Use of b. ovatus protein duf3109 in preparation of anti-tumor drugs

Abstract

The application discloses application of DUF3109 protein in preparation of an antitumor drug, wherein the DUF3109 protein is Bacteroides ovatus DUF3109 protein. The DUF3109 protein can inhibit the progression of liver cancer by regulating CD4+ T cell secretion of CXCL13 and promoting the formation of intratumoral tertiary lymphoid structures. In specific embodiments, the DUF3109 protein exhibits significant inhibitory effect on liver orthotopic tumors and significant promotion of CD4+ T lymphocyte and Jurkat cell secretion of CXCL13. The technical scheme of the application not only expands the use of the DUF3109 protein, but also provides a potential innovative solution for tumor treatment and a new research direction for development of an antitumor drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of Bacteroides ovatus protein DUF3109 in the preparation of an antitumor drug. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, with a five-year survival rate of less than 20%. Existing treatment methods face multiple challenges in advanced HCC, including high tumor heterogeneity, active vascular invasion, and complex immune escape mechanisms. Although immunotherapy represented by PD-1 / PD-L1 inhibitors has made some progress, the objective response rate of patients is still only 12-25%, and there is a 6.2% risk of super-progression. Therefore, it is urgent to develop new immune regulation strategies targeting the tumor microenvironment to reconstruct the dynamic balance of anti-tumor immune response.

[0003] Tertiary lymphoid structures (TLS) are immune activation hubs in the tumor microenvironment, recruiting CD4+ T cells and B cells to form germinal center microregions through CXCL13 / CCL21. Its anti-tumor mechanisms include promoting the differentiation of effector T cells, enhancing the presentation of new antigens, and inducing plasma cells to produce targeted antibodies. Clinical studies have shown that the five-year survival rate and PD-1 inhibitor response rate of liver cancer patients with high-density TLS are significantly improved.

[0004] Existing methods to promote TLS formation include phototherapy and nanomaterials, cytokines and chemokines, chemotherapy and radiotherapy, etc. Phototherapy and nanomaterials have limited effect in cold tumors, with insufficient laser penetration depth; cytokines and chemokines can trigger systemic inflammation; chemotherapy and radiotherapy are associated with significant side effects. These methods have certain effects, but have limitations, therefore, finding new therapeutic drugs targeting TLS formation is of great significance for improving the current situation of liver cancer immunotherapy and developing new and effective immunotherapy methods. SUMMARY

[0005] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the object of the present application is to provide the application of Bacteroides ovatus protein DUF3109 in the preparation of an antitumor drug.

[0006] To achieve the above-mentioned object, the technical solution adopted by the present application is as follows:

[0007] In a first aspect of the present application, the application of DUF3109 protein in the preparation of a drug for preventing and / or treating tumors is provided.

[0008] In some embodiments of the present application, the amino acid sequence of the DUF3109 protein is shown in SEQ ID NO: 1.

[0009] In some embodiments of the present application, the amino acid sequence of the DUF3109 protein comprises a polypeptide derivative with the same function obtained by modifying the amino acid sequence shown in SEQ ID NO: 1 at the N-terminus, C-terminus, amino acid skeleton, and / or amino acid side chain group.

[0010] In some embodiments of the present application, the modification comprises one or more of glycosylation, phosphorylation, N-methylation, myristoylation, palmitoylation, biotinylation, fluorescent labeling, polyethylene glycol modification, polyantigenic peptide, isoprenylation cyclization, acetylation, or amidation.

[0011] In some embodiments of the present application, the tumor comprises melanoma, breast cancer, non-small cell lung cancer, liver cancer, or ovarian cancer.

[0012] In some embodiments of the present application, the dosage form of the drug comprises tablets, injections, powders, oral liquids, or injections.

[0013] In some embodiments of the present application, the drug achieves the treatment of the tumor by promoting the formation of tertiary lymphoid structures within the tumor and / or promoting the secretion of CXCL13 by T lymphocytes.

[0014] In some embodiments of the present application, the T lymphocytes comprise CD4+ T lymphocytes.

[0015] In some embodiments of the present application, the CD4+ T lymphocytes comprise Jurkat cells.

[0016] In a second aspect of the present application, the use of a biomaterial in the preparation of a drug for preventing and / or treating a tumor, characterized in that the biomaterial comprises:

[0017] (1) a nucleic acid molecule encoding the DUF3109 protein of the above aspect;

[0018] (2) an expression cassette containing the nucleic acid molecule of (1);

[0019] (3) a recombinant vector containing the nucleic acid molecule of (1) or the expression cassette of (2);

[0020] (4) a recombinant cell containing the nucleic acid molecule of (1), the expression cassette of (2), the recombinant vector of (3), or the DUF3109 protein of the above aspect.

[0021] In a third aspect of the present application, a pharmaceutical composition is provided, comprising at least one of the DUF3109 protein of the above aspect and the biomaterial.

[0022] In some embodiments of the present application, the DUF3109 protein comprises a modified or unmodified DUF3109 protein.

[0023] In some embodiments of the present application, the modification comprises adding a tag sequence to the N-terminus or C-terminus of the amino acid sequence of the DUF3109 protein shown in SEQ ID NO: 1.

[0024] In some embodiments of the present application, the tag sequence comprises at least one of a signal peptide, a targeting peptide, a tag peptide, a fluorescent protein, or a transmembrane peptide.

[0025] In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0026] In some embodiments of the present application, the excipient comprises one or more of a diluent, a stabilizer, an osmotic pressure regulator, a pH regulator, a preservative, and an antioxidant, but is not limited thereto.

[0027] In some embodiments of the present application, the pH regulator comprises at least one of citric acid / sodium citrate, glucose, sodium hydroxide, and sodium bicarbonate, but is not limited thereto.

[0028] In some embodiments of the present application, the antioxidant comprises at least one of vitamin C, sodium pyrosulfite, sodium sulfite / hydrogen sulfite, but is not limited thereto.

[0029] In some embodiments of the present application, the pharmaceutical composition is administered at a dose of 1-5 mg of the DUF3109 protein contained therein per kg of body weight of the patient.

[0030] The present application has the following beneficial effects:

[0031] The present application provides a use of a DUF3109 protein in the preparation of an anti-tumor drug. The DUF3109 protein promotes the formation of tertiary lymphoid structures in tumors by regulating the secretion of CXCL13 by CD4+ T cells, thereby inhibiting the progression of liver cancer. In the examples, the DUF3109 protein exhibits a significant inhibitory effect on liver orthotopic tumors and significantly promotes the secretion of CXCL13 by CD4+ T lymphocytes and Jurkat cells compared to the control group. The technical solution of the present application not only expands the use of the DUF3109 protein, but also provides a potential innovative solution for tumor treatment and a new research direction for the development of anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A schematic diagram for the construction of the recombinant plasmid of Example 1 of the present application.

[0033] Figure 2Figure of SDS-PAGE electrophoresis and coomassie blue staining for identifying expression and purification result of DUF3109 protein in embodiment 1 of the present application, wherein M is standard molecular weight protein, 1 represents protein before induction, 2 represents protein after induction, 3 is sample flow liquid, 4-6 represent protein after purification.

[0034] Figure 3 Figure of inhibitory effect of DUF3109 protein on liver orthotopic tumor model according to the present application. A is a schematic diagram of experimental steps; B is a result figure of in vivo imaging of tumor tissue of mice after intervention of DUF3109 protein on liver orthotopic tumor model, wherein PBS is blank control group; C is a columnar contrast figure of infrared radiation infrared quantity of B figure, *: p<0.05; D is a result figure of detecting intratumoral TLS content by multiplex immunofluorescence staining experiment, expression level of CD3 represents density of T cells in tumor tissue (red fluorescence), expression level of CD19 represents density of B cells in tumor tissue (yellow fluorescence), and blue fluorescence represents DAPI stained nucleus; E is a columnar contrast figure of proportion of TLS area to total area in D figure, **: p<0.01; F is a columnar contrast figure of TLS quantity per square millimeter in D figure, **: p<0.01.

