A universal method for preparing exosomes that present tumor antigens and highly activate t cells

Abstract

The present application relates to a kind of universal preparation method for presenting tumor antigen and highly activated T cell exosome, belong to biomedical and immunotherapy technical field.The present application described method is first synthesized the tumor antigen specificity nano stimulant capable of activating dendritic cell, makes dendritic cell carry IFN-β, expresses specific tumor-related antigen and costimulatory molecule, to solve the problems, such as low antigen presentation efficiency, T cell activation deficiency and high cost of individualized treatment in the existing tumor immunotherapy method.Exosome is extracted from the supernatant of the above dendritic cell culture.The preparation method of the present application not only significantly improves the efficiency of exosome as antigen presentation carrier, effectively and specifically stimulates T cell, but also reduces the preparation cost, provides more effective general solution for tumor immunotherapy, has wide application prospect and clinical value.

Description

Technical Field

[0001] This invention relates to a universal method for preparing exosomes that present tumor antigens and highly activate T cells, belonging to the field of biomedical and immunotherapy technology. Background Technology

[0002] Dendritic cells (DCs), as crucial antigen-presenting cells in the body's immune system, play a central role in initiating and regulating immune responses. Particularly in the field of tumor immunotherapy, the functions of DCs are being further explored and utilized. Among them, dendritic cell-derived exosomes (DExs) have shown great potential and significance as a novel immunotherapy carrier. Specifically, exosomes are nanoscale lipid bilayer vesicles secreted by cells, carrying various bioactive molecules from the mother cell, including proteins, RNA, and DNA fragments, and mediating intercellular communication and substance exchange. DExs are particularly rich in MHC complexes, co-stimulatory molecules, and tumor-associated antigen information, making them a natural "immunoeducation tool." In tumor immunotherapy, the applications of DExs are mainly reflected in the following aspects:

[0003] In terms of antigen presentation, the tumor antigens carried by DEx can be directly presented to T lymphocytes, activating specific T cells, especially CD8 cells. + Cytotoxic T cells enable them to recognize and kill tumor cells bearing the same antigen markers, achieving precise targeting. Regarding breaking immune tolerance, the tumor microenvironment often induces immunosuppression, limiting T cell function. DEx, by carrying immune activation signals such as cytokines and co-stimulatory molecules, can reverse this state and reactivate the immune response at the tumor site. In terms of establishing immune memory, DEx not only promotes immediate anti-tumor immune responses but also helps form long-term immune memory, preventing tumor recurrence. By mimicking the natural immune process, it promotes the generation of memory T cells, providing continuous protection for patients. In terms of safety and convenience, compared to traditional DC cell therapy, using DEx as a treatment method offers higher safety. Exosomes avoid the risks associated with direct cell transfer, and the preparation process is more standardized, facilitating quality control and large-scale production, increasing the feasibility and accessibility of the treatment.

[0004] Despite the immense potential of DEx in tumor immunotherapy, its clinical application remains in the research stage, facing challenges such as determining the optimal dosage, optimizing the effective payload, and developing personalized treatment strategies. Therefore, continuous exploration and improvement of the preparation, modification, and utilization methods of DEx are needed to maximize its benefits in tumor immunotherapy and provide cancer patients with safer and more effective treatment options. Summary of the Invention

[0005] To address the problems of low antigen presentation efficiency, insufficient T cell activation, and high cost of personalized treatment in existing tumor immunotherapy methods, the present invention aims to provide a universal method for preparing exosomes that present tumor antigens and highly activate T cells. First, a tumor-specific nanostimulant that stimulates dendritic cells is prepared. Then, it is co-incubated with dendritic cells, causing the dendritic cells to secrete exosomes that can present tumor antigens and highly activate T cells. Compared with other engineered exosomes, the method described in this invention is simpler and more cost-effective, while still achieving the aforementioned functional objectives.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] A universal method for preparing exosomes that present tumor antigens and highly activate T cells, comprising the following steps:

[0008] (1) Remove the cell wall of Escherichia coli expressing the IFN-β gene to obtain protoplasts, mix the protoplasts with tumor antigens, collect the supernatant containing nanoprotoplasts with a particle size of 100-900 nm, and then add it to the tumor-specific nanostimulant.

[0009] (2) Tumor-specific nanostimulants were added to a complete culture medium, dendritic cells were incubated, and exosomes were extracted to obtain an exosome that presented tumor antigens and highly activated T cells.

[0010] Preferably, in step (1), when preparing the tumor-specific nanostimulant: an engineered Escherichia coli strain carrying the IFN-β gene is inoculated into M9 medium containing 0.05–0.2 mM isopropyl-β-D-thiogalactoside for culture, and the E. coli precipitate is collected by centrifugation; the E. coli precipitate is added to lysozyme cell wall disruption buffer, incubated and the E. coli cell wall is removed, and the protoplasts are collected by centrifugation; the protoplasts and tumor antigens are mixed in Tris-HCl buffer at a total protein concentration ratio of 2:1–6:1, and sonicated at 30–40 W for 10–30 minutes, large particles are removed by centrifugation, and the supernatant containing nanoprotoplasts with a particle size of 100–900 nm is collected to obtain the tumor-specific nanostimulant.

[0011] Preferably, in step (1), the culture conditions are: shaking at 180-220 rpm for 15-20 hours at 37°C.

[0012] Preferably, in step (1), the composition of the lysozyme cell disruption buffer is: 50mM Tris-HCl, 5mM EDTA-2Na, 4mg / ml lysozyme, and 0.9wt% glucose; the pH of the buffer is 8.0.

[0013] Preferably, in step (1), the concentration of Tris-HCl buffer is 50 mM and the pH is 8.0.

[0014] Preferably, in step (1), the incubation conditions are: incubation at 37°C for 0.5 to 2 hours; removal of the Escherichia coli cell wall is carried out at 4°C.

[0015] Preferably, in step (1), the conditions for centrifuging to collect E. coli precipitate and centrifuging to collect protoplasts are: centrifuging at 3000-6000g for 10-30 minutes at 4℃.

[0016] Preferably, in step (1), the conditions for centrifugation to remove large particles are: centrifugation at 100-200g for 5-10 minutes at 4°C.

[0017] Preferably, in step (2), when extracting exosomes that present tumor antigens and highly activate T cells: the tumor-specific nanostimulant is prepared to a concentration of 40-100 μg / ml in complete culture medium, and dendritic cells are incubated for 24-48 hours, and the cell culture supernatant is collected; then, the cells are centrifuged at 1000-1500 rpm for 3-5 minutes to remove suspended cells or other large particles, the supernatant is collected, and exosomes are extracted using a total exosome separation kit, filtered, and exosomes that present tumor antigens and highly activate T cells are obtained.

[0018] Preferably, in step (2), the tumor-specific nanostimulant is prepared to a concentration of 50-80 μg / ml using a complete culture medium.

[0019] Preferably, in step (2), a water-based microporous filter membrane with a specification of 0.22μm is used for filtration.

[0020] An exosome that presents tumor antigens and highly activates T cells was prepared using the above method.

[0021] Beneficial effects

[0022] This invention provides a universal method for preparing exosomes that present tumor antigens and highly activate T cells. After removing the cell wall of IFN-β-expressing Escherichia coli, protoplasts are obtained. The protoplasts are mixed with tumor antigens to ensure the dosage and optimal loading efficiency of the tumor antigens. Then, the mixture is sonicated and centrifuged to remove large particles, and the supernatant containing protoplasts with a particle size of 100-900 nm is collected to ensure successful uptake by dendritic cells, thereby enabling the dendritic cells to secrete exosomes with these specific functions.

[0023] The method described in this invention has the following advantages, specifically:

[0024] (1) Enhanced immune recognition: This method can effectively increase the level of tumor-specific antigens expressed on the surface of exosomes, thereby significantly enhancing the T cells' ability to recognize tumor cells. This not only increases the specificity of immunotherapy but also improves the therapeutic effect.

[0025] (2) Enhancing T cell activation efficiency: By optimizing the preparation conditions of exosomes, this invention can promote the interaction between exosomes and T cells, significantly increase the activation rate of T cells, and thus improve the intensity and persistence of the immune response. Specifically, mixing protoplasts with tumor antigens under specific conditions can ensure the dosage and optimal loading efficiency of tumor antigens; sonicating and centrifuging to remove large particles under specific conditions, and collecting the supernatant containing protoplasts with a particle size of 100-900 nm, can ensure successful uptake by dendritic cells, thereby enabling dendritic cells to secrete exosomes with these specific functions.

[0026] (3) Reduce non-specific activation: Traditional methods may cause non-specific immune responses when activating T cells, while the present invention effectively avoids non-specific activation by precisely controlling the composition and structure of exosomes, thereby reducing side effects during treatment.

[0027] (4) Simplified preparation process: Compared with the existing complex and time-consuming preparation methods, the present invention proposes a simpler and more efficient preparation process, which greatly shortens the production cycle, reduces costs, and is conducive to large-scale production and clinical application.

[0028] (5) Adaptable to multiple tumor types: Since the preparation method of the present invention has high versatility, it can be applied to a variety of different types of tumor antigens, which means that it has broad clinical application prospects and can meet the needs of different patients.

[0029] (6) Promoting personalized medicine: Based on antigens extracted from individual tumor cells, the preparation method of the present invention can prepare personalized exosome vaccines, which further improves the personalization of treatment and enhances efficacy. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the process of generating exosomes that present tumor antigens and highly activate T cells in Example 1 of the present invention.

[0031] Figure 2 This section describes the partial physicochemical properties of the tumor-specific nanostimulant used to stimulate dendritic cells in Example 1 of the present invention.

[0032] Figure 3 This is a partial physicochemical characterization of the exosomes secreted by dendritic cells after stimulation in Example 1 of the present invention, which present tumor antigens and highly activate T cells.

[0033] Figure 4 The activation effect of exosomes secreted in Example 1 of this invention, which present tumor antigens and highly activate T cells, on T cells. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the raw materials and operating techniques involved in the following embodiments and comparative examples are all conventional raw materials and techniques in the prior art.

[0035] like Figure 1 As shown, a universal method for preparing exosomes that present tumor antigens and highly activate T cells includes the following steps:

[0036] (1) Preparation of tumor-specific nanostimulants: Engineered Escherichia coli strains carrying the IFN-β gene were inoculated into M9 medium containing 0.05–0.2 mM isopropyl-β-D-thiogalactoside and cultured. The E. coli precipitate was collected by centrifugation. The E. coli precipitate was added to lysozyme cell wall disruption buffer, incubated and the cell wall of E. coli was removed. The protoplasts were collected by centrifugation. The protoplasts and tumor antigens were mixed in Tris-HCl buffer at a total protein concentration ratio of 2:1–6:1. The mixture was sonicated at 30–40 W for 10–30 minutes. Large particles were removed by centrifugation. The supernatant containing nanoprotoplasts with a particle size of 100–900 nm was collected to prepare tumor-specific nanostimulants.

[0037] (2) Secretion of exosomes that present tumor antigens and highly activate T cells: The tumor-specific nanostimulant was prepared to a concentration of 40-100 μg / ml in complete culture medium. After incubating dendritic cells for 24-48 hours, the cell culture supernatant was collected. Then, the cells were centrifuged at 1000-1500 rpm for 3-5 minutes to remove suspended cells or other large particles. The supernatant was collected, and exosomes were extracted using a total exosome separation kit. After filtration, exosomes that present tumor antigens and highly activate T cells were obtained.

[0038] Example 1

[0039] (1) Preparation of tumor-specific nanostimulants: An engineered Escherichia coli strain carrying the IFN-β gene (PET-22b-IFN-β-His) was inoculated into M9 medium containing 0.1 mM isopropyl-β-D-thiogalactoside and shaken at 200 rpm for 18 hours at 37 °C. The E. coli pellet was collected by centrifugation at 4,500 g for 15 minutes at 4 °C. This pellet was added to 50 mM Tris-HCl (pH 8.0, 5 mM EDTA-2Na, 4 mg / ml lysozyme, 0.9% glucose) and incubated at 37 °C for 1 hour. The bacterial cell walls were removed at 4 °C, and the protoplasts were collected by centrifugation at 4,500 g for 15 minutes at 4 °C. Protoplasts and tumor antigen (extracted EG7-OVA tumor cell total protein) were mixed in 20 ml Tris-HCl (50 mM, pH 8.0) at a total protein concentration ratio of 5:1, and the mixture was sonicated at 35 W for 20 minutes. The mixture was then centrifuged at 150 g for 5 minutes at 4 °C to remove large particles, and the supernatant containing nanoprotoplasts was collected.

[0040] (2) Secretion of exosomes that present tumor antigens and highly activate T cells: The above-mentioned tumor-specific nanostimulant was prepared to a final concentration of 50 μg / ml in complete culture medium. After incubating dendritic cells for 48 hours, all cell culture supernatants were collected. The cells were centrifuged at 1000 rpm for 5 minutes to remove suspended cells or other large particles. All supernatants were collected, and exosomes were extracted using a total exosome isolation kit (brand: Thermo Fisher Scientific, catalog number: 4478359). The exosomes were then filtered through a 0.22 μm aqueous microporous membrane to obtain an exosome that presents tumor antigens and highly activates T cells.

[0041] Figure 2 The particle size and surface potential of the tumor-specific nanostimulant (nano-P-IO group in the figure) obtained in this embodiment were characterized. The results showed that the particle size of the nanostimulant was about 600-800 nm and the surface had a negative potential.

[0042] Figure 3 The particle size and surface potential of the exosomes (exo-IO group in the figure) that presented tumor antigens and highly activated T cells obtained in this embodiment were characterized. The results showed that the particle size of the nanostimulant was about 40-50 nm and the surface had a negative potential.

[0043] Figure 4 The results of the T cell activation effect test of exosomes that present tumor antigens and highly activate T cells obtained in this embodiment show that CD4 cells highly express IFN-γ. + and CD8 + T cells (i.e., activated CD4 cells) + and CD8 +The significant increase in T cells indicates that exosomes have a direct activating effect on T cells.

[0044] Example 2

[0045] (1) Preparation of tumor-specific nanostimulants: An engineered Escherichia coli strain carrying the IFN-β gene (PET-22b-IFN-β-His) was inoculated into M9 medium containing 0.1 mM isopropyl-β-D-thiogalactoside and shaken at 180 rpm for 20 hours at 37°C. The E. coli precipitate was collected by centrifugation at 3,000 g for 30 minutes at 4°C. This precipitate was added to 50 mM Tris-HCl (pH 8.0, 5 mM EDTA-2Na, 4 mg / ml lysozyme, 0.9% glucose) and incubated at 37°C for 0.5 hours. The bacterial cell walls were removed at 4°C, and the protoplasts were collected by centrifugation at 3,000 g for 30 minutes at 4°C. Protoplasts and tumor antigen (extracted EG7-OVA tumor cell total protein) were mixed in 20 ml Tris-HCl (50 mM, pH 8.0) at a total protein concentration ratio of 6:1, and sonicated at 30 W for 30 minutes. The mixture was then centrifuged at 150 g for 5 minutes at 4 °C to remove large particles, and the supernatant containing nanoprotoplasts was collected.

[0046] (2) Secretion of exosomes that present tumor antigens and highly activate T cells: The above-mentioned tumor-specific nanostimulant was prepared to a final concentration of 40 μg / ml in complete culture medium. After incubating dendritic cells for 48 hours, all cell culture supernatants were collected. The cells were centrifuged at 1000 rpm for 5 minutes to remove suspended cells or other large particles. All supernatants were collected, and exosomes were extracted using a total exosome isolation kit (brand: Thermo Fisher Scientific, catalog number: 4478359). The exosomes were then filtered through a 0.22 μm aqueous microporous membrane to obtain an exosome that presents tumor antigens and highly activates T cells.

[0047] The particle size and surface potential characterization results of the tumor-specific nanostimulant obtained in this embodiment show that the particle size of the nanostimulant is approximately between 600 and 800 nm, and the surface has a negative potential.

[0048] The particle size and surface potential characterization results of the exosomes that present tumor antigens and highly activate T cells obtained in this embodiment show that the particle size of the nanostimulant is about 40-50 nm and the surface has a negative potential.

[0049] The results of the T cell activation effect test of exosomes that present tumor antigens and highly activate T cells obtained in this embodiment showed that CD4+ highly expresses IFN-γ. + and CD8 + T cells (i.e., activated CD4 cells) + and CD8+ The exosomes significantly increased T cells, indicating a direct activating effect on T cells.

[0050] Example 3

[0051] (1) Preparation of tumor-specific nanostimulants: An engineered Escherichia coli strain carrying the IFN-β gene (PET-22b-IFN-β-His) was inoculated into M9 medium containing 0.1 mM isopropyl-β-D-thiogalactoside and shaken at 220 rpm for 15 hours at 37 °C. The E. coli precipitate was collected by centrifugation at 6000 g for 10 minutes at 4 °C. This precipitate was added to 50 mM Tris-HCl (pH 8.0, 5 mM EDTA-2Na, 4 mg / ml lysozyme, 0.9% glucose) and incubated at 37 °C for 2 hours. The bacterial cell walls were removed at 4 °C, and the protoplasts were collected by centrifugation at 6000 g for 10 minutes at 4 °C. Protoplasts and tumor antigen (extracted EG7-OVA tumor cell total protein) were mixed in 20 ml Tris-HCl (50 mM, pH 8.0) at a total protein concentration ratio of 2:1, and sonicated at 40 W for 10 minutes. The mixture was then centrifuged at 150 g for 10 minutes at 4 °C to remove large particles, and the supernatant containing nanoprotoplasts was collected.

[0052] (2) Secretion of exosomes that present tumor antigens and highly activate T cells: The above-mentioned tumor-specific nanostimulant was prepared to a final concentration of 100 μg / ml in complete culture medium. After incubating dendritic cells for 24 hours, all cell culture supernatants were collected. The cells were centrifuged at 1500 rpm for 3 minutes to remove suspended cells or other large particles. All supernatants were collected, and exosomes were extracted using a total exosome isolation kit (brand: Thermo Fisher Scientific, catalog number: 4478359). The exosomes were then filtered through a 0.22 μm aqueous microporous membrane to obtain an exosome that presents tumor antigens and highly activates T cells.

[0053] The particle size and surface potential characterization results of the tumor-specific nanostimulant obtained in this embodiment show that the particle size of the nanostimulant is approximately between 600 and 800 nm, and the surface has a negative potential.

[0054] The particle size and surface potential characterization results of the exosomes that present tumor antigens and highly activate T cells obtained in this embodiment show that the particle size of the nanostimulant is about 40-50 nm and the surface has a negative potential.

[0055] The results of the T cell activation effect test of exosomes that present tumor antigens and highly activate T cells obtained in this embodiment showed that CD4+ highly expresses IFN-γ. + and CD8 + T cells (i.e., activated CD4 cells) +and CD8 + The exosomes significantly increased T cells, indicating a direct activating effect on T cells.

