Use of a caerin polypeptide in combination with a dendritic cell vaccine in the preparation of a medicament for the treatment of breast cancer

Abstract

The application belongs to the field of biological pharmacy, and particularly relates to application of a Caerin polypeptide combined with a dendritic cell vaccine in preparation of a drug for treating breast cancer. The application finds that F1 / F3 significantly inhibits proliferation of 4T-1 cells and induces cell death in vitro, significantly inhibits growth of a primary tumor, reduces lung metastasis, and prolongs overall survival in vivo, and inoculation of a DC vaccine loaded with a tumor-related antigen can reduce tumor growth and enhance T cell activation in draining and non-draining lymph nodes. Compared with a vaccine loaded with a whole antigen (DCV1), a DC vaccine (DCV2) combined with F1 / F3 induced 'programmed death antigen' exhibits stronger anti-tumor activity, although TNF-alpha and IL-12 secretion thereof is lower. Therefore, the application combines the F1 / F3 polypeptide with the DCV2 to prepare a drug for treating breast cancer, effectively improves the treatment effect on the tumor, and highlights the importance of a combined immunotherapy strategy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biopharmaceuticals, and particularly relates to application of a Caerin polypeptide combined with a dendritic cell vaccine in preparation of a drug for treating breast cancer. BACKGROUND

[0002] Breast cancer is caused by malignant transformation of breast epithelial cells under the influence of various carcinogenic factors, leading to a gradual increase in its prevalence. This disease has become the main cause of death from malignant tumors in women, posing a major threat to women's health. Although great progress has been made in the early detection and treatment of breast cancer, the incidence of breast cancer is still on the rise. The heterogeneity of this disease poses a great challenge to its treatment and management. To date, surgery, chemotherapy and radiotherapy are still the basic treatment methods for breast cancer, but poor postoperative prognosis, high recurrence rate and induced drug resistance make it refractory, and there is an urgent need for a treatment to improve the current situation of breast cancer.

[0003] In recent years, immunotherapy has become one of the emerging treatment approaches for cancer and has become the standard of care for many cancer types (such as lung cancer, melanoma, etc.) and specific TNBC patients. Among them, the immune checkpoint inhibitors of PD-1 / PD-L1 such as pembrolizumab and atezolizumab have been approved for the treatment of solid tumors, including breast cancer. Although some effects have been achieved in the treatment of breast cancer, most triple-negative breast cancer patients have poor response to monotherapy. Combination therapy is considered the most promising immunotherapy strategy for cancer and has successfully overcome some drug resistance. The classic combination of PD-1 inhibitors and CTLA-4 inhibitors has been approved for the treatment of various cancers.

[0004] Therapeutic cancer vaccines can promote tumor regression, eliminate minimal residual disease (MRD), and establish long-term anti-tumor memory and avoid non-specific or adverse reactions. Dendritic cells (DCs) are the most effective functional antigen-presenting cells in vivo and are the core of the initiation and regulation of innate immunity and adaptive immunity in the tumor microenvironment. DC vaccines take advantage of the superior antigen-presenting mechanism of DCs by loading DCs with tumor-associated antigens (TAAs) before autologous reinfusion into cancer patients and promoting maturation in vitro to bypass the immunosuppressive mechanisms of the tumor microenvironment (TME) and generate TAA-specific CTLs to initiate tumor cell killing and induce long-term immune memory. DC vaccines are widely used in cancer, such as non-small cell lung cancer (NSCLC), ovarian cancer, prostate cancer, melanoma, renal cell carcinoma and glioblastoma, etc. However, DC vaccines are rarely used in the clinical treatment of breast cancer.

[0005] Therefore, how to use therapeutic vaccines in the clinical treatment of breast cancer is of great significance for the future treatment of breast tumors. SUMMARY

[0006] To solve the above problems, the application improves the inhibition of 4T-1 tumor by studying the inhibition and killing effect of Caerin1.1 / Caerin1.9 on breast cancer cell line 4T-1 in vitro and the inhibition effect on tumor growth and lung metastasis in vivo, and by combining DC vaccine and other immunotherapy.

[0007] For the above technical effects, the application adopts the following specific schemes:

[0008] The application of a Caerin polypeptide combined with a dendritic cell vaccine in the preparation of a drug for treating breast cancer.

[0009] Preferably, the Caerin polypeptide comprises Caerin1.1 and Caerin1.9, wherein the amino acid sequence of Caerin1.1 (i.e., F1) is shown in SEQ ID NO. 1, and the amino acid sequence of Caerin1.9 (i.e., F3) is shown in SEQ ID NO. 2.

[0010] GLLSVLGSVAKHVLPHVVPVIAEHL-NH2 (SEQ ID NO. 1);

[0011] GLFGVLGSIAKHVLPHVVPVIAEKL-NH2 (SEQ ID NO. 2).

[0012] Preferably, the dendritic cell vaccine is a DC vaccine induced by F1 / F3 (i.e., Caerin1.1 and Caerin1.9, the same below) programmed death antigen, which is composed of DC cells and F1 / F3 induced programmed death antigen.

[0013] Preferably, the preparation process of the dendritic cell vaccine is as follows: animal bone marrow cells are extracted, cultured in RPMI1640 culture medium containing IL-4 and GM-CSF, half-volume liquid exchange is performed during the culture, DC cell purification is performed after the culture, then the DC cells are co-cultured with the polypeptide induced programmed death antigen at a volume ratio of 1:3-4, TNFα, IL-1β, IL-6, PEG2 are added during the culture process, and the DC cells are collected, washed and centrifuged, resuspended with PBS, and then obtained.

[0014] Preferably, the preparation process of the F1 / F3 induced programmed death antigen is as follows: tumor cells are stimulated with F1 / F3 polypeptide, then the cells are collected, washed and resuspended, then the cell suspension is subjected to 4-7 freeze-thaw cycle treatments, the cell debris is removed by centrifugation, the supernatant is extracted, and the supernatant is stored at -80℃ for standby.

[0015] Preferably, the process of treating the cell suspension with 4-7 freeze-thaw cycles is: repeatedly placing the cell suspension at -75 to -85 DEG C, and at 36 to 38 DEG C for freeze-thaw cycles 4-7 times.

[0016] Preferably, the drug further comprises a physiological saline and a pharmaceutically acceptable carrier.

[0017] Preferably, the drug is a non-carrier drug.

[0018] Preferably, the dosage form of the non-carrier drug includes but is not limited to injections, tablets, capsules.

[0019] Preferably, the drug is a carrier drug, and the carrier includes but is not limited to viruses, bacterial carriers, nucleic acids, exogenous cell carriers, liposomes, and exosomes.

[0020] Compared with the prior art, the technical advantages of the present application are as follows: the present application proves that F1 / F3 can significantly inhibit the proliferation of 4T-1 tumor cells in vitro, and can significantly inhibit the tumor growth and lung metastasis in vivo, thereby prolonging the survival of mice. In order to further improve the treatment efficiency of 4T-1 tumor, the application of DC vaccine is also studied. The results show that the use of DC vaccine alone does not significantly enhance the inhibition effect of 4T-1, and after combined with F1 / F3, its treatment effect is obviously better than that of F1 / F3 or DC vaccine alone. Therefore, the present application realizes the combined use of DC vaccine and F1 / F3 for treating tumors for the first time, which has important application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 F1 / F3 in vitro detection of cell proliferation chart;

[0022] Figure 2 Different treatment conditions of 4T-1 cell microscopic real-time observation result chart;

[0023] Figure 3 Different treatment conditions of cell apoptosis result chart;

[0024] Figure 4 F1 / F3 inhibits the growth of 4T-1 tumor in vivo result chart;

[0025] Figure 5 F1 / F3 inhibits the metastasis of 4T-1 tumor in vivo result chart;

[0026] Figure 6 Different samples affect T cell proliferation result chart;

[0027] Figure 7 DC vaccine inhibits the growth of 4T-1 tumor in vivo chart;

[0028] Figure 8Figure for INF-γ expression in draining lymph nodes;

[0029] Figure 9 Figure for INF-γ expression in non-draining lymph nodes;

[0030] Figure 10 Figure for immune cell proportion in DCV group in draining lymph nodes;

[0031] Figure 11 Figure for immune cell proportion in DCV group in non-draining lymph nodes;

[0032] Figure 12 Figure for effector cell distribution in draining lymph nodes;

[0033] Figure 13 Figure for effector cell distribution in non-draining lymph nodes;

[0034] Figure 14 Figure for naive T cell distribution in draining lymph nodes;

[0035] Figure 15 Figure for naive T cell distribution in non-draining lymph nodes;

[0036] Figure 16 Figure for effector memory cell distribution in draining lymph nodes;

[0037] Figure 17 Figure for effector memory cell distribution in non-draining lymph nodes;

[0038] Figure 18 Figure for flow cytometry analysis of immune cell changes in UN group and DCV group in mouse spleen;

[0039] Figure 19 Figure for the effect of different samples on inhibiting tumor growth;

[0040] Figure 20 Figure for anti-tumor activity of different test samples;

[0041] Figure 21 Figure for PD-L1 expression after co-culturing DC, T and Tumour by flow cytometry. DETAILED DESCRIPTION

[0042] The application will be further explained below with reference to specific examples. However, it should be noted that the following examples are only used to explain the application and cannot be used to limit the application. All technical solutions that are the same as or similar to the application are within the protection scope of the application. The specific techniques or conditions not mentioned in the examples are operated according to the conventional technical methods and instrument instruction book. The reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased in the market.

[0043] The polypeptides in the experiments of the application were synthesized by Mimotopes Proprietary Limited (Wuxi, China), with a purity of more than 99% and a lipopolysaccharide concentration of less than 0.44 EU / mL. They were stored in a -80℃ refrigerator, dissolved and prepared with PBS after being divided into small portions, mixed uniformly by a vortex instrument, and stored at -20℃ for standby.

[0044] The experimental mice in the application were approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University (Ethics Approval No.: GYFYG2R202326). The tumor size was strictly controlled in the experiment, and the ethical standards were met: mice with tumor diameters exceeding 15 mm were determined to be ethically dead. Adult female Balb / c mice of 6 to 8 weeks old without specific pathogens (SPF) were ordered from the Animal Resources Center of Guangdong Province, China, and weighed about 20 g. The mice were raised under SPF conditions in the animal facilities of the First Affiliated Hospital of Guangdong Pharmaceutical University. Five mice were kept in each cage, the temperature was 22℃ and the humidity was 60%, and sterile standard mouse food and water were provided, as well as a 12-hour light / 12-hour dark cycle.

[0045] The breast cancer cells 4T-1 in the application were purchased from Procell Life Science & Technology Co., Ltd. in Wuhan. The 4T-1 cells were cultured in DMEM medium (Gibco) with the addition of 10% fetal bovine serum (FBS; Gbico) and 100 mM penicillin / streptomycin (Gbico). The cells were cultured in a humidified incubator at 37℃ with 5% CO2.

[0046] The results obtained in the experiments were analyzed using GraphPad Prism 9 and FlowJo-v10.8.1 software. The results were expressed as mean ± standard deviation by T test, two-way ANOVA analysis, and Kaplan-Meier method analysis, and the difference was statistically significant when P<0.05.