[0035] Figure 4 Figure of flow cytometry result of influence of DUF3109 protein on CXCL13 expression of Jurkat cells and human CD4+T cells according to the present application. A is a flow cytometry result figure of CXCL13 expression of human CD4+T cells; B is a flow cytometry result figure of CXCL13 expression of Jurkat cells; both take PBS as blank control group, ****: p<0.0001, ***: p<0.001.

[0036] Figure 5 Figure of enzyme-linked immunosorbent assay result of influence of DUF3109 protein on CXCL13 expression of mouse CD4+T cells according to the present application, wherein PBS is blank control group, ***: p<0.001. DETAILED DESCRIPTION

[0037] The content of the present application is further described in detail through specific embodiments. Raw materials, reagents or devices used in the embodiments can be obtained from conventional commercial channels or can be obtained by prior art methods, unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0038] Example 1: Purification of DUF3109 recombinant protein

[0039] The present embodiment provides a purification method of DUF3109 recombinant protein, and the amino acid sequence of the DUF3109 protein is: MIQIGDVVVSLDVFQEKFLCDLGACKGACCIEGDAGAPVELDEVMELEEVLPVIWDELAPEARAVIEKQGVVYTDQEGDLVTSIVNNKDCVFTCYDENGCCYCAIEKAYRAGKTAFYKPVSCHLYPIRIGDYGPYKAVNYNRWDICKAAVLLGKKENLPVYQFLKEPLIRKFGEEWYKELVTVAEELKKQQYI (SEQ ID NO: 1). The specific experimental steps are as follows.

[0040] (1) The sequence encoding the DUF3109 protein is digested by PaeR7I and XhoI endonuclease, and then inserted into a commercially available pET-42b vector to obtain a recombinant plasmid. After sequencing, the recombinant plasmid is transformed into a host bacterium Escherichia coli to obtain a single clone.

[0041] (2) Prepare a sterile kanamycin (KAN) solid LB medium plate, and coat the single clone obtained in (1) for overnight culture.

[0042] (3) Take the plate after (2) culture, and select a single colony to LB liquid medium (10 mL) added with KAN (50 μg / mL) for culture for 24 h.

[0043] (4) The bacterial solution obtained by culture in (3) is cultured in 200 mL of LB liquid medium added with KAN (50 μg / mL) at 37°C until the OD600 value is about 0.6-0.8. IPTG is added to a final concentration of 1 mM, and induced at 37°C for 6 h.

[0044] (5) The bacterial solution induced in (4) is centrifuged at 8000g for 10 min, the bacterial body is collected, washed with PBS once, the supernatant is removed, and 10 mL of PBS, 100 μL of protease inhibitor (Solarbio, P6730) and lysozyme (1 mg / mL) are added to the precipitate. Incubate on ice for 30 min.

[0045] (6) The solution after incubation in (5) is placed on ice, and treated with an ultrasonic lysis instrument. The ultrasonic power is set to 225W, each ultrasonic treatment is 5 seconds, the interval is 10 seconds, and the total ultrasonic treatment is 20 min, until the solution is clear.

[0046] (7) The clear solution obtained in (6) is centrifuged at 4°C, 12000g for 10 min, the supernatant is sucked, and filtered (0.45 μm) into a new centrifuge tube. Add 4 mL of 50% BeyoGoldTM His-tag Purification Resin (resistant to reduction chelating type), 4℃ slowly shaking on a shaker for 60 min.

