Tumor whole tissue source antigen polypeptide tumor nano vaccine and preparation method thereof
By extracting tumor whole tissue antigen peptides and preparing nanoparticle vaccines through high-temperature hydrothermal method, the problems of insufficient tumor vaccine coverage and low delivery efficiency were solved, and efficient tumor inhibition effect and low-cost preparation were achieved.
Patent Information
- Application Number
- CN202510808191.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-17
- Publication Date
- 2025-09-26
AI Technical Summary
Existing tumor vaccines lack coverage diversity and stability, personalized vaccines are expensive and complex to prepare, and traditional delivery systems are inefficient, resulting in limited immunotherapy effects.
The high-temperature hydrothermal method was used to extract tumor tissue antigen peptides, and nanoparticle vaccines with uniform particle size were prepared by ethanol precipitation. BSA and adjuvants were combined to form a peptide-BSA complex to improve the efficiency of antigen presentation.
It achieves efficient coverage of tumor antigens and significantly inhibits tumor growth in tumor-bearing mice. It is simple to prepare, low-cost, has a significant immune response and good safety.
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Figure CN120695170A_ABST
Abstract
Description
Field of the Invention
[0001] The present invention belongs to the field of tumor treatment. Specifically, the present application provides a tumor whole tissue derived antigen polypeptide tumor nanovaccine and a preparation method thereof. Background Art
[0002] Although tumor vaccines, as an emerging immunotherapy, show potential, they still face multiple technical bottlenecks and clinical challenges.
[0003] Single antigen (bacterial or cell-derived) tumor vaccine: A single antigen is difficult to cover all tumor cells, especially subpopulation differences caused by mutation accumulation or clonal evolution, which can easily lead to immune escape; secondly, single antigens of bacterial or cell origin are only expressed in some patients or tumor types, and are therefore applicable to a narrow population.
[0004] Peptide vaccines have problems: Peptides are unstable and easily degraded by proteases, resulting in a short half-life that affects their immune stimulation. They require adjuvants or delivery systems (such as liposomes and nanoparticles) to enhance their stability. Furthermore, tumors are highly heterogeneous, making it difficult for a single peptide to cover all subclones.
[0005] Problems with personalized tumor vaccines: Personalized vaccine algorithms are highly algorithmic, with only approximately 10%-30% of predicted neoantigens being truly immunogenic. Patient-to-patient variability makes universal prediction models inapplicable to all populations. Tumor heterogeneity can lead to missed antigens, resulting in incomplete vaccine coverage and impacting vaccine effectiveness. Furthermore, the process from tumor sampling, sequencing, neoantigen prediction, to vaccine production typically takes 4-8 weeks, and the complex manufacturing process makes it costly.
[0006] Issues with tumor-derived whole-cell vaccines: Most tumor-derived whole-cell vaccines have low single-agent response rates (typically <10%) and require combination therapy with other therapies. If the vaccine is derived from allogeneic tumor cells, it may lack patient-specific antigens, limiting efficacy. Summary of the Invention
[0007] In one aspect, the present application provides a tumor tissue-derived antigen polypeptide tumor nanovaccine, wherein the preparation method of the vaccine comprises:
[0008] (1) Extraction of tumor tissue antigen peptides using high-temperature hydrothermal method;
[0009] (2) Formation of peptide-BSA complex and preparation of nanovaccine using ethanol-protein precipitation method
[0010] Furthermore, in step (1), 1-10 g of fresh tumor tissue is taken, chopped, and placed in a high-pressure reactor; deionized water is added to fully infiltrate the tumor tissue; the high-pressure reactor is placed in a 100-150° C. oven to react for 6-12 hours; after the reaction is completed, the product is filtered using filter paper and the filtrate is collected; the filtrate is freeze-dried to obtain a tumor whole tissue antigen polypeptide powder.
[0011] Furthermore, step (2) includes:
[0012] (2-1) using deionized water to prepare the tumor whole tissue antigen polypeptide obtained in step (1) into a 10-30 mg / mL polypeptide solution;
[0013] (2-2) Take 1 mL of the peptide solution, add 10 mL of a 20 mg / mL BSA solution, and stir magnetically for 5-10 minutes to form a peptide-BSA complex;
[0014] (2-3) adding an adjuvant solution to the product of step (2-2) and mixing uniformly;
[0015] (2-4) quickly adding ethanol to the product of step (2-3) to precipitate the protein and precipitate the nanoparticles;
[0016] (2-5) The product of step (2-4) is concentrated by rotary evaporation, dispersed evenly by ultrasonication in a water bath, centrifuged to remove free polypeptides and adjuvants, and then freeze-dried.