[0047] Test Example 1 F1 / F3 induced programmed death of 4T-1 cells in vitro

[0048] 1. Test Samples: F1 peptide, F3 peptide, F1 / F3 peptide, P3 peptide (GTELPSPPSVWFEAEFK-OH); 2. Test Procedure: MTT assay: 4T-1 cells were cultured at 1×10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured for 24 hours. Then, F1 peptide, F3 peptide, and F1 / F3 peptide were added sequentially at concentration gradients of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg / mL, and cultured for 18 hours, with three replicates for each concentration. Next, 10 μL of MTT (Beyotime) was added to each well, and the cells were cultured for 4 hours. Finally, 50 μL of dimethyl sulfoxide (DMSO; Sigma-Aldirich) was added. The absorbance was measured at 540 nm using a microplate reader. Cell viability was calculated using the formula [(Experimental well OD value - Blank well OD value) / (Control well OD value - Blank well OD value)]. The half-maximal inhibitory concentration (HIC) of the drug was then calculated.

[0049] Apoptosis detection: Apoptosis was assessed using an apoptosis detection kit (Annexin V-FITC Apoptosis Detection Kit, Beyotime). 4T-1 cells were cultured to the logarithmic growth phase, and then multiplied at 5 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured for 24 hours. Then, 10 μg / mL of F1 / F3 peptide was added, with 10 μg / mL of P3 peptide as a control. An untreated group (UN group as a control) was also included. All cells were cultured for 18 hours. Cells were digested with EDTA-free trypsin (Gibco), stained with 5 μL Annexin V-FITC and 10 μL propidium iodide staining solution, incubated at room temperature in the dark for 20 minutes, and immediately analyzed by flow cytometry.

[0050] Morphological inspection: A 12-well plate was used, with 1×10⁻⁶ ohms per well. 5 Cells were seeded with 4T-1 and cultured for 24 hours. Cells were washed twice with PBS, and 10 μg / mL of F1 / F3 peptide was added. P3 peptide at 10 μg / mL was used as a control. An untreated group (UN group as a control) was also set up. Cells were cultured for 18 hours, and cell morphology changes were observed in real time under bright field conditions using a fluorescence microscope.

[0051] 3. Test Results: The MTT test results are as follows: Figure 1 As shown, F1, F3, and F1 / F3 all significantly inhibited the proliferation of 4T-1 cells. The half-maximal inhibitory concentrations (IC50) of F1, F3, and F1 / F3 were 9.607 μg / ml, 15.91 μg / ml, and 7.472 μg / ml, respectively. The combined use of F1 and F3 showed the best inhibitory effect. Figure 1A~C). By real-time observation under microscope, it was found that the morphology of 4T-1 cells was significantly changed under the action of 10 μg / ml F1 / F3, while the morphology of cells in the untreated group (UN group) and P3 group was basically not changed. Figure 2

[0052] The apoptosis kit was used to detect the apoptosis degree of 4T-1 cells under the action of F1 / F3. It was found by experiment that the addition of 10 μg / ml F1 / F3 could significantly induce the apoptosis of 4T-1 cells, and the apoptosis rate was 61.7%, while the apoptosis rates of the untreated group and P3 group were 27.5% and 32.8% respectively. Figure 3 The above studies all show that F1 / F3 can significantly inhibit 4T-1 cells in vitro and induce apoptosis.

[0053] Test Example 2 F1 / F3 can inhibit the growth of 4T-1 tumor and lung metastasis in vivo

[0054] 1. Test sample: F1 / F3 polypeptide, P3 polypeptide, PBS solution;

[0055] 2. Test process: 4T-1 cells were cultured to the second generation using DMEM medium, and then 4T-1 tumor-bearing model and 4T-1 lung metastasis model were constructed.

[0056] First, the 4T-1 tumor-bearing model was constructed: 4T-1 cells with 70% confluence were collected by trypsin digestion and washed repeatedly with PBS, and 5×10 5 / 200 μL of 4T-1 cells were subcutaneously inoculated in the flank of each mouse, recorded as Day 0, and the tumor size of each mouse was measured every 2 days at the end of the test on the same day, and the tumor volume was calculated as length × (short diameter / 2) 2 . When the tumor diameter reached 3-5 mm, the mice were grouped according to the experimental requirements, with 8 mice in each group.

[0057] Then, P3 polypeptide and Caerin 1.1 / Caerin 1.9 (i.e. F1 / F3) polypeptide solution were prepared. Or 100 μL of PBS for injection containing 30 μg of P3 polypeptide was injected into each mouse, or 100 μL of PBS for injection containing F1 / F3 (F1, F3 each 30 μg) was injected into each mouse, or only 100 μL of PBS was injected, and injected into the tumor on Day 3. The injection was continued for 7 days. The length and width of the tumor of each mouse were measured with a vernier caliper, and then the tumor volume was calculated by the formula [length × (width / 2) 2 ]. When the length or width of the tumor of the mouse was greater than or equal to 15 mm, the mouse was sacrificed by cervical dislocation, and the weight of the tumor was measured.​

[0058] Construction of a 4T-1 lung metastasis model: 4T-1 cells cultured to 70% confluence were digested and collected using trypsin, washed repeatedly with PBS, and seeded at a concentration of 5 × 10⁶ cells per mouse under the subcutaneous fat pad of the fourth pair of mammary glands. 5 4T-1 cells were administered at a rate of 200 μL. Tumor size in mice was measured every 2 days using calipers. Tumor volume was calculated as major axis × (minor axis / 2). 2 After about 30 days, the mouse lungs were removed for staining, and the number of lung nodules was counted. Seven mice were used in each group.