[0047] (8) The solution obtained in (7) was added to a chromatography empty column tube (Biyun Tian, FCL06), slowly loaded, and the loading flow was collected. Gradient elution was performed using 50, 200 and 500 mM imidazole solutions to obtain DUF3109 protein.

[0048] (9) The obtained DUF3109 protein was subjected to SDS-PAGE (10%) electrophoresis and Coomassie blue staining to verify the size of DUF3109. Meanwhile, a 14 kDa dialysis bag was immersed in PBS dialysis solution for 16 h to remove salt. The protein concentration was measured using a BCA protein concentration determination kit (according to the instructions), and the sample was frozen and dried overnight and stored at -80℃.

[0049] The constructed recombinant plasmid map is shown in Figure 1 , the experimental results are shown in Figure 2 , and the purified DUF3109 protein is obtained in this embodiment.

[0050] Example 2 Inhibition of DUF3109 protein on liver orthotopic tumor model

[0051] This embodiment provides detection of the inhibition of DUF3109 protein on liver orthotopic tumor model, and the experimental steps are shown in Figure 3 A. One test cycle is ten days, and treatment starts on the third day after the mice are inoculated with tumor cells. There are two groups in total: a control group (given PBS buffer) and a treatment group (given DUF3109 protein). The specific experimental steps are as follows.

[0052] (1) Construction of liver orthotopic tumor mouse model: Take mouse hepatoma cells Hepa1-6 (Wuhan Punuo Life Science and Technology Co., Ltd.) in logarithmic growth phase and prepare a single cell suspension. Wash with PBS buffer for 3 times; female C57BL / 6J mice, 6-8 weeks old, 1% sodium pentobarbital solution, 5 mL / kg standard intraperitoneal injection of anesthetized mice, sterile scissors cut the mouse abdominal skin, expose the viscera, find the liver, and inject 20 μL (5 x 10 5 cells) of cell suspension into each mouse under the liver capsule.

[0053] (2) Starting from the third day of inoculation of hepatoma cells, inject DUF3109 protein PBS buffer into the liver orthotopic tumor mouse model through the tail vein every day. The amount of DUF3109 protein used is according to the ratio of DUF3109 protein: mouse = 4 mg / kg. The control group is injected with the same amount of PBS buffer.

[0054] (3) Ten days after the establishment of the liver orthotopic tumor model, mice were anesthetized with 1% sodium pentobarbital and injected with D-fluorescein potassium salt solution via intraperitoneal injection. Five minutes later, fluorescence level was detected using a small animal in vivo imaging instrument to record the growth of the orthotopic liver tumor in mice.

[0055] (4) Simultaneously, after euthanizing the mice, free liver cancer tissue was collected and paraffin sections were prepared. The sections were dewaxed in fresh xylene for 10 min, repeated 3 times, and then gradually hydrated with a gradient of ethanol (100%, 95%, 70%) for 5 min, 5 min, and 2 min, respectively. The sections were washed 3 times with PBS buffer for 1 min each time. The sections were placed in EDTA antigen retrieval solution (Solepro, C1034), heated to boiling on high (700W) in a microwave oven, then on low (150W) for 15 minutes, and then allowed to cool to room temperature. The sample area was delineated with an immunohistochemical pen, and 5% BSA blocking solution was added. The sections were then incubated at room temperature with shaking for 10 minutes. After removing the blocking solution, diluted CD3 primary antibody solution (1:1000, abcam, ab237721) was added and incubated at 37°C for 2 hours. The sections were washed with TBST buffer for 3 minutes, repeated once. Add fluorescently labeled secondary antibody (1:600, Yaxin, LF102) and incubate at room temperature for 30 minutes. After washing again with TBST, add TSA+ enhancer (five-color multiplex fluorescent staining kit, GuduoBiological, HYDS0045) (diluted 1:100) and incubate in a moist environment for 15 minutes. Wash three times with TBST buffer, 3 minutes each time. Then repeat the above steps and perform a second round of staining with CD19 primary antibody solution (1:1000, abc am, ab317335). After staining, mount with anti-fluorescence quenching mounting solution (containing DAPI) (Beyotime, P0131), and observe and photograph the expression of CD3 and CD19 using a Nikon inverted fluorescence microscope.