[0017] Furthermore, in (2-1), the tumor whole tissue antigen polypeptide obtained in step (1) is prepared into a 20 mg / mL polypeptide solution using deionized water.
[0018] Furthermore, in (2-3), 1 mL of 1 mg / mL R848 ethanol solution was added to the product of step (2-2).
[0019] Furthermore, in (2-4), 20 mL of ethanol was quickly added to the product of step (2-3) to precipitate the protein and separate the nanoparticles.
[0020] When using the vaccine, add an appropriate amount of PBS and use water bath ultrasound to disperse and resuspend it.
[0021] The tumor whole tissue antigen polypeptide powder and vaccine powder can be stored frozen, preferably at -80°C.
[0022] On the other hand, the present application provides the use of the above-mentioned tumor whole tissue derived antigen polypeptide tumor nanovaccine in the preparation of drugs for preventing or treating tumors.
[0023] Furthermore, the drug for preventing or treating tumors is a drug for preventing or treating breast cancer.
[0024] Furthermore, the drug is a freeze-dried powder preparation or a suspension prepared from a freeze-dried powder preparation.
[0025] The vaccine of the present application can be prepared using tumor tissue removed from a tumor patient, or can be prepared using a culture of the patient's own tumor cells, or can be prepared using tumor tissue from other sources after appropriate testing and evaluation.
[0026] On the other hand, the present application provides a method for preparing the above-mentioned tumor tissue-derived antigen polypeptide tumor nanovaccine, the preparation method comprising:
[0027] (1) Take 1-10g of fresh tumor tissue, mince it, and place it in an autoclave; add deionized water to fully infiltrate the tumor tissue; place the autoclave in an oven at 100-150°C for 6-12 hours; after the reaction, filter the product with filter paper and collect the filtrate; freeze-dry the filtrate to obtain tumor whole tissue antigen polypeptide powder;
[0028] (2-1) using deionized water to prepare the tumor whole tissue antigen polypeptide obtained in step (1) into a 10-30 mg / mL polypeptide solution;
[0029] (2-2) Take 1 mL of the peptide solution, add 10 mL of a 20 mg / mL BSA solution, and stir magnetically for 5-10 minutes to form a peptide-BSA complex;
[0030] (2-3) adding an adjuvant solution to the product of step (2-2) and mixing uniformly;
[0031] (2-4) quickly adding ethanol to the product of step (2-3) to precipitate the protein and precipitate the nanoparticles;
[0032] (2-5) The product of step (2-4) is concentrated by rotary evaporation, dispersed evenly by ultrasonication in a water bath, centrifuged to remove free polypeptides and adjuvants, and then freeze-dried.
[0033] Beneficial effects:
[0034] In response to the difficulty in selecting antigens for traditional vaccines, protein antigens derived from whole-tissue tumors are taken from all the antigens of the tumor itself and contain rich target antigens (peptide information).
[0035] The high-temperature hydrothermal method for extracting polypeptides is simple, has abundant raw material sources, and has a significantly higher yield than other methods; it can be used to mass-produce polypeptide antigens with a molecular weight between 100-5000.
[0036] To address the low efficiency of traditional vaccine delivery systems, the ethanol sedimentation method is used to directly convert polypeptide-BSA into nanoparticles with uniform particle size and good dispersion. Due to their size advantage, nanovaccines are more conducive to uptake and internalization by antigen-presenting cells, which can effectively improve antigen presentation efficiency.
[0037] The nanovaccine has significant anti-tumor efficacy in tumor-bearing mice, with a tumor inhibition efficiency of 100%.
[0038] To address the high cost of personalized preparation, the nanovaccine material is taken from individual patients, with the tumor removed after surgery as the raw material, and the hydrothermal method is used as the extraction method for tumor antigen peptides. It has the significance of personalized design, and the subsequent nanovaccine preparation procedure is simple, short and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 Schematic diagram and actual picture of the preparation method of tumor whole tissue-derived antigen polypeptides, as well as a comparison of the effects of various extraction methods.
[0040] Figure 2 It is the electrophoresis diagram and molecular weight characteristic data of antigen polypeptides derived from whole tumor tissue.
[0041] Figure 3 Schematic diagram of the nanovaccine preparation method and morphology characterization.