[0059] Four days after inoculating the subcutaneous fat pads of the fourth pair of mammary glands in mice with 4T-1, when the tumor diameter reached 3-5 mm, the mice were injected intratumorally with 100 μL of PBS solution containing 30 μg F1 / F3 peptides, or 100 μL of PBS solution containing 30 μg P3 peptides, or only 100 μL of PBS solution, for 7 consecutive days. On Day 30, the mice were sacrificed and injected with Indian ink, then the lungs were removed, washed in PBS, and then placed in Fekete's solution (70 mL anhydrous ethanol, 5 mL glacial acetic acid, 10 mL formalin, 30 mL pure water) for 20 minutes. Finally, the lung tumor nodules were counted.

[0060] 3. Test Results: The test results are as follows Figures 4-5 As shown, the data represents a single independent experiment, while Figure 5 C summarizes data from two independent experiments, replicated once. Results are expressed as mean ± standard deviation or mean ± standard error. ns, no significant difference; *P<0.05; **P<0.01; ****P<0.0001. Statistical analysis was performed using two-way ANOVA (…). Figure 4 B, 5A), Kaplan-Meier survival analysis ( Figure 4 C, 5B), one-way ANOVA ( Figure 4 D) and Student's t test ( Figure 5 C).

[0061] Given that F1 / F3 induces programmed cell death in 4T-1 cells in vitro, a Balb / c mouse lateral ventral 4T-1 tumor model was further constructed and F1 / F3 were administered intratumorally. Figure 4 A). The results showed that, compared with the PBS group and the P3 group, the F1 / F3 group significantly inhibited the growth of mouse tumors (A). Figure 4 B), and significantly prolonged the survival time of mice, with a median survival time of 37 days in the F1 / F3 group, and median survival times of 27 days and 27 days in the PBS group and P3 group, respectively. Figure 4C). The tumors were removed from the mice around Day 18 and weighed. It was found that the tumor weights of the F1 / F3 group were significantly smaller than those of the PBS group and the P3 group. The average tumor weight of the F1 / F3 group was 0.0471 g, and the average tumor weights of the PBS group and the P3 group were 0.1221 g and 0.1256 g, respectively Figure 4 D, Table S1A).

[0062] In clinical practice, breast cancer has a tendency to metastasize to distant sites, such as the lung, bone, liver, brain, and the like. Therefore, the F1 / F3 was used to study the inhibition of 4T-1 metastasis by constructing a Balb / c mouse orthotopic 4T-1 tumor-bearing treatment model. The results showed that, compared with the UN group and the P3 group, the F1 / F3 group could significantly inhibit the growth of tumors and prolong the survival cycle of mice. The median survival cycle of the F1 / F3 group was 36 days, and the median survival cycles of the UN group and the P3 group were 28 days and 30 days, respectively Figure 5 A-B), and the lung staining results showed that the number of lung tumor nodules in the F1 / F3 group was significantly reduced compared with the UN group and the P3 group. The average number of tumor nodules in the F1 / F3 group was 2, and the average numbers of tumor nodules in the UN group and the P3 group were 7 and 11, respectively Figure 5 C). The above studies showed that F1 / F3 could significantly inhibit the growth of 4T-1 tumors and prolong the survival cycle of mice in vivo, and could also significantly reduce the metastasis to the lung.

[0063] Preparation of tumor antigens

[0064] Preparation of whole antigen (TAA1): 4T-1 cells in the logarithmic phase were collected, washed with PBS, and then resuspended in RPMI1640 medium containing 400 μL of G418 antibiotic (purchased from Gibco) to a concentration of 1 x 10 7 / mL. The cell suspension was subjected to 5 cycles of freeze-thawing at -80°C and 37°C. Then, the cell fragments were removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant was extracted and stored at -80°C.

[0065] Preparation of polypeptide-induced programmed death antigen (TAA2): 4T-1 or TC-1 cells (TC-1 cells were purchased from Shanghai Institutes for Biological Sciences) were stimulated with 10 μg / mL of F1 / F3 (the concentrations of F1 and F3 were both 10 μg / mL) polypeptide for about 1 hour, and then the cells were collected, washed with PBS, and resuspended in RPMI1640 medium to a concentration of 1 x 10 7The cell suspension was treated with 5 cycles of freeze-thawing at -80°C and 37°C. Then the cell debris was removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant was extracted and stored at -80°C.

[0066] Preparation of DC vaccine: The mouse bone marrow cells were extracted and cultured in a culture dish at 1 x 10 7 DC cells were collected on the 7th day, washed twice with solution 1 (2.5 mL PBS + 1 mL 100 mM EDTA + 46.5 mL PBS) prepared in advance, centrifuged at 540 g for 5 minutes, resuspended the cells with 6 mL of solution 1, and finally added to solution 2 (solution 2 was prepared with 2.3 volumes of solution A + 9.7 volumes of solution B, A was OptiPrep reagent, and B was prepared with 0.88% (w / v) + sodium chloride + 1 mM EDTA + 0.5% bovine serum albumin + 0 mM Tricine-NaOH), then centrifuged at 700 g for 25 minutes, with a requirement of 5 up and 0 brake, and after the end, the middle separation layer liquid was taken as the purified DC cells (labeled as DCV).

