A targeting transmembrane peptide for triple-negative breast cancer and its application

Abstract

The present invention discloses a transmembrane peptide that targets and binds to triple-negative breast cancer (MDA-MB-231) and its uses. The triple-negative breast cancer-targeting transmembrane peptide is a polypeptide, and its amino acid sequences are respectively: FKQDAWEAVDIR, APTTWFNSDSIT, FNSDSRSTHQED, IPLENQHKIYST, GSGFNESSVARP, DGSMLNRMRGFS; it also includes a bioactive substance formed on the surface of microorganisms and polymer mixtures with the triple-negative breast cancer-targeting transmembrane peptide. The present invention can efficiently target triple-negative breast cancer tissues in mice, penetrate the cell membrane of triple-negative breast cancer cells, and enter the interior of triple-negative breast cancer cells. Therefore, it can guide cancer treatment drugs to effectively reach the lesion site and achieve targeted treatment of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to a polypeptide and its application in the field of biomedicine, and in particular to a polypeptide capable of targeting tumor tissue of triple-negative breast cancer (MDA-MB-231) and entering tumor cells and its application. Background Art

[0002] Triple-negative breast cancer refers to breast cancer in which the expression of estrogen receptors, progesterone receptors and proto-oncogenes are all negative. Due to the loss of multiple receptors, it is difficult to achieve targeted drug delivery in the treatment of triple-negative breast cancer. In addition, the targets of most tumor-targeting molecules are located on the surface of tumor tissues or tumor cells, making it difficult for targeted drug molecules to enter the interior of tumor cells.

[0003] Therefore, constructing biological molecules that can target tumor tissues of triple-negative breast cancer and enter tumor cells is a scientific problem that urgently needs to be solved in the biomedical community. Summary of the invention

[0004] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a targeted transmembrane polypeptide for triple-negative breast cancer. The polypeptide can be targeted and accumulated in the tumor tissue site of triple-negative breast cancer in vivo, and can pass through the cell membrane of tumor cells and enter the interior of tumor cells. It provides the possibility of achieving efficient and precise treatment for triple-negative breast cancer.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] 1. A targeted transmembrane peptide for triple-negative breast cancer

[0007] The targeted cell-penetrating peptide is a polypeptide, and the amino acid sequence is selected from one of SEQ ID No.1 to SEQ ID No.6.

[0008] The amino acid sequences of the targeted cell-penetrating peptides are:

[0009] FKQDAWEAVDIR, APTTWFNSDSIT, FNSDSRSTHQED, IPLENQHKIYST, GSGFNESSVARP, DGSMLNRMRGFS

[0010] Table 1 Amino Acid Sequence Listing

[0011] Code Amino acid sequence Code Amino acid sequence No.1 FKQDAWEAVDIR No.4 IPLENQHKIYST No.2 APTTWFNSDSIT No.5 GSGFNESSVARP No.3 FNSDSRSTHQED No.6 DGSMLNRMRGFS

[0012] 2. A biologically active substance:

[0013] Contains targeted transmembrane peptides for triple-negative breast cancer;

[0014] It includes microorganisms, a polymer mixture, and the surface is formed with the targeted transmembrane peptide.

[0015] The polymer mixture includes covalently linked compounds or nanoparticles, and the microorganisms include engineered phages.

[0016] If it is a nanoparticle, the surface is modified with a targeted transmembrane peptide against triple-negative breast cancer;

[0017] If it is a covalently linked compound, the surface is modified with a targeted transmembrane peptide against triple-negative breast cancer through a chemical bond;

[0018] If it is an engineered phage, the targeted transmembrane peptide against triple-negative breast cancer is displayed on the phage surface through genetic engineering.

[0019] The bioactive substance formed in this way has the same biomedical function as the targeted transmembrane peptide against triple-negative breast cancer, that is, it is targeted and enriched at the tumor tissue site of triple-negative breast cancer and enters the interior of the cells of triple-negative breast cancer.