[0056] In summary, the invention includes, but is not limited to, the above embodiments. Any equivalent substitutions or partial improvements made under the spirit and principles of this invention shall be considered to be within the protection scope of this invention.

Claims

1. A method for the general preparation of exosomes presenting tumor antigens and highly activating T cells, characterized in that: The method steps include: (1) removing the cell wall of E. coli expressing the IFN-β gene to obtain protoplasts, mixing the protoplasts with tumor antigens, collecting supernatant containing protoplasts with a particle size of 600-800 nm to obtain tumor-specific nano stimulants; (2) adding the tumor-specific nano stimulants to complete culture medium, incubating dendritic cells, and extracting exosomes to obtain exosomes that present tumor antigens and highly activate T cells.

2. The general preparation method of exosomes for presenting tumor antigens and highly activating T cells according to claim 1, characterized in that: In step (1), when preparing the tumor-specific nano stimulants: the engineered E. coli strain carrying the IFN-β gene is inoculated into M9 culture medium containing 0.05-0.2 mM isopropyl-β-D-thiogalactoside for culture, and E. coli is collected by centrifugation; the E. coli precipitate is added to a zymolyase cell wall breaking buffer, incubated to remove the cell wall of E. coli, and protoplasts are collected by centrifugation; the protoplasts and tumor antigens are mixed in Tris-HCl buffer at a total protein concentration ratio of 2:1-6:1, ultrasonicated at a power of 30-40 W for 10-30 minutes, centrifuged to remove large particles, and supernatant containing protoplasts with a particle size of 600-800 nm is collected to obtain tumor-specific nano stimulants.

3. The general method for preparing exosomes presenting tumor antigens and highly activating T cells according to claim 2, characterized in that: In step (1), the culture conditions are: 37°C, 180-220 rpm shaking for 15-20 hours.

4. The general preparation method of exosomes for presenting tumor antigens and highly activating T cells according to claim 2, characterized in that: In step (1), the zymolyase cell wall breaking buffer consists of 50 mM Tris-HCl, 5 mM EDTA-2Na, 4 mg / ml zymolyase, and 0.9wt% glucose; and the pH of the buffer is 8.

0.

5. The general preparation method of exosomes for presenting tumor antigens and highly activating T cells according to claim 2, characterized in that: In step (1), the concentration of the Tris-HCl buffer is 50 mM, and the pH is 8.

0.

6. The general preparation method of exosomes for presenting tumor antigens and highly activating T cells according to claim 2, characterized in that: In step (1), the incubation conditions are: 37°C for 0.5-2 hours; and the removal of the cell wall of E. coli is performed at 4°C.

7. The general method for preparing exosomes presenting tumor antigens and highly activating T cells according to claim 2, characterized in that: In step (1), the conditions for centrifuging to collect the E. coli precipitate and centrifuging to collect the protoplasts are: 4°C, 3000-6000 g centrifugation for 10-30 minutes; The conditions for centrifuging to remove large particles are: 4°C, 100-200 g centrifugation for 5-10 minutes.

8. The general method of preparing exosomes presenting tumor antigens and highly activating T cells according to claim 1, characterized in that: In step (2), when extracting the exosomes that present tumor antigens and highly activate T cells: the tumor-specific nano stimulants are configured to a concentration of 40-100 μg / ml with complete culture medium, and after incubating dendritic cells for 24-48 hours, the cell culture supernatant is collected; then the supernatant is collected after removing suspended cells or other large particles by centrifugation at 1000-1500 rpm for 3-5 minutes, and exosomes are extracted using a total exosome separation kit, filtered, and the exosomes that present tumor antigens and highly activate T cells are obtained.

9. The general method of preparing exosomes presenting tumor antigens and highly activating T cells according to claim 2, characterized in that: In step (2), the tumor-specific nano stimulants are configured to a concentration of 50-80 μg / ml with complete culture medium; A water-based microporous filter with a size of 0.22 μm is used for filtration.

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