[0042] Figure 4 These are the live-dead staining results used in the in vitro safety evaluation.
[0043] Figure 5 The results of in vivo safety evaluation are shown in Figure 2 (Part a is the hemolysis test results; Part b is a schematic diagram of the experimental scheme; Part c is the pathological sections of important organs).
[0044] Figure 6A These are the results of various cell proportions detected in routine blood tests in the in vivo safety evaluation results.
[0045] Figure 6B These are the results of routine blood test parameters in the in vivo safety evaluation results.
[0046] Figure 7 The results are for in vivo anti-tumor effect evaluation. DETAILED DESCRIPTION
[0047] Example 1 Experimental Materials and Basic Methods
[0048] Chemical reagents:
[0049] Bovine serum albumin (BSA, ≥96%) and resiquimod (R848, ≥98%) were purchased from Aladdin (Shanghai, China). Ethanol (96%) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
[0050] Experimental animals and cell lines:
[0051] Female Balb / c mice (7 weeks old) were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were housed in individually ventilated cages, five per cage, with a 12-h light cycle (08:00–20:00; dark cycle 20:00–08:00), at a constant room temperature (21 ± 1°C) and a relative humidity of 40%–70%. All mice had free access to food and water. Rabbits were housed in individually ventilated cages, one per cage, with the same light cycle and ambient temperature and humidity as mice. All animal experiments followed the animal experiment protocol approved by the Laboratory Animal Center of Tsinghua University (approval number 24-ZLY1).
[0052] The formula for calculating tumor volume is: V = a × b 2 / 2, where a is the long diameter of the tumor and b is the short diameter.
[0053] The 4T1 cell line was provided by the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. All cells were authenticated by short tandem repeat analysis and tested for mycoplasma contamination.
[0054] Example 2 Antigen extraction and SDS-PAGE analysis
[0055] Preparation method of tumor tissue-derived antigen polypeptides Figure 1 (parts a and b) shown.
[0056] To obtain tumor tissue-derived peptides, place 1-10g of fresh tumor tissue (from tumor-bearing mice, removed after euthanasia) in a polytetrafluoroethylene autoclave, add 20mL of deionized water, and react at 150°C for 6 hours. After the reaction, filter the filtrate through filter paper to collect the tumor peptide solution. After determining the peptide concentration using the BCA assay, dilute with deionized water to 20mg / mL and store at -80°C until ready for use.
[0057] The amount of tumor tissue-derived antigen peptides obtained by the hydrothermal method of the present application is significantly better than that obtained by freeze-thaw, boiling and other methods (see Figure 1 Part c of the study included the following methods: freeze-thaw method: mince 1 g of fresh tumor tissue, add 1 mL of deionized water, and quickly add the minced tissue to liquid nitrogen. After standing for 1 min, remove the tissue and thaw it in a 37°C water bath. The tissue was then rapidly cooled in liquid nitrogen, and this process was repeated five times. After thawing, the supernatant was collected and assayed for protein content using BCA. The boiling method: mince 1 g of fresh tumor tissue, add 10 mL of deionized water, and boil at 100°C for 30 min. The supernatant was collected and assayed for protein content using the BCA assay.
[0058] Take an equal amount of sample (antigen obtained by ultrasonic treatment of 4T1 cells as a control group) in a centrifuge tube, add 5μL 4× loading buffer and 2% SDS to a total volume of 20μL. Load 1μL prestained protein marker and 20μL protein sample in sequence, and add 20μL 1× loading buffer to the blank well. Run electrophoresis on the stacking gel at 15mA until the protein is concentrated into a line (about 15 minutes), and run electrophoresis on the separation gel at 35mA until the dye reaches the bottom of the gel. After removing the gel, perform fixation, sensitization and silver staining steps in sequence, and finally transfer to the developer for color development for about 10 minutes. After the bands are clear, add the stop solution to terminate the reaction.
[0059] The results are as follows Figure 2 As shown, the molecular weight of tumor tissue antigen polypeptides is concentrated between 95-5600 Da, and its molecular weight is normally distributed.
[0060] Example 3 Nanoparticle Preparation
[0061] To prepare the nanoparticles, 1 mL of tumor protein peptide was added to 10 mL of BSA solution (20 mg / mL) and magnetically stirred for 5 minutes to form a peptide-BSA complex. Subsequently, 1 mL of R848 ethanol solution (1 mg / mL) was added and mixed thoroughly. Immediately after mixing, 20 mL of ethanol (analytical grade) was added to precipitate the protein nanoparticles, resulting in a turbid nanovaccine suspension. The ethanol / water solution containing the nanovaccine was concentrated to approximately 10 mL by rotary evaporation. After sonication in a water bath for 5 minutes, the solution was centrifuged (8000 rpm for 5 minutes) to remove free peptide and R848. The final product was stored at 4°C until ready for use.
[0062] The preparation process of adjuvant-free nanomedium is the same as that of nanovaccine, except that the step of adding R848 solution is omitted; no tumor peptide is added during the preparation of polypeptide-free nanomedium.
[0063] like Figure 3 As shown, tumor whole tissue antigen polypeptide nanovaccine was obtained by nanoprecipitation method, and its particle size was about 200nm, which was a uniform spherical particle.
[0064] Example 4 In vitro and in vivo biosafety evaluation of nanovaccines
[0065] In vitro safety evaluation:
[0066] In accordance with and referring to the relevant provisions of the international medical device biological evaluation standard ISO10993-5 (Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity) and the national standard of the People's Republic of China GB / T16886.5 (Biological evaluation of medical devices Part 5: In vitro cytotoxicity test), the in vitro biosafety evaluation of nanoparticles was carried out using mouse fibroblast L929 cells as the research object.
[0067] L929 cells were first inoculated into a 96-well plate (5000 cells per well), incubated for 12 hours to allow the cells to adhere, and the old culture medium was discarded. The experimental group was replaced with fresh culture medium containing 1 mg / mL nanovaccine, and the control group was replaced with complete culture medium. The cells were continued to be cultured in a cell culture incubator. After 24 hours, the cells were stained for live and dead, and photographed and recorded using a laser confocal microscope.
[0068] In vivo safety evaluation:
[0069] Nine healthy 7-week-old Balb / c mice were subcutaneously injected with 100 μL of the nanovessel (1 mg / mL; three media, including antigen-free, adjuvant-free, and nanoparticle-free, were administered in parallel groups) and continued to be fed normally. Three days later, the mice were euthanized, and the hearts, livers, spleens, lungs, and kidneys were removed, fixed in paraformaldehyde, embedded, and sliced for H&E. Six healthy 7-week-old Balb / c mice were also subcutaneously injected with 100 μL of the nanovaccine (1 mg / mL) in three mice, while the other three mice were left untreated as a control group. Blood was collected from the mice's orbits 30 days later, and various blood routine indicators were measured.
[0070] The results are as follows Figure 4 - As shown in Figure 6, the vaccine had no detectable cytotoxicity ( Figure 4 ); vaccine without hemolysis ( Figure 5 Part a) and pathological sections of important organs showed no abnormalities ( Figure 5 One month after the nanovaccine was administered, the blood routine indicators of the mice showed no significant differences from those of the healthy mice (Figure 6).
[0071] Example 5 Evaluation of anti-tumor effects in vivo
[0072] The tumor model was established by inoculating 4T1 cells into the flank of Balb / c mice, and the experiment was started when the tumor diameter reached about 5 mm. The tumor-bearing mice were randomly divided into four groups, with 5 mice in each group: (1) blank control group (2) no peptide group (1 mg / mL) (3) no adjuvant group (1 mg / mL) (4) nanovaccine group (1 mg / mL), and each group was subcutaneously injected with 100 μL of the corresponding preparation. The body weight and tumor volume of the mice were monitored daily. The animals were killed on the 12th day, and the tumor tissue and spleen were taken and weighed. Afterwards, the spleen was ground into a single cell suspension, and DCs were labeled with flow cytometric antibodies CD11c-FITC, CD80-APC and CD86-PE; T cells were labeled with CD3-FITC, CD4-APC and CD8-PE, and flow cytometric analysis and statistics were performed. At the same time, the blood of the mice was collected, and the serum was collected. The levels of TNF-α and IFN-γ in the mouse serum were detected using ELISA kits.
[0073] The results are as follows Figure 7As shown, compared with the control group, the non-peptide group and the non-adjuvant group, the mice showed a significant weight loss in the early stage of nanovaccine administration ( Figure 7 Part b of the study) may be attributed to the acute inflammation caused by it. After three or four days, the weight of the mice gradually returned to normal levels without long-term toxic side effects. In addition, during the treatment cycle, the tumors of the mice in the nanovaccine group were completely eliminated ( Figure 7 Part c), spleen weight returned to normal level ( Figure 7 Part d) shows the excellent tumor treatment effect of nano vaccines. Analysis of immune cells in the spleen showed that the nano vaccine group increased the levels of mature DC (CD80+CD86+DC) and cytotoxic T cells (CD3+CD8+T) ( Figure 7 e), elevated levels of pro-inflammatory factors TNF-α and IFN-γ were detected in the serum, indicating that the nanovaccine group activated the anti-tumor immune effect ( Figure 7 (part f of the ).
[0074] Obviously, the above embodiments of the present invention are merely examples for the purpose of illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will readily appreciate that other variations or modifications based on the above description are possible. It is not necessary and impossible to enumerate all embodiments here. However, such obvious variations or modifications arising from the spirit of the present invention remain within the scope of protection of the present invention.
Claims
1. A tumor tissue-derived antigen polypeptide tumor nanovaccine, characterized in that: The preparation method of the vaccine comprises: (1) Extraction of tumor tissue antigen peptides using high-temperature hydrothermal method; (2) Peptide-BSA complexes were formed and nanovaccines were prepared using the ethanol-protein precipitation method.
2. The vaccine according to claim 1, wherein in step (1), 1-10 g of fresh tumor tissue is taken, chopped, and placed in an autoclave; deionized water is added to fully infiltrate the tumor tissue; the autoclave is placed in an oven at 100-150° C. to react for 6-12 hours; after the reaction is completed, the product is filtered using filter paper and the filtrate is collected; the filtrate is freeze-dried to obtain a whole-tissue tumor antigen polypeptide powder.
3. The vaccine according to claim 1 or 2, wherein step (2) comprises: (2-1) using deionized water to prepare the tumor whole tissue antigen polypeptide obtained in step (1) into a 10-30 mg / mL polypeptide solution; (2-2) Take 1 mL of the peptide solution, add 10 mL of a 20 mg / mL BSA solution, and stir magnetically for 5-10 min to form a peptide-BSA complex; (2-3) adding an adjuvant solution to the product of step (2-2) and mixing uniformly; (2-4) quickly adding ethanol to the product of step (2-3) to precipitate the protein and precipitate the nanoparticles; (2-5) The product of step (2-4) is concentrated by rotary evaporation, dispersed evenly by ultrasonication in a water bath, centrifuged to remove free polypeptides and adjuvants, and then freeze-dried.
4. The vaccine according to claim 3, wherein in (2-1), the tumor whole tissue antigen polypeptide obtained in step (1) is prepared into a 20 mg / mL polypeptide solution using deionized water.
5. The vaccine according to claim 3, wherein in (2-3) 1 mL of a 1 mg / mL R848 ethanol solution is added to the product of step (2-2).
6. The vaccine according to claim 3, wherein in (2-4), 20 mL of ethanol is quickly added to the product of step (2-3) to precipitate the protein and separate the nanoparticles.
7. Use of the vaccine according to any one of claims 1 to 6 in the preparation of a medicament for preventing or treating tumors.
8. The use according to claim 7, wherein the drug for preventing or treating tumors is a drug for preventing or treating breast cancer.
9. The use according to claim 7, wherein the drug is a freeze-dried powder preparation or a suspension prepared from a freeze-dried powder preparation.
10. A method for preparing a tumor nanovaccine containing antigenic polypeptides derived from whole-tissue tumors, characterized in that: The preparation method comprises: (1) Take 1-10g of fresh tumor tissue, mince it, and place it in an autoclave; add deionized water to fully infiltrate the tumor tissue; place the autoclave in an oven at 100-150°C for 6-12 hours; after the reaction, filter the product with filter paper and collect the filtrate; freeze-dry the filtrate to obtain tumor whole tissue antigen polypeptide powder; (2-1) using deionized water to prepare the tumor whole tissue antigen polypeptide obtained in step (1) into a 10-30 mg / mL polypeptide solution; (2-2) Take 1 mL of the peptide solution, add 10 mL of a 20 mg / mL BSA solution, and stir magnetically for 5-10 min to form a peptide-BSA complex; (2-3) adding an adjuvant solution to the product of step (2-2) and mixing uniformly; (2-4) quickly adding ethanol to the product of step (2-3) to precipitate the protein and precipitate the nanoparticles; (2-5) The product of step (2-4) is concentrated by rotary evaporation, dispersed evenly by ultrasonication in a water bath, centrifuged to remove free polypeptides and adjuvants, and then freeze-dried.