[0020] The targeted transmembrane peptide or bioactive substance against triple-negative breast cancer of the present invention is used in the preparation of drugs.

[0021] III. A polynucleotide sequence capable of encoding the targeted transmembrane peptide against triple-negative breast cancer.

[0022] IV. A polynucleotide sequence capable of encoding the bioactive substance.

[0023] V. A polypeptide drug for treating diseases, comprising the targeted transmembrane peptide against triple-negative breast cancer.

[0024] The drug targets the tumor tissue of triple-negative breast cancer, enters the interior of tumor cells, and can achieve the diagnosis and treatment of triple-negative breast cancer.

[0025] The targeted transmembrane peptide, bioactive substance or polypeptide drug against triple-negative breast cancer is targeted and enriched at the tumor tissue of triple-negative breast cancer and enters the interior of tumor cells. Specifically, it is applied in the efficient and precise cancer targeted diagnosis and treatment.

[0026] The targeted transmembrane peptide against triple-negative breast cancer obtained by the present invention can be targeted and enriched at the tumor tissue site of triple-negative breast cancer in vivo and enter the interior of tumor cells. It helps to achieve the targeted diagnosis and treatment of triple-negative breast cancer and has a wide application prospect in the biomedical field.

[0027] The inventors used a phage 12 - peptide library and, through an alternating method of in - vivo screening using a mouse tumor model and in - vitro screening of tumor cells, conducted 5 rounds of screening for triple - negative breast cancer (MDA - MB - 231). After monoclonal sequencing and exploratory verification, a targeting transmembrane polypeptide against triple - negative breast cancer was finally obtained.

[0028] Compared with other tumor - targeting molecules, the advantages of the targeting transmembrane polypeptide of the present invention are as follows:

[0029] (1) The polypeptide sequence is short, and the synthesis production cost is low;

[0030] (2) The polypeptide fragment has a stable structure, reducing the cost and difficulty of transportation and storage;

[0031] (3) It can be administered in multiple ways;

[0032] (4) It specifically targets triple - negative breast cancer (MDA - MB - 231);

[0033] (5) While targeting and enriching in the tumor tissue site, it can penetrate through the tumor cell membrane and enter the interior of tumor cells.

[0034] The beneficial effects of the present invention are as follows:

[0035] The polypeptide of the present invention can target and enrich in the tumor tissue site of triple - negative breast cancer in vivo, and at the same time penetrate through the cell membrane of tumor cells and enter the cell interior.

[0036] The targeting ability and cell - membrane - penetrating ability of the polypeptide of the present invention against triple - negative breast cancer provide new ideas for targeted diagnosis and treatment of triple - negative breast cancer clinically. Description of the Drawings

[0037] Figure 1 It is a graph of the output / input ratio for each round during the combined screening of triple - negative breast cancer tumor tissue and tumor cells using a phage 12 - peptide library in Example 1.

[0038] Figure 2 It is a graph of 10 polypeptide sequences with relatively high frequencies and their corresponding frequencies after analyzing the sequencing results using DNAStar software in Example 1.

[0039] Figure 3 It is an in - vivo fluorescence imaging graph of a tumor - bearing mouse after injecting a fluorescently labeled polypeptide molecule through the tail vein in Example 2.

[0040] Figure 4Ex vivo imaging of major organs and tumor tissues (i: spleen; ii: kidney; iii: heart; iv: lung; v: liver; vi: tumor) of a tumor-bearing mouse after injecting a fluorescently labeled polypeptide molecule into the mouse via the tail vein in Example 2. Detailed implementation mode

[0041] The present invention will be further described below in conjunction with examples and drawings. The following examples are only preferred examples of the present invention and do not limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0042] Examples of the present invention are as follows:

[0043] Example 1

[0044] Using a phage 12-mer library constructed by phage display technology, 5 rounds of alternating in vivo and in vitro screening were performed on a mouse tumor model and tumor cells of triple-negative breast cancer. Due to the large abundance of the phage 12-mer library, 10 μL of the phage 12-mer library was taken for screening during the screening process. Through the following operating steps, a targeting transmembrane polypeptide against triple-negative breast cancer was successfully obtained.