[0056] Experimental results are as follows Figure 3 As shown, Figure 3 The results showed that the tumor volume in mice treated with DUF3109 was significantly reduced compared to the control group, and this reduction was statistically significant. Figure 3 (C) Figure 3 In the middle (D), multiplex immunofluorescence assays were performed to detect the abundance of TLS in tumor tissues of a mouse orthotopic liver xenograft model after treatment with DUF3109 protein and PBS buffer. CD3 expression level represents the density of T cells in the tumor tissue (red indicates positive); CD19 expression level represents the density of B cells in the tumor tissue (yellow indicates positive). Regions enriched by both cell types can be defined as TLS structures. The abundance of TLS in tumors of mice treated with DUF3109 was significantly increased compared to the control group. Figure 3 (E and F in the middle).

[0057] Example 3 DUF3109 protein promotes the expression of CXCL13 by CD4+ T cells

[0058] This example provides the effect of DUF3109 protein on the expression of chemokine CXCL13 by Jurkat cells and human CD4+ T lymphocyte cells, using PBS buffer as a control group, and detecting the effect of DUF3109 protein on the ability of cells to secrete CXCL13 after treating mouse CD4+ T lymphocytes for 48 h by enzyme-linked immunosorbent assay. The specific experimental steps are as follows.

[0059] 1. Extraction and culture of mouse CD4+ T cells

[0060] (1) After euthanizing the mouse, the mouse spleen was removed in a sterile environment, and the spleen was gently ground in PBS buffer containing 2% FBS to prepare a single nuclear cell suspension;

[0061] (2) The cell suspension was treated with red blood cell lysis solution at room temperature for 10 min to remove red blood cells. After lysis, the cells were washed by centrifugation (300g, 5 min) to remove the lysis solution, and then resuspended in PBS buffer;

[0062] (3) The cell suspension obtained in (2) was filtered through a 70 μm cell filter to remove tissue debris;

[0063] (4) The cell suspension obtained in (3) was sorted for CD4+ T cells using a mouse CD4+ T cell magnetic bead sorting kit;

[0064] (5) Mouse CD3 stimulator (BioGems, 05112-25) and CD28 stimulator (BioGems, 10312-25-500) were added to the CD4+ T cells obtained in (4) to a final concentration of 3 μg / mL in solution, and mouse IL-2 cytokine (PEPROTECH, 212-12-5) was added to a final concentration of 20 nM in solution, and then placed in a 37°C, 5% CO2 cell incubator for further culture.

[0065] 2. Sorting and culturing human CD4+ T lymphocytes

[0066] (1) Fresh anticoagulated human peripheral blood was collected and diluted with PBS buffer at a ratio of 1:1 and mixed well by inverting;

[0067] (2) Add 5 mL lymphocyte separation medium to a 15 mL centrifuge tube, and gently overlay 10 mL of diluted blood in (1) on the lymphocyte separation medium, and centrifuge at 400g at room temperature for 30 min with 0 acceleration and deceleration;

[0068] (3) After centrifugation, the tube was taken out smoothly, and the lymphocyte layer was carefully sucked out using a Pasteur tube. PBS buffer was added, and centrifugation was performed at 300g for 5min at room temperature. Peripheral blood mononuclear cells were collected;

[0069] (4) The cells obtained in (3) were subjected to CD4+ T lymphocyte separation using a human CD4+ T lymphocyte separation kit according to the instructions;

[0070] (5) The CD4+ T lymphocytes obtained in (4) were washed once using PBS solution (300g, 5min), resuspended using RPMI-1640 culture medium containing 10% FBS, and 25μL CD3 / CD28 combined activator (Thermo Fisher, 11161D) was added, and mouse IL-2 cytokine (PEPROTECH, 212-12-5) was added to make the final concentration in the solution 20nM. It was placed in a cell incubator at 37℃, 5% CO2 for further culture.

