Preparation method of engineered B cell vaccine, engineered B cell vaccine prepared thereby and application of engineered B cell vaccine

Abstract

The invention relates to a preparation method of an engineered B cell vaccine, the engineered B cell vaccine prepared by the preparation method and application of the engineered B cell vaccine. The B cell vaccine agent stimulated by the immunogenic death tumor cell lysate and the aluminum adjuvant is used for preventing occurrence and development of tumors, the treatment effect of the obtained engineered B cell vaccine on the tumors can be improved, and the engineering B cell vaccine has huge potential in the aspect of preventing occurrence and development of the tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing an engineered B-cell vaccine, the engineered B-cell vaccine prepared therefrom, and its applications. Background Technology

[0002] Malignant melanoma (MM) is an epithelial tumor caused by melanocytes. In recent years, various anti-tumor immunotherapies have made significant progress in cancer treatment, among which tumor vaccines are a research hotspot in immunotherapy.

[0003] However, whole tumor cell antigens have problems such as poor immunogenicity and insufficient stimulation of immune cells. Summary of the Invention

[0004] This study utilized the chemotherapy drug doxorubicin (DOX) to induce immunogenic death in melanoma cells. Tumor cell lysates were then extracted using a freeze-thaw method and used as a tumor-specific antigen. This lysate was mixed with aluminum adjuvant to activate B cells in vitro. The activated B cells were then used as a cell vaccine to inhibit melanoma development and progression. The results showed that inducing immunogenic death in tumor cells with doxorubicin effectively enhanced the immunogenicity of the weighted six-cell antigen. The combined use of aluminum adjuvant further enhanced its stimulatory effect on B cells, improving the therapeutic efficacy of the resulting engineered B-cell vaccine against tumors, thus completing this invention.

[0005] Therefore, one object of the present invention is to provide a method for preparing an engineered B-cell vaccine.

[0006] Another object of the present invention is to provide an engineered B-cell vaccine prepared by the above method.

[0007] Another object of the present invention is to provide the application of engineered B-cell vaccines.

[0008] Therefore, on the one hand, the present invention provides a method for preparing an engineered B-cell vaccine, comprising: S1, Preparation of B cells: Isolated lymphocytes were cultured with interleukin-4 (IL-4) and CD3 monoclonal antibody to eliminate T cells and obtain purified cultured B cells; S2, Preparation of immunogenic dead tumor cell lysate particles: Cultured tumor cells are subjected to immunogenic death treatment to obtain immunogenic dead tumor cell lysate particles. S3, B cell activation: The B cells obtained in S1 and the tumor cell lysate particles obtained in S2 are co-cultured at a ratio of 5:1 to 50:1 to obtain activated B cells, thereby obtaining an engineered B cell vaccine.

[0009] In this invention, there is no particular restriction on the order of the above steps S1 and S2. S1 can be performed first and then S2; S2 can be performed first and then S1; or S1 and S2 can be performed simultaneously.

[0010] In some embodiments, the lymphocytes isolated in S1 are not particularly limited; for example, in some embodiments, they may be lymphocytes isolated from blood. In other embodiments, the isolated lymphocytes may be lymphocytes isolated from the spleen.

[0011] In some embodiments, in S1, the lymphocytes may be derived from mammals, such as humans, mice, rats, dogs, rabbits, pigs, horses, cattle, sheep, etc.

[0012] In some embodiments, in S1, the CD3 monoclonal antibody can refer to an antibody that specifically binds to CD3. Examples include, but are not limited to, GMP Monoclonal Anti-Human CD3 Antibody (OKT3).

[0013] In some implementations, the culture time in S1 can be 3-5 days, preferably 4 days.

[0014] In some embodiments, in S1, the separated lymphocytes are... 9 From 1.0 × 10⁻⁶ units / L to 1.0 × 10⁻� 10 Cells / L, preferably 6.0×10 9 B cells were suspended at a concentration of 1 / L in culture medium (e.g., RPMI 1640 medium), and 10-30 µg / L (preferably 20 µg / L) of IL-4 and 3-10 mg / L (preferably 5 mg / L) of CD3 monoclonal antibody were added. The cells were then cultured, and every other day the original culture medium was discarded and an equal volume of the above-mentioned culture medium, IL-4, and CD3 monoclonal antibody were added to continue culturing until purified B cells were obtained. In a further embodiment, the culture was carried out in an incubator at 37°C with 5% CO2.

[0015] In this invention, tumor cells refer to proliferating cells isolated from tumor tissue, or commercially available tumor cells. In some embodiments, in S2, tumor cells may refer to proliferating cells isolated from melanoma tissue, or tumor cells may be commercially available melanoma cells.

[0016] In some embodiments, in S1, the tumor cells may be derived from mammals, such as humans, mice, rats, dogs, rabbits, pigs, horses, cattle, sheep, etc.

[0017] In some embodiments, in S2, an immunogenic death treatment can be performed using a drug capable of inducing tumor immunogenic death (e.g., a chemotherapy drug, such as doxorubicin (DOX)). In a further embodiment, the dosage of the drug capable of inducing tumor immunogenic death can be 0.5-1.5 μM.

[0018] In some embodiments, in S2, tumor cell lysate particles are extracted using a freeze-thaw method.

[0019] In some embodiments, in S3, the B cells obtained in S1 and the tumor cell lysate particles obtained in S2 are co-cultured at a ratio of 10:1 (cells:particles).

[0020] In some embodiments, in S3, an aluminum adjuvant is further used to activate B cells upon co-stimulation with immunogenic dead tumor cell lysate particles and the aluminum adjuvant. In some embodiments, alum adjuvant, particularly Thermo Scientific, can be used as the aluminum adjuvant. TM Imject TM Alum adjuvant. In some embodiments, the amount of aluminum adjuvant used may be 3-10 μg / mL, preferably 5 μg / mL.

[0021] In some implementations, the co-cultivation time in S3 can be 24-48 hours, preferably 36 hours.

[0022] On the other hand, the present invention provides an engineered B-cell vaccine prepared by the above method.

[0023] Furthermore, this invention relates to the application of the aforementioned engineered B-cