Peptides that specifically target human liver cancer cells and their applications

Abstract

This invention belongs to the field of biopharmaceuticals, specifically relating to a polypeptide that specifically targets human liver cancer cells and its uses. Addressing the insufficient targeting specificity of existing liver cancer-targeting polypeptides to tumor cells in vivo, and the need to develop polypeptides with better in vivo targeting, this invention provides a liver cancer targeting peptide F11, obtained through phage display technology, with its amino acid sequence shown in SEQ ID NO:1. This polypeptide exhibits targeted affinity for HepG2 human liver cancer cells. In HepG2 human liver cancer-bearing mice, this polypeptide demonstrates specific affinity for liver cancer tumor tissue, achieving targeted delivery. The targeting polypeptide of this invention exhibits high specificity and targeting ability, showing great application potential in the fields of early tumor diagnosis and targeted therapy.

Description

Technical Field

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

[0002] Cancer treatment technology has always been a hot topic and a challenge in medical research. Thanks to the development of chemotherapy drugs and the implementation of immunotherapy, the success rate of cancer treatment has continued to increase. However, the mortality rate of some cancers remains high. Therefore, we need to find new targeted therapy strategies for liver cancer to improve treatment efficiency and reduce drug side effects. Meanwhile, given the difficulty in early diagnosis of liver cancer, the invention of fluorescent dye-labeled targeted liver cancer peptide imaging technology offers a new approach to discovering even smaller lesions, achieving early detection and treatment. In conclusion, exploring and screening high-affinity peptides targeting liver cancer cells is an effective way to solve the challenges of liver cancer treatment.

[0003] Currently, the main method for screening peptides targeting liver cancer cells is phage display technology. Phage display technology is a new technique that allows the fusion expression of exogenous peptides or proteins with proteins on the surface of phages. By introducing various exogenous genes into phages, a phage display library expressing various proteins on its surface can be constructed. For screening tumor-targeting antigens, by specifically binding the phage display peptide library to tumor cells, highly specific and affinity-based tumor-targeting peptides can be screened. Currently, existing technologies have screened targeted peptides with a certain affinity for liver cancer cells. For example, patent CN101918433A discloses a peptide that specifically binds to HCC cells. Patent CN201110451767.2 discloses a peptide that specifically binds to the HepG2 liver cancer cell line; although it can target the HepG2 liver cancer cell line, it does not disclose the targeting properties of this targeted peptide in vivo.

[0004] In actual targeted peptide screening processes, some methods involve synthesizing receptor cells in vitro or using tumors in model mice to screen for liver cancer-targeting peptides. However, these methods cannot effectively simulate the tumor growth environment in liver cancer patients, resulting in significant differences from clinical applications. Therefore, it is necessary to develop liver cancer cell-targeting peptides that exhibit better targeting specificity for liver cancer cells in the human body. Summary of the Invention

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

[0006] The technical solution of this invention to solve the above-mentioned technical problem is: to provide a polypeptide that specifically targets human liver cancer cells. The amino acid sequence of the polypeptide that specifically targets human liver cancer cells described in this invention is shown in SEQ ID NO:1.

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

[0008] FYLEHPSGGLAV.

[0009] Among them, the liver cancer cells targeted by the aforementioned peptide that specifically targets human liver cancer cells are the HepG2 cell line.

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

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

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

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

[0014] TTTTATCTGGAACATCCGAGCGGCGGCCTGGCGGTG.

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

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

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

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

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

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

[0021] Furthermore, the liver cancer cells mentioned are the HepG2 cell line.

[0022] The present invention also provides the use of the above-mentioned polypeptide that specifically targets human liver cancer cells in the preparation of anti-liver cancer drugs or in the preparation of imaging agents for diagnosing liver cancer.

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

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

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

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

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

[0028] This invention provides a polypeptide, named F11 polypeptide, capable of specifically targeting the HepG2 human liver cancer cell line. In vivo studies have demonstrated that this polypeptide exhibits a strong affinity for HepG2 liver cancer cells. The targeting polypeptide of this invention possesses high specificity, specifically targeting HepG2 human liver cancer cells, while showing no targeting ability against human LO2 cells and human peripheral blood cells. The high specificity and targeting ability of this invention's targeting polypeptide indicate significant potential for application in early tumor diagnosis and targeted therapy. Attached Figure Description

[0029] Figure 1 The chemical structural formula of the F11 polypeptide of this invention is shown.