[0045] 1.1 Construction of a nude mouse triple-negative breast cancer model

[0046] Suspend 1x10 5 MDA-MB-231 cells in PBS solution and inject them into the axillary region of nude mice through a syringe. When the diameter of the tumor tissue reaches 10 mm, the construction of the model is completed.

[0047] 1.2 Resuscitation and culture of the host bacterium E.coil.ER2738

[0048] Prepare an LB solid culture plate and preheat it in a 37 °C incubator for 1 hour. Use a bacterium picking stick to dip into the melted E.coil.ER2738 bacterial solution and spread it on the surface of the LB plate in a "Z" shape, and then culture it in a 37 °C incubator for 12 hours. Add 5 mL of liquid LB medium containing tetracycline to a shaking flask, use a sterile pipette tip to pick a monoclonal colony from the plate, and place the pipette tip in the medium. Place the culture tube in a 37 °C shaker and shake it at 220 rpm until the bacteria are in the mid-logarithmic growth phase.

[0049] 1.3 Binding

[0050] Take a sterile EP tube, add 100 μL of PBS solution and 10 μL of phage 12-mer peptide library, and mix by vortexing. Inject it into the nude mice through the tail vein so that the phage reaches the tumor site through blood circulation.

[0051] 1.4. Washing

[0052] One hour later, anesthetize the nude mice. Perfuse the nude mice with sterile normal saline through the heart to wash away the phages that are not bound or weakly bound at the tumor site.

[0053] 1.5. Elution

[0054] Dissect the tumor tissue and add it to a tissue grinder. Add 1 mL of non-denaturing tissue lysate and fully lyse the tumor tissue. Aspirate the tissue homogenate into a sterile EP tube, centrifuge at 10000 rpm for 5 min to collect the supernatant, and perform phage titer determination, phage amplification and purification.

[0055] 1.6. Determination of phage titer

[0056] Preheat the LB / IPTG / Xgal plate in a 37 °C incubator for 1 hour. Gradient dilute the supernatant of the tissue homogenate, take 10 μL of the diluted phage solution and add it to 200 μL of E. coil. ER2738 bacterial solution in the logarithmic growth phase. Let it stand for 15 min. Add the infected phage to the surface of the LB / IPTG / Xgal plate and spread it evenly with a sterile coating rod. Invert the coated plate and culture it overnight in a 37 °C incubator. Check the phage blue plaques on the plate and count them the next day.

[0057] 1.7. Preparation of bacterial solution

[0058] Place 20 mL of LB medium in a 250 mL sterile conical flask, and then add 1 mL of the host bacterium E. coil. ER2738 in the mid-logarithmic growth phase. Place the conical flask in a 37 °C shaker and shake it at 220 rpm until the bacteria are in the mid-logarithmic growth phase.

[0059] 1.8. Phage amplification and purification

[0060] The supernatant of the tissue homogenate was added to 20 mL of E. coli ER2738 bacterial solution in the logarithmic growth phase. After standing at room temperature for 20 min, the bacterial solution was placed in a shaker at 37 °C and cultured with shaking at 220 rpm for 4.5 hours. Then the bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 12,000×g for 20 min. The supernatant was poured into a centrifuge tube containing 4 mL of PEG / NaCl solution, placed in a refrigerator at 4 °C, and allowed to settle overnight. The next day, the centrifuge tube was centrifuged at 12,000×g for 20 min, and the supernatant was discarded. 1 mL of PBS solution was added to the centrifuge tube to redissolve the precipitate, and the resuspended solution was transferred to a 1.5 mL EP tube and placed in a shaker at 37 °C and shaken for 1 hour. Then the EP tube was centrifuged at 12,000×g for 10 min, and the supernatant was transferred to an EP tube containing 200 μL of PEG / NaCl solution. The EP tube was placed at 4 °C and allowed to stand for 1 hour to precipitate the phages. Then the EP tube was centrifuged at 12,000×g for 20 min, the supernatant was discarded, and 100 μL of PBS was added to resuspend the precipitate.