[0067] Then the 24-well plate was taken out, and the DCV group and the DCV(TAA) group were labeled respectively. The DCV(TAA) group was added with DC cells and TAA, with a volume ratio of DC cells: TAA (TAA1 or TAA2) = 1:3, and was placed in the incubator for overnight culture. The next day, TNFα (10 ng / mL), IL-1β (10 ng / mL), IL-6 (100 ng / mL), and PEG2 (0.2 mg / mL) were added for stimulation for more than 8 hours. Finally, the collection was washed with PBS once, then centrifuged and added with RPMI1640 complete culture medium for standby. The cells were counted and quantified with PBS at about 1 x 10 6 / 100 μL, and the DCV1 and DCV2 vaccines were obtained. The one loaded with TAA1 was recorded as DCV1, and the one loaded with TAA2 was recorded as DCV2.

[0068] T cell collection: Take 4 healthy C57BL / 6 mice (C57BL / 6 mice source and feeding method are the same as Balb / c mice), after the mice are killed, use the dissecting instruments to take out the mouse spleen, and immerse it in RPMI1640 medium for standby. Crush the spleen with the needle handle of a 5 mL syringe, then collect the cells and pass through a 70 μm filter screen, centrifuge to remove the upper liquid part, add 4 mL of red blood cell lysis solution (biosharp, model BL503B) to lyse the cells for 3 minutes, then add Wash buffer and centrifuge again, then add an appropriate amount of Wash buffer to count the cells, and finally use CD4 / CD8 (TIL) MicroBeads magnetic bead separation kit (Miltenyi Biotec) to separate T cells, and after separation, add an appropriate amount of Wash buffer to count the cells.

[0069] Test Example 4 Flow cytometry detection

[0070] I. The specific detection process is as follows:

[0071] 1. Plating and co-culturing. After obtaining the required DC cells and T cells on the same day, prepare the 24-well plate in advance, label the T, DCV+T, and DCV(TAA1)+T groups on the well plate, then add the DC cells and T cells to the corresponding wells according to the volume ratio of 1:5, and finally place it in the incubator for 3 days of culture.

[0072] 2. Flow detection. After 3 days of co-culture, collect the cells into the flow tube labeled in advance, centrifuge and count the cells to ensure that there are 1x10 6 cells in the flow tube for machine, then perform staining according to the flow scheme in Table 1. Since the INFγ antibody is an intracellular antibody, it needs to be added after the staining of the remaining extracellular antibodies is complete, and then 1 mL of Wash buffer is used to wash the cells, followed by the addition of 250 μL of flow intracellular fixation and membrane permeabilization buffer (purchased from eBioscience) per tube, and placed on ice for 60 min. After the end, wash twice with the above-mentioned flow intracellular fixation and membrane permeabilization buffer, each time with 1 mL, then stain the INFγ antibody, incubate on ice for 30 min in the dark, finally centrifuge with 1 mL of PBS, and immediately add 300 μL of Wash buffer to the machine.

[0073] Table 1 Flow scheme

[0074]

[0075] Then construct a 4T-1 tumor-bearing DCV-treated mouse model:

[0076] After successfully constructing the mouse tumor-bearing model (i.e., the 4T-1 tumor-bearing model) using the method described in Experiment Example 2, on Day 3, mice were randomly assigned to the untreated group (UN), DCV1 group (DCV+TAA1), DCV2 group (DCV+TAA2), and F1 / F3 group. Tumor size was measured and recorded. Subsequently, the F1 / F3 group received 30 μg / 100 μL Caerin peptide injections (F1 / F3) for 7 consecutive days, with an injection volume of 100 μL. DCV1 and DCV2 vaccines were administered at doses of 1×10⁻⁶ on Day 3 and Day 9. 6 One cell / 100μL per mouse. The untreated group received no treatment. Tumor growth and mouse survival were observed. The injection volume was 100μL for all mice.

[0077] The expression of INFγ in the lymph nodes and spleen of mice treated with DC vaccine was analyzed by flow cytometry:

[0078] After successfully constructing the animal tumor-bearing and treatment model using the above method, the mice were given a third dose of DCV1 or DCV2 vaccine on Day 23. Drained and non-drained lymph nodes and spleens were removed, and single-cell suspensions were prepared from the lymph nodes and spleen. 24-well plates were prepared, labeled with UN and DCV groups, and incubated with anti-CD3e (2 mg / mL) for 2 hours. Then, 2 × 10⁻⁶ cells were added to each well. 6 Add cells to the corresponding wells and add Cellstimulation Cocktail Plus Protein Transport Inhibitor (500x, eBioscience) to culture for 10 hours. Finally, collect the cells, stain them according to the staining protocol in Table 2, and perform the above-mentioned procedures before testing.

[0079] Table 2 Flow Cytometry Scheme

[0080]

[0081] Flow cytometry was used to analyze the changes in the number of various T cells in the lymph nodes and spleen of mice after treatment with DC vaccine:

[0082] After successfully constructing the animal tumor-bearing and treatment model according to the above method, the mice were given a third dose of DCV1 or DCV2 vaccine on Day 23. The drained and non-drained lymph nodes and spleen were removed, and the lymph nodes and spleen were prepared into single-cell suspensions. Then, they were stained according to the staining protocol in Table 3 and analyzed by machine.