[0030] Figure 2 The results show the verification of the affinity of F11 peptide for different cells in Experiment 1; a represents the fluorescence imaging result; b represents the flow cytometry result.

[0031] Figure 3 The figure shows the distribution of F11 peptide targeting liver cancer cells in mice.

[0032] Figure 4 The image shows the immunohistochemical staining effect of F11 peptide on cancerous and adjacent tissues of liver cancer patients. Detailed Implementation

[0033] This invention utilizes phage display technology to screen for a highly specific targeting polypeptide against HepG2 liver cancer cells, named the F11 polypeptide. The amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0034] The phage peptide library screened in this invention is randomly generated with a capacity greater than 10. 9The dodecapeptide library underwent three rounds of screening before finally yielding peptide F11. In the screening process using HepG2 cells, this invention also selected adsorbent cells to remove peptides with affinity for other cells. This ensures that the final peptide F11 can bind to HepG2 cells while failing to recognize LO2 cells and human peripheral blood cells.

[0035] Furthermore, the present invention also provides the encoding nucleotides, vectors, and host cells of the above-mentioned polypeptides that specifically target human liver cancer cells.

[0036] Furthermore, the present invention also provides the use of the above-mentioned peptides that specifically target human liver cancer cells in targeting liver cancer cells.

[0037] The present invention also provides the use of the above-mentioned polypeptide that specifically targets human liver cancer cells in the preparation of drugs for the prevention or treatment of liver cancer.

[0038] The present invention also provides the use of the above-mentioned polypeptide that specifically targets human liver cancer cells in the preparation of diagnostic reagents for liver cancer.

[0039] The polypeptide of the present invention that specifically targets human liver cancer cells was obtained by screening using the following method:

[0040] Human hepatocellular carcinoma cells (HepG2 and LO2 cells) in good growth condition were seeded into 24-well plates. Blocking buffer (4% milk + PBS) was added, and the cells were blocked at 37°C for 1 h. The blocking buffer was removed, and the cells were washed three times with PBS. A 10 μl phage library was mixed with 1 ml PBS and incubated with LO2 cells at 37°C for 1 h. The supernatant was then incubated with the blocked HepG2 cells at 37°C for 2 h. The phages bound to the cells were collected and amplified using ER2738 host bacteria. The amplified phages were used for the next screening of HepG2 cells.

[0041] The above operation was repeated twice. The phages obtained in the third round of screening were titered and identified using LB / IPTG / X-Gal plates. The screened phage clones were identified using ELISA technology. Phage clones with OD values ​​greater than 0.8 in the ELISA test results were selected for purification and sequencing to obtain the specific polypeptide F11 of the present invention that can recognize HepG2.

[0042] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0043] The preparation methods for each reagent in the following examples or test cases are as follows:

[0044] 0.2M Gly-HCl pH2.2: Weigh 1.5014 g g of glycine, dissolve it in water, adjust the pH to 2.2 with HCl, and then bring the volume to 100 mL with water.

[0045] 1M Tris-HCl pH 9.1: Weigh 121.1 g Tris, dissolve it in water, adjust the pH to 9.1 with HCl, and then bring the volume to 100 mL with water.

[0046] 20% PEG / 2.5M NaCl: Dissolve 20 g of polyethylene glycol and 14.6 g of NaCl in water, then add water to a final volume of 100 mL.

[0047] All other reagents are commercially available products.