[0061] 1.9, Second-round tumor cell screening

[0062] MDA-MB-231 cells were seeded in a 6-well plate. The amplified phage solution was diluted with serum-free medium and added to the cell well plate. The cell well plate was placed in a cell culture incubator for 1 hour. The cells were washed multiple times with PBST buffer to remove the phages not bound to the cell surface; the phages bound to the cell surface were removed with the eluent; finally, the cells were collected using a cell scraper, and the cells were lysed with the lysis solution to collect the phages inside the cells. Then, the phage titer was tested and the phage solution was amplified according to the steps in 1.6 and 1.8.

[0063] 1.10 Third, fourth, and fifth rounds of screening

[0064] The third-round in vivo tumor tissue screening in nude mice, the fourth-round in vitro tumor cell screening, and the fifth-round in vivo tumor tissue screening in nude mice were carried out according to the same steps. The amplified and purified phage solution in each round was used as the secondary phage peptide library for the next-round screening. The input amount of phages in each round of screening was kept consistent. The titer of the elution product in each round of screening was measured as the output amount of phages in each round, and the output / input ratio of phages in each round was calculated.

[0065] The output / input ratio results are as Figure 1 shown, Figure 1 for the output / input ratios of each round of in vivo tumor screening and in vitro tumor cell screening of triple-negative breast cancer using a phage 12-mer peptide library. The output / input ratio of the first-round screening was 1.65×10 -4 ; the output / input ratio of the second-round screening was 1.40×10 -5; The output / input ratio of the third round is 1.25×10 -3 ; The output / input ratio of the fourth round is 3.2×10 -4 ; The output / input ratio of the fifth round is 5.63×10 -3 . The results of the root experiment show that the output / input ratio increases alternately round by round. Therefore, the targeted transmembrane peptide for triple-negative breast cancer achieves effective enrichment.

[0066] 1.11. Phage positive monoclonal

[0067] Titer determination was performed on the elution products of the third, fourth, and fifth rounds. Select plates with no more than 100 plaques, randomly pick 100 phage blue plaques, add them to 100 shaking tubes containing 5 mL of LB medium, and culture them at 37°C with shaking at 220 rpm for 24 hours. Take 700 μL of the culture from each monoclonal and add it to an EP tube containing 300 μL of glycerol (50%) solution. After mixing, store it in a -80°C refrigerator. The remaining bacterial solution was subjected to sequencing.

[0068] Analyze the DNA sequences of phage monoclonal

[0069] According to the sequencing results, find the inserted foreign DNA sequence and translate the corresponding amino acid sequence according to the principle of the triple codon.

[0070] The results are as Figure 2 shown. Figure 2 The polypeptide sequences with a high frequency of occurrence of positive monoclonal phages and their repetition times are shown. Among them, the phage showing the polypeptide sequence FKQDAWEAVDIR appears 40 times, which is the phage with the highest frequency of occurrence.

[0071] Next, in combination with the examples, verify the targeting effect of the polypeptide molecule in mice.

[0072] Example 2

[0073] 2.1 Construction of a nude mouse triple-negative breast cancer model

[0074] Suspend 1x10 5 MDA-MB-231 cells in PBS solution and inject them into the axillary region of nude mice through a syringe. When the diameter of the tumor tissue reaches 10 mm, the construction of the model is completed.