[0083] Table 3 Flow Cytometry Scheme

[0084]

[0085]

[0086] Construct 4T-1 tumor-bearing mice model treated with Caerin1.1 / Caerin1.9 combined with DCV:

[0087] After the model is successfully constructed by the method of the mouse tumor-bearing model of Experimental Example 2 (i.e., 4T-1 tumor-bearing model), the mice are randomly divided into cages on Day 3, and the PBS group, the F1 / F3 group, the DCV1+F1 / F3 group, and the DCV2+F1 / F3 group are set up. After the mice are randomly placed, the tumor size is recorded. The F1 / F3 group is continuously given Caerin1.1 / Caerin1.9 (F1 and F3 are both 30 μg / 100 μL, and the injection volume is 100 μL) from Day 3, and the injection is performed for 7 days. The DCV1+F1 / F3 group is continuously given F1 / F3 (F1 and F3 are both 30 μg / 100 μL, and the injection volume is 100 μL) from Day 3, and the injection is performed for 7 days. At the same time, DCV1 is given on Day 3 and Day 9, and the injection volume is 100 μL. The DCV2+F1 / F3 group is similar to the DCV1+F1 / F3 group, and DCV1 is replaced by DCV2. The PBS group is continuously given 100 μL of PBS buffer for 7 days, and the tumor size and the survival period of the mice are observed. The co-culture of tumor cells is performed, and the killing of tumor cells and the proliferation of T cells are analyzed by flow cytometry:

[0088] The DC cells (which need to be loaded with TAA1 / TAA2) and T cells are treated by the above method. The T cells are dyed with eFlour dye, and the above cells are temporarily placed in a culture box for standby. In a 24-well plate, the DCV1+T+Tumour group, the DCV2+T+Tumour group, and the Tumour group are set up. The 4T-1 and TC-1 cells are digested and dyed with CFSE dye. Finally, the DC cells, T cells, and 4T-1 / TC-1 cells are co-cultured at a cell number ratio of 2:10:1, and the culture is performed for 3 days.

[0089] Then, the supernatant of the cells in the well plate is collected in a 1.5 mL tube, and the corresponding label is prepared. The tube is placed in a-80℃ refrigerator for standby. The cells in the well plate are washed once with PBS, and then 300 μL of trypsin without EDTA is added for digestion. Then, the cells are centrifuged and counted, 1×10 6 cells per tube. Finally, the cells are dyed according to the dyeing scheme in Table 4 and Table 5, and finally analyzed by flow cytometry.

[0090] Table 4 Flow Cytometry Scheme 1

[0091]

[0092]

[0093] Table 5 Flow Cytometry Scheme 2

[0094]

[0095] Detection of the secretion of immune-related factors in the co-culture supernatant by an ELISA kit

[0096] The previously frozen co-culture supernatant at -80°C was taken out for thawing, and the operation was performed according to the instructions of the LISA kit (purchased from Biolegend), and the secretion of TNFα, IL-12 and IL-10 was detected, respectively.

[0097] II. Experimental results:

[0098] 1. The DC vaccine can effectively inhibit the growth of 4T-1 tumor and prolong the survival cycle of mice

[0099] The literature research results show that the 4T-1 tumor has no unique specific antigen, and the existing therapeutic vaccine is mostly a vaccine targeting or dependent on DC cells. The combination of F1 / F3 and anti-PD-1 cannot further inhibit the growth of 4T-1 tumor, and the present application further selects the DC vaccine as a potential combination therapy strategy for F1 / F3. The results are shown in Figure 6 (A) CD4+T cells (CD45.2+CD3+CD4+) and (B) CD8+T cells (CD45.2+CD3+CD8+) expression, and (C) IFNγ (CD45.2+CD3+CD8+IFNγ+) secretion. The data of (A-C) represent an independent experiment, each experiment is repeated once, and the data are expressed as mean ± SD. ns: not significant; *P<0.05; ***P<0.001; ****P<0.0001. (A-C) were tested by one-way ANOVA.

[0100] It can be seen that, compared with DC cells without antigen (DCV) and T cell groups only, DC cells loaded with tumor antigen (DCV(TAA1, same below)) can more effectively stimulate T cell proliferation, making the number of CD4 + T cells and CD8 + T cells significantly increased (77.5% vs. 72.2% vs. 64.4% for CD4+T cells, and 26.1% vs. 18.8% vs. 15.7% for CD8+T cells) ( Figure 6 A, B), and at the same time, the secretion of INF-γ was increased (1.43% vs. 0.96% vs. 0.71%) ( Figure 6 C).

[0101] To further verify the therapeutic feasibility of the whole antigen DC vaccine in 4T-1 tumor-bearing mice in vivo, a DC vaccine loaded with 4T-1 tumor whole antigen (TAA) was constructed, and the results are shown in Figure 7 Figure 7 ​A, middle, is a 4T-1 tumor-bearing mouse model established in the flank, comparing tumor growth in untreated group (UN), DC vaccine group (DCV) and F1 / F3 group; B is the tumor growth curve; C is the mouse survival cycle, using Kaplan-Meier analysis, Student's t test. ns, no significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. The results show that the DC vaccine can effectively inhibit the growth of 4T-1 tumor and prolong the survival cycle of mice; among them, the median survival time of DC vaccine group (DCV group) and untreated group (UN group) loaded with tumor antigen is 41 days and 30 days, respectively, but there is no significant difference compared with F1 / F3 group alone (median survival time 39 days) Figure 7 B, C).

[0102] 2. DC vaccine loaded with tumor antigen can induce strong T cell response

[0103] Subsequently, flow cytometry was used to analyze the changes in the immune microenvironment, including draining lymph nodes, non-draining lymph nodes and spleen. The results showed that in the draining lymph nodes, the expression of INF-γ in the DC vaccine group (10.2%) was lower than that in the UN group (27.1%), while in the non-draining lymph nodes, the expression of INF-γ in the DCV group (DC+TAA1, same below) was significantly higher than that in the UN group (11.5% vs. 1.5%) Figure 8 )( Figure 9 ) Further analysis found that in the draining lymph nodes, the proportion of immune cells CD45.2, CD3 + CD4+T cell subsets in the DCV group were significantly higher than those in the UN group (99.7% vs. 98.5%, 50.9% vs. 36.5%, 60.2% vs. 42.8%), while the CD8+T cell was significantly lower (35.1% vs. 48.7%) Figure 10 ) Similar results were observed in non-draining lymph nodes Figure 11 ).

[0104] In further analysis of various T cells, the effector T cells (CD45.2+CD3e+CD44 high CD62L-) in the DCV group were significantly higher than those in the UN group in the draining lymph nodes (66.8% vs. 55.4%), but significantly lower in the non-draining lymph nodes (67.1% vs. 84.3%). In addition, the effector CD4 + T cells (CD45.2+CD3e+CD44 high CD62L-CD4+) in the DCV group (82.9%) were significantly higher than those in the UN group (63.3%), while the effector CD8 +T cells (CD45.2+CD3e+CD44 high CD62L-CD8+) were decreased (40.2% vs. 78.5%) in DCV group Figure 12 ) in non-draining lymph nodes. + CD4 + T cells were slightly changed, while effector CD8 high T cells were significantly up-regulated (16.5% vs. 12.1%) Figure 13 ) in DCV group.

[0105] Further, in draining lymph nodes, the proportion of naive T cells (CD45.2+CD3e+CD62L + CD44-) and its subtype naive CD4 high T cells (CD45.2+CD3e+CD62L + CD44-CD4+) were significantly higher in DCV group (47.6% vs. 6.59%), while naive CD8 high T cells (CD45.2+CD3e+CD62L high CD44-CD8+) were significantly decreased (40.2% vs. 78.5%) in DCV group Figure 14 ) in non-draining lymph nodes. Figure 15 ) in DCV group. Meanwhile, effector memory CD4 + T cells (CD45.2+CD3e+CD44 high CD62L-CCR7-CD4+) were significantly increased in draining lymph nodes (82.7% vs. 58.8%), while effector memory CD8 + T cells (CD45.2+CD3e+CD44 high CD62L-CCR7-CD8+) were decreased in DCV group. In non-draining lymph nodes, effector memory CD4 + T cells were slightly decreased, while effector memory CD8 + T cells were significantly up-regulated (14.7% vs. 11%) in DCV group Figure 16 ) in non-draining lymph nodes. Figure 17 ) in non-draining lymph nodes.

[0106] Finally, the same detection was performed on spleen, and the results were shown in Table 5. Figure 18Figure 1 shows the results of the experiment. The results are shown in (A) INF-γ (CD45+CD3e+CD8+INF-γ+) secretion and T cell and T cell subtypes expression, including: (B) CD45.2 cells, T cells (CD45.2+CD3e+), CD4 T cells (CD45+CD3e+CD4+), CD8 T cells (CD45+CD3e+CD8+), (C) Effector T cells (CD45.2+CD3e+CD44 high CD61L-), Effector CD4 T cells (CD45.2+CD3e+CD44 high CD61L-CD4+), Effector CD8 T cells (CD45.2+CD3e+CD44 high CD61L-CD8+), (D) Naive T cells (CD45.2+CD3e+CD62L high CD44-), Naive CD4 T cells (CD45.2+CD3e+CD62L high CD44-CD4+), Naive CD8 T cells (CD45.2+CD3e+CD62L high CD44-CD8+), (E) Effector Memory T cells (CD45.2+CD3e+CD44 high CD61L-CCR7-), Effector Memory CD4 T (CD45.2+CD3e+CD44 high CD61L-CCR7-CD4+), Effector Memory CD8 T (CD45.2+CD3e+CD44 high CD61L-CCR7-CD8+), 3-6 mice per group, data in (A-E) represent one independent experiment, each experiment was repeated once, expressed as mean ± SD or mean ± SEM. ns: not significant; ***: P value <0.001; ****: P value <0.0001. T test analysis (A-E). It can be seen that there is no statistical difference between the DC vaccine group and the untreated group in terms of INF-γ, immune cell CD45.2 and expression of various T cells Figure 18 In summary, the DC vaccine loaded with 4T-1 tumor whole antigen can enhance the immune response of mice in vivo and produce significant inhibition on 4T-1 tumor.

[0107] 3. DC vaccine loaded with Caerin polypeptide-induced programmed death whole antigen is superior to whole antigen DC vaccine

[0108] The intratumoral injection treatment effect of DCV1 and DCV2 was evaluated in 4T-1 tumor-bearing mouse model. The results are as follows Figure 19Figure 6. DCV1 and DCV2 can inhibit tumor growth and prolong survival of 4T-1 tumor-bearing mice. A) Mice were treated with DCV1, DCV2 or F1 / F3 alone, and tumor growth was recorded. B) Survival rate of mice treated with DCV1, DCV2 or F1 / F3 alone. Then, to determine whether DC vaccine can enhance the efficacy of F1 / F3, mice were treated with DCV1+F1 / F3 or DCV2+F1 / F3, and tumor growth was measured. C) Survival rate of mice treated with DCV1+F1 / F3 or DCV2+F1 / F3. Each group contained 3-6 mice. Data in (A-C) represent a single independent experiment, each repeated once. Results are expressed as mean ± SD or mean ± SEM. ns, not significant; ***P < 0.001; ****P < 0.0001. Statistical significance was assessed by two-way ANOVA (B, D) and Kaplan-Meier survival analysis (C).