[0048] Example 1: Screening of phage polypeptide libraries for HepG2 liver cancer cells

[0049] HepG2 and LO2 hepatocytes (from ATCC) in good growth condition were passaged and seeded into 24-well cell culture plates. The plates were incubated at 37°C, saturated humidity, and 5% CO2 for 24 hours, then the medium was changed. Cells were cultured until they adhered well and showed good growth. When confluence reached over 90% and formed a monolayer, the culture medium was aspirated, and the cells were gently washed twice with PBS. Serum-free medium was added, and the cells were incubated at 37°C and 5% CO2 for 1 hour. The culture medium was then aspirated, and blocking buffer (4% milk + PBS) was added, and the cells were blocked at 37°C for 1 hour. The same procedure was repeated to block HepG2 hepatocytes. After blocking, the blocking buffer was aspirated, and the cells were washed three times with PBS. 10 μl of a phage library was mixed with 1 ml of PBS and incubated with LO2 cells at 37°C for 1 hour to allow for negative cell adsorption. After adsorption, the supernatant was aspirated, transferred to a 1.5 ml sterile centrifuge tube, centrifuged at 1000 rpm for 5 min, transferred to a new centrifuge tube, and centrifuged again to remove any cells that might be present in the supernatant. Immediately incubate the supernatant with the blocked and washed HepG2 liver cancer cells at 37°C for 2 hours. After incubation, discard the supernatant and wash five times with PBS. Elute with 1 ml of 0.2M Gly-HCl (pH 2.2), then neutralize with 150 μl of 1M Tris-HCl (pH 9.1) and collect the phages adsorbed on the HepG2 cells.

[0050] The collected phages were infecting 20 ml of ER2738 bacterial suspension (1:100 inoculation after overnight shaking) at 37°C for 4.5 h. After centrifugation at 12000 g for 10 min, the supernatant was collected and 1 / 6 volume of 20% PEG / 2.5M NaCl was added, and the phages were extracted at 4°C for 2 h. The phage solution was centrifuged at 12000 g at 4°C for 15 min, the supernatant was discarded, and 1 mL of culture medium was added to obtain the phage solution. The above steps were repeated for the second and third rounds of screening.

[0051] Example 2: ELISA identification of phage clones obtained through screening

[0052] After the third round of screening, HepG2 / LO2 cells were cultured in 96-well cell culture plates. Once the cells adhered, the culture medium was removed, and the cells were washed twice with PBS. The cells were then blocked with 4% PBSM blocking buffer at 37°C for 1 h, the blocking buffer was removed, and the cells were washed once with PBS. 50 μl of 4% PBSM blocking buffer and 50 μl of the selected phage solution were added to each well, and the cells were incubated at 37°C for 1 h. After incubation, the cells were washed five times with PBS, anti-P8 / HRP antibody was added, and the cells were incubated at 37°C for 1 h. After washing five times with PBS, 100 μl of TMB substrate was added to each well, and the reaction was terminated after 15 min in the dark. The absorbance was measured using a microplate reader. Phage clones with absorbance greater than 0.8 were selected for sequencing; the sequence was FYLEHPSGGLAV. The polypeptide of this sequence was named the F11 polypeptide.

[0053] Example 3 Synthesis and purification of F11 polypeptide

[0054] Weigh a certain amount of 2Cl resin and add it to the reactor. Then, add amino acids and base according to the ratio of resin:Fmoc-Cys(Trt)-OH:DIEA 1:3:6. React with DCM as solvent for 3 hours, then add chromatographic grade methanol for 30 minutes to cap the resin. Finally, wash and dry to synthesize the resin. Select and accurately weigh the appropriate amount of resin and add it to a clean reactor. Add 2 times the volume of DMF (DCM) to swell for 60-90 minutes. Remove the DMF (DCM), add 3 times the volume of 20% piperidine solution and react for 30 minutes. Remove the 20% piperidine solution and wash with DMF 5 times. Take a small amount of resin in a detection tube, add two drops each of ninhydrin solution (solutions A, B, and C), and heat at 110℃ for 3 minutes. A positive result indicates that Fmoc has been removed. After removal, add the resin to the reactor in the order of 3 times AA: 6 times NMM: 2.85 times HBTU (HATU), and add DMF (just enough to allow the resin to be fully stirred). Take a small amount of resin in a test tube, add two drops each of ninhydrin solution (solutions A, B, and C), heat at 110℃ for 3 minutes. If the resin shows a negative result, the reaction is complete; if the resin shows a positive result, the reaction is incomplete. Repeat the process starting from the point where three times the volume of 20% piperidine solution is added and the reaction is continued for 30 minutes. After all peptide sequences are assembled, wash the resin with DMF*3, MeOH*2, DCM*2, and MeOH*3, then place it in a desiccator overnight to dry. Add the dried resin to the cutting buffer (solution E / F) at 8-10 ml per gram, and react on a shaker for 2 hours. After the reaction is complete, filter with a stencil to obtain the filtrate. Add ice-cold ether to precipitate the crude product, centrifuge to collect the precipitate, and wash three times with ether. Place the washed crude product in a desiccator overnight to dry, obtaining the target peptide F11, with the structure shown below. Figure 1 As shown.

