker effect. Therefore, this experiment tested the in vivo transfer inhibition effects of the two fusion proteins, TRAIL-NGR and RGD-TRAIL-NGR, with the TRAIL group serving as a control. HT1080LUC cells were constructed in our laboratory, and BALB / c nude mice were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. HT1080LUC cells were collected and diluted to 7.5 × 10⁻⁶. 6 Cell suspension. 200 μl of cell suspension was injected into each nude mouse via the tail vein. One week later, the luciferase substrate D-luciferin (150 mg / kg) was injected intraperitoneally. -1 Animals were placed in a sealed dark chamber using the Xenogen in vivo animal imaging system to observe the growth of lung tumors. Nude mice were randomly divided into four groups based on fluorescence intensity: control group, TRAIL group, TRAIL-NGR group, and RGD-TRAIL-NGR group, with five mice in each group. The fusion protein group received the same dose as the TRAIL-NGR group (10 mg / kg), while the TRAIL and RGD-TRAIL-NGR groups received the same molar dose. Administration began on day 8 post-inoculation via tail vein injection, every three days for a total of four injections. Animals were sacrificed after in vivo imaging on day 29, and intact lung tissue was collected from each mouse. The tissue was fixed in Bouin's solution for 48 hours. Normal lung tissue appeared brownish-yellow, while cancerous lesions appeared as white raised nodules. The number of metastatic nodules on the lung surface was counted.

[0075] The results are as follows Figure 8 As shown, the inhibitory effects of the TRAIL group and the TRAIL-NGR group were similar, with a significant difference compared to the control group (P<0.05). The RGD-TRAIL-NGR group, however, significantly inhibited tumor cell metastasis compared to both the TRAIL group and the TRAIL-NGR group (P<0.05). This demonstrates that the RGD-TRAIL-NGR multi-target fusion protein has good in vivo therapeutic efficacy.

Claims

1. A preparation method of a TRAIL-related anti-tumor invasion and metastasis multi-target fusion protein, characterized in that, The fusion protein contains an RGD peptide, an NGR peptide and an sTRAIL protein fragment, and has a structure of RGD peptide - linker peptide - TRAIL functional peptide segment - linker peptide - NGR peptide; The fusion protein has the amino acid sequence shown in SEQ ID NO:1, or the fusion protein RGD - linker peptide - TRAIL - linker peptide - NGR has the nucleotide sequence shown in SEQ ID NO:2; The preparation method of the fusion protein comprises the following steps: 1) Synthesize the gene sequence of the fusion protein RGD - linker peptide - TRAIL - linker peptide - NGR; 2) Use restriction enzyme digestion, PCR, and ligation molecular biology techniques to construct a recombinant plasmid pHBM - rgd - trail - ngr using a pHBM expression vector; 3) After linearizing the recombinant plasmid pHBM - rgd - trail - ngr with SalI restriction endonuclease, electrotransform the competent cells of Pichia pastoris GS115, and screen to obtain an expression strain; the strain is named GS115 - RTN, and was deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 19, 2016, and its accession number is CGMCC No. 13016; 4) Inoculate the expression strain of the fusion protein into yeast - peptone - YNB (amino - acid - free yeast nitrogen base) - biotin, 100 mM potassium phosphate buffer (pH 6.0) - glycerol medium, culture at 30 °C for 36 h, let it stand at room temperature or centrifuge to collect the thallus, transfer it to yeast - peptone - YNB - biotin, 100 mM potassium phosphate buffer (pH 6.0) - methanol medium, continue to culture at 20 °C for 48 - 96 h, add methanol to induce the expression of the fusion protein; 5) After the fusion protein in the Pichia pastoris culture broth was filtered through a 0.45 μm filter membrane, it was purified using a Ni affinity column from GE. 2+ column.

2. An anti - tumor invasion and metastasis multi - target fusion protein prepared by the preparation method described in claim 1.

3. Use of the anti - tumor invasion and metastasis multi - target fusion protein described in claim 2 in the preparation of anti - tumor drugs; The anti - tumor drug is any one or more of an anti - large cell lung cancer drug, an anti - non - small cell lung cancer drug, an anti - pancreatic cancer drug, or an anti - fibrosarcoma drug.

4. A pharmaceutical composition comprising the anti - tumor invasion and metastasis multi - target fusion protein described in claim 2 as an active ingredient and a pharmaceutically acceptable carrier.

5. Use of the composition described in claim 4 in the preparation of anti - tumor drugs; The anti - tumor drug is any one or more of an anti - large cell lung cancer drug, an anti - non - small cell lung cancer drug, an anti - pancreatic cancer drug, or an anti - fibrosarcoma drug.

Images