[0075] 2.2 Chemical synthesis of polypeptides

[0076] Chemically synthesize six polypeptide molecules No.1 to No.6 and connect biotin to the carboxyl terminus of the polypeptides. In addition, select the polypeptide molecule with the sequence KGYGVGLRFPAW as the control group (CP), and also connect biotin to the carboxyl terminus of this polypeptide molecule.

[0077] 2.3 Labeling with Fluorescein

[0078] Dissolve the biotinylated polypeptide molecule and Cy5-labeled streptavidin in physiological saline and stir at room temperature for 1 h. Then, remove the excess Cy5-streptavidin by dialysis to obtain the polypeptide molecule labeled with Cy5 fluorescein.

[0079] 2.4 In Vivo Fluorescence Imaging of Mice

[0080] Inject the polypeptide molecule labeled with Cy5 fluorescein into the tumor-bearing mice via the tail vein. After 24 h, collect the in vivo fluorescence images of the mice and the ex vivo fluorescence images of the main tissues and organs using an in vivo fluorescence imager. According to the results of the fluorescence images, all six different polypeptide molecules, No.1 to No.6, can effectively target and accumulate in the triple-negative breast cancer tissues in the tumor-bearing mice ( Figure 3 ).

[0081] Figure 4 Figure showing ex vivo imaging of the main visceral organs and tumor tissues (i: spleen; ii: kidney; iii: heart; iv: lung; v: liver; vi: tumor) of mice after injecting the fluorescence-labeled polypeptide molecule into the tumor-bearing mice via the tail vein in Example 2.

[0082] It can be seen from this implementation that the present invention can efficiently target triple-negative breast cancer tissues in mice, penetrate the cell membrane of triple-negative breast cancer cells, and enter the interior of triple-negative breast cancer cells. Therefore, it can guide cancer therapeutic drugs to effectively reach the lesion site and achieve targeted therapy of triple-negative breast cancer.

[0083] The genes and protein sequences involved in the present invention are as follows:

[0084] SEQ ID No.1;

[0085] Name: Amino acid sequence of a transmembrane peptide targeting triple-negative breast cancer

[0086] DNA type: other DNA

[0087] Source: Artificial Sequence

[0088] FKQDAWEAVDIR

[0089] SEQ ID No.2;

[0090] Name: Amino acid sequence of a transmembrane peptide targeting triple-negative breast cancer

[0091] DNA type: other DNA

[0092] Source: Artificial Sequence

[0093] APTTWFNSDSIT

[0094] SEQ ID No.3;

[0095] Name: Amino acid sequence of a targeting cell-penetrating peptide against triple-negative breast cancer DNA Type: other DNA

[0096] Source: Artificial Sequence

[0097] FNSDSRSTHQED

[0098] SEQ ID No.4;

[0099] Name: Amino acid sequence of a targeting cell-penetrating peptide against triple-negative breast cancer DNA Type: other DNA

[0100] Source: Artificial Sequence

[0101] IPLENQHKIYST

[0102] SEQ ID No.5;

[0103] Name: Amino acid sequence of a targeting cell-penetrating peptide against triple-negative breast cancer DNA Type: other DNA

[0104] Source: Artificial Sequence

[0105] GSGFNESSVARP

[0106] SEQ ID No.6;

[0107] Name: Amino acid sequence of a targeting cell-penetrating peptide against triple-negative breast cancer DNA Type: other DNA

[0108] Source: Artificial Sequence

[0109] DGSMLNRMRGFS。

Claims

1. A targeting transmembrane peptide for triple-negative breast cancer MDA-MB-231, characterized in that: The targeted transmembrane peptide is a polypeptide with an amino acid sequence of SEQ ID No.

5.

2. A polynucleotide, characterized in that: It can encode the targeted transmembrane peptide for triple-negative breast cancer described in claim 1.

3. A polypeptide drug, characterized in that, It contains the targeted transmembrane peptide for triple-negative breast cancer described in claim 1.

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