[0055] Example 4 Synthesis and purification of FITC-F11 peptide

[0056] Weigh a certain amount of 2Cl resin and add it to the reactor. Then, add amino acids and alkali according to the resin:Fmoc-Lys(Dde)-OH:DIEA ratio of 1:3:6. React with DCM as solvent for 3 hours, then add chromatographic grade methanol for 30 minutes to cap the resin. Finally, wash and dry to synthesize the resin. Select and accurately weigh the appropriate amount of resin and add it to a clean reactor. Add 2 times the volume of DMF (DCM) to swell for 60-90 minutes. Remove the DMF (DCM), add 3 times the volume of 20% piperidine solution and react for 30 minutes. Remove the 20% piperidine solution and wash with DMF 5 times. Take a small amount of resin in a detection tube, add two drops each of ninhydrin solution (solutions A, B, and C), and heat at 110℃ for 3 minutes. A positive result indicates that Fmoc has been removed. After removal, add the resin to the reactor in the order of 3 times AA: 6 times NMM: 2.85 times HBTU (HATU), and add DMF (just enough to allow the resin to be fully stirred). Take a small amount of resin in a detection tube, add two drops each of ninhydrin solution (solutions A, B, and C), heat at 110℃ for 3 minutes. If the resin shows a negative result, the reaction is complete; if the resin shows a positive result, the reaction is incomplete. Repeat the operation starting from the point where three times the volume of 20% piperidine solution is added and reacted for 30 minutes. After all linear peptide sequences are assembled, remove Dde with 4% hydrazine hydrate / DMF solution. Add the resin to the reactor in the order of 4 times NMM: 2 times FITC. Add DMF, take a small amount of resin in a detection tube, add two drops each of ninhydrin solution (solutions A, B, and C), heat at 110℃ for 3 minutes. If the resin shows a positive result, the reaction is incomplete. Repeat the previous step. If the resin shows a negative result, the reaction is complete. Wash with methanol and place in a desiccator overnight to dry. Add 8-10 ml of cutting buffer (solution E / F) per gram of dried resin and react on a shaker for 2 hours. After the reaction was complete, the solution was filtered through a sintered glass filter to obtain the filtrate. Iced ether was added to precipitate the crude product, which was then centrifuged to form the precipitate. The precipitate was washed three times with ether. The washed crude product was then placed in a desiccator and dried overnight to obtain the target peptide FITC-F11.

[0057] Experimental Example 1: F11 Peptide Cell Targeting Affinity Verification Assay

[0058] To investigate the targeted uptake of the liver cancer peptide F11 on HepG2 cells in vitro, an in vitro assay was established for verification. First, HepG2 cells, LO2 cells, and 293T cells were seeded into Millicell® EZ cell culture slides at a density of 5 x 10⁴ cells per well. After 24 hours of complete cell adhesion, FITC-labeled F11 peptide (prepared in Example 4) was added to the culture wells. Five minutes later, 5 μl of Hoechst-stained nuclei were added to each well. After 5 minutes, the cell culture supernatant was aspirated, the cells were washed three times with PBS, the cell culture trays were removed, and coverslips were placed on top. The uptake of FITC-F11 peptide on each cell type was observed under a microscope and photographed. Figure 2As shown in Figure a, F11 peptide extensively occupies the surface of HepG2 cells, while it is almost absent on the surface of LO2 cells in the 293T nucleus. Subsequently, cells from each well were collected for flow cytometry analysis, and the results are as follows: Figure 2 As shown in b, the affinity of the F11 peptide for HepG2 cells is significantly greater than that for 293T cells and LO2 cells. These results demonstrate the targeting affinity of the F11 peptide for HepG2 cells.