[0109] Results showed that both DCV1 and DCV2 alone can significantly inhibit tumor growth and prolong the survival of mice compared with the untreated group (UN group), but there is no significant difference between the two, and there is no statistical difference compared with the F1 / F3 alone group Figure 19 A, B). Specifically, the median survival time of the DCV1 group was 49 days, the DCV2 group was 48 days, the F1 / F3 group was 43 days, and the UN group was only 31 days. Given that the DC vaccine and F1 / F3 alone showed similar tumor inhibition, combined with the previous flow cytometry results of F1 / F3 alone and DCV1 improving tumors, to further explore whether the combination of DC vaccine and F1 / F3 can achieve better efficacy Figure 19 C). Results showed that both DCV1+F1 / F3 and DCV2+F1 / F3 groups had better tumor inhibition effect than the F1 / F3 alone group, among which the difference between DCV2+F1 / F3 and F1 / F3 alone reached statistical significance, while DCV1+F1 / F3 also showed a better trend but did not reach a significant level Figure 19 D), DCV2+F1 / F3 is better than DCV1+F1 / F3.

[0110] 4. Compared with DCV1, DCV2 can stimulate stronger T cell response

[0111] To study the mechanism of DCV2 having better tumor inhibition effect than DCV1, the present application co-cultured DCV1 or DCV2 with T cells and TC-1 or 4T-1 tumor cells in vitro. The results are shown in Figure 20 Figure 20 ​The graph shows that DCV2 exhibits superior antitumor activity compared to DCV1. A shows the establishment of a 4T-1 tumor-bearing mouse model in the flank. Mice were treated with DCV1, DCV2, or F1 / F3 alone, and the results were recorded; B shows the tumor growth results; C shows the survival rate. Next, to determine whether the DC vaccine could enhance the efficacy of F1 / F3, mice were treated with DCV1+F1 / F3 or DCV2+F1 / F3, and the results were measured; D shows the tumor growth. Each group contained 3-6 mice. Data in (AD) represent single independent experiments, each repeated once. Results are expressed as mean ± standard deviation or mean ± standard error. NS, no significant difference; ***P < 0.001; ****P < 0.0001. Statistical significance was assessed by two-way ANOVA (B, D) and Kaplan-Meier survival analysis (C). Therefore, compared with the control groups (Tumor, T+Tumor), the experimental groups (DCV1+T+Tumor, DCV2+T+Tumor) showed cytotoxic effects on 4T-1 cells, with DCV2 being more effective than DCV1 (93.8% vs. 94.2%). Furthermore, compared with DCV1, DCV2 significantly promoted T cell proliferation (2.61% vs. 2.3%). Figure 20 A, B). A similar trend was also observed in the TC-1 model.

[0112] ELISA analysis of the culture supernatant showed that in the 4T-1 model, both experimental groups significantly enhanced the ability of T cells to secrete immune-related inflammatory factors (such as TNF-α and IL-12). However, the secretion levels of these factors in the DCV2+T+Tumor group were lower than those in the DCV1+T+Tumor group (20C, D). In the 4T-1 model, IL-10 production increased in both experimental groups, but the IL-10 secretion level in the DCV2+T+Tumor group was significantly lower than that in the DCV1+T+Tumor group (20C, D). Figure 20 E). A similar trend was observed in the TC-1 system.

[0113] These results indicate that, compared to DCV1, DCV1 induced a stronger T-cell response, manifested as enhanced cytotoxicity and proliferation, but with reduced IL-10 secretion, which may explain its superior anti-tumor activity. Further analysis, using a T-test, showed that both experimental groups significantly upregulated PD-L1 expression on 4T-1 cells compared to the control group (0.085% vs. 0.13% vs. 1.95% for DCV1 and DCV2, respectively), and a similar trend was observed in TC-1 cells. Figure 21 ).

[0114] These findings suggest that DCV2 induced stronger T cell responses with enhanced cytotoxicity and proliferative capacity but lower IL-10 secretion, which might be the reason for its superior anti-tumor activity. In addition, the upregulation of PD-L1 on tumor cells suggests a potential benefit of combining DCV2 with anti-PD-Ll therapy to further improve anti-tumor efficacy.

Claims

1. The application of a Caerin peptide combined with a dendritic cell vaccine in the preparation of a drug for treating breast cancer; wherein the dendritic cell vaccine is a DC vaccine induced by F1 / F3 programmed death antigen, consisting of DC cells and F1 / F3 induced programmed death antigen; the preparation process of the F1 / F3 induced programmed death antigen is as follows: tumor cells are stimulated with F1 / F3 peptide, then the cells are collected, washed and resuspended, then the cell suspension is subjected to 4-7 freeze-thaw cycles, centrifuged to remove cell debris, the supernatant is extracted and stored at -80℃ for later use; The preparation process of the dendritic cell vaccine is as follows: animal bone marrow cells are extracted and cultured in RPMI 1640 medium containing IL-4 and GM-CSF. Half of the medium is replaced during the culture. After culture, the DC cells are purified. Then, the DC cells are co-cultured with F1 / F3 peptide-induced programmed death antigen at a volume ratio of 1:3-4. During the culture, TNFα, IL-1β, IL-6, and PEG2 are added for stimulation. The cells are collected, washed, centrifuged, and resuspended in PBS to obtain the vaccine. The Caerin polypeptides are Caerin 1.1 and Caerin 1.9, wherein, Caerin1.1 is designated as F1, and Caerin1.9 as F3. The amino acid sequence of the Caerin1.1 polypeptide is shown in SEQ ID NO.1, and the amino acid sequence of the Caerin1.9 polypeptide is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The process of subjecting the cell suspension to 4-7 freeze-thaw cycles is as follows: repeatedly place the cell suspension at -75 to -85°C and 36 to 38°C for 4-7 freeze-thaw cycles.

3. The application as described in claim 1, characterized in that, The drug also includes physiological saline and a pharmaceutically acceptable carrier.

4. The application as described in claim 1, characterized in that, The drug is either a non-carrier drug or a carrier drug.

5. The application as described in claim 4, characterized in that, The dosage form of the non-carrier drug is one of injection, tablet, or capsule.

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