[0071] 3. Flow cytometry experiment

[0072] (1) 1×10 6 6 human CD4+ T lymphocytes obtained in the above steps and 1×10 6 6 Jurkat cells (Wuhan Punsai Life Science and Technology Co., Ltd., item number CL-0129) were added with 100μg of the DUF3109 protein and incubated for 24h, and then centrifuged at 300g, 4℃ for 10min to collect the cells for subsequent antibody staining steps;

[0073] (2) The above cells were incubated with anti-CXCL13 antibody (Abmart, PC2553S) at 4℃ for 1h, washed with PBS for 2 times, then added with CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L) diluted at 1:200 and incubated at 4℃ for 30min, washed with PBS for 2 times, collected the cells, fixed with 4% paraformaldehyde, and detected on a flow cytometer.

[0074] 4. Enzyme-linked immunosorbent assay

[0075] Enzyme-linked immunosorbent assay was performed using an enzyme-linked immunosorbent kit (enzyme, MM-45471M2). The specific experimental steps are as follows.

[0076] 1×10 7The mouse CD4+ T lymphocytes are added with 100 μg DUF3109 for 24 h, and the cell culture medium is collected and centrifuged at 300 g for 10 min at room temperature, and the supernatant of the culture medium is collected, the sample diluent is used to dilute the supernatant of the cells to be tested, the standard solution is diluted, and the standard solution, the sample to be tested, the negative control and the positive control are sequentially added, and then incubated at 37 ℃ for 30 min; the plate is washed for 5 times, the enzyme-labeled reagent is added and incubated at 37 ℃ for 30 min; the plate is washed for 5 times, color developing solution A and B are added, and then reacted at 37 ℃ for 15 min in the dark, the stop solution is added, and the OD value of the sample hole is detected by using an enzyme-labeled instrument within 15 min.

[0077] The experimental results are shown in Figure 4 and 5 As shown in A of Figure 4 , in the human CD4+ T lymphocytes, the positive rate of DUF3109 for promoting the expression of CXCL13 by the cells reaches 52.8%, which is significantly higher than that of the control group (26.5%); Figure 4 As shown in B of Figure 5 , in the Jurkat cells, the positive rate of the negative control for promoting the expression of CXCL13 by the cells is 9.16%, and the positive rate of DUF3109 for promoting the expression of CXCL13 by the cells is 52.1%, which is significantly higher than that of the control group; Figure 5 It is shown that the DUF3109 protein has a significant stimulating effect on the secretion of CXCL13 by the mouse CD4+ T lymphocytes.

[0078] In summary, the DUF3109 protein described in the present application promotes the formation of intratumoral TLS and inhibits the progression of liver cancer by regulating the secretion of CXCL13 by CD4+ T cells, the protein exhibits significant anti-tumor activity, has broad application potential, and can be used for developing anti-tumor protein drugs, and provides an innovative treatment idea for the current tumor treatment field.

[0079] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. The application of DUF3109 protein in the preparation of drugs for treating liver cancer, characterized in that, The amino acid sequence of the DUF3109 protein is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, injections, powders, and oral liquids.

3. The application according to claim 1, characterized in that, The dosage form of the drug includes injections.

4. The application of biomaterials in the preparation of drugs for treating liver cancer, characterized in that, The biomaterials include: (1) An expression cassette containing a nucleic acid molecule encoding the DUF3109 protein of claim 1; (2) A recombinant vector containing the expression cassette described in (1); (3) Recombinant cells containing the expression cassette of (1), the recombinant vector of (2), or the DUF3109 protein of claim 1.

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