[0059] Experiment Example 2: Verification of the Targeted Distribution Ability of F11 Peptide in Mice

[0060] To verify the targeting affinity of the hepatocellular carcinoma peptide F11 for hepatocellular carcinoma tissue in vivo, a HepG2 subcutaneous tumor model was established in Balb / c-nude mice (4-5 weeks old, female). HepG2 cells cultured in vitro were digested with trypsin and brought to a constant volume in dual-DmEm-free medium. Each mouse was inoculated with 1×102 7 Cells. Wait until the subcutaneous tumor reaches a volume of 1000 mm. 3 Subsequently, tumor-bearing mice were intravenously injected with 200 μg of FITC-F11 solution. Two hours later, the mice were sacrificed, and subcutaneous tumors, heart, liver, spleen, lungs, and kidneys were harvested. The tissues were minced and placed in collagenase solution for digestion at 37°C for 4 hours. The solution was then filtered through a 70 μm cell sieve, and the filtrate was washed three times with PBS buffer and analyzed by flow cytometry. Flow cytometry data for mouse tumors and major organs are shown below. Figure 3 As shown, the FITC-labeled peptide F11 is distributed significantly higher in tumor tissue than in other organs, demonstrating the targeting affinity of F11 peptide for tumor tissue in mice.

[0061] Experiment 3: Immunohistochemical staining verification of F11 peptide in liver cancer patient tissue sections

[0062] To verify whether the F11 peptide might have a liver cancer targeting effect in liver cancer patients, immunohistochemistry was used to stain liver cancer patient tissue sections. First, the liver cancer patient sections were baked, dewaxed, and hydrated for antigen retrieval. After blocking with goat serum for 20 minutes, the tissue was coated with a 10 μg / ml FITC-F11 peptide solution as the primary antibody and incubated overnight at 4°C. The next day, the tissue was washed three times with PBS, and the nuclei were stained with DAPI, followed by three more washes with PBS. After nucleus staining, an anti-fluorescence quencher was added, and the slides were mounted. Representative images of the staining results are shown below. Figure 4 As shown, compared to adjacent tissues, liver cancer tissues from liver cancer patients showed a more significant positive result, demonstrating the targeting affinity of F11 peptide for liver cancer tissues in patients.

[0063] This invention screened and obtained a polypeptide that can successfully target liver cancer cells. Studies have shown that it has a good affinity for HepG2 liver cancer cells and can specifically target HepG2 cells. It also showed good targeting effects in mice. It is suitable for the preparation of anti-liver cancer drugs or imaging agents for the diagnosis of liver cancer, and has great application prospects. sequence list <110> West China Hospital of Sichuan University <120> Peptides that specifically target human liver cancer cells and their applications <130> A210274K (Preface) <141> 2021-04-29 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <400> 1 Phe Tyr Leu Glu His Pro Ser Gly Gly Leu Ala Val 1 5 10 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <400> 2 ttttatctgg aacatccgag cggcggcctg gcggtg 36

Claims

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

1.

2. The polypeptide specifically targeting human liver cancer cells according to claim 1, characterized in that: The liver cancer cells mentioned are the HepG2 cell line.

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

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

2.

5. An expression vector containing the encoding nucleotide as described in claim 3 or 4.

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

7. A host cell containing the polypeptide of claim 1, the encoding nucleotide of claim 3 or 4, and the expression vector of claim 5 or 6.

8. Use of the polypeptide of claim 1 in the preparation of an imaging agent for diagnosing liver cancer.

9. The use according to claim 8, characterized in that: The polypeptide is prepared as a solution and used at a concentration of 1 mg / mL.

10. Use of the encoding nucleotide of claim 3 or 4, the expression vector of claim 5 or 6, and the host cell of claim 7 in the preparation of an imaging agent for diagnosing liver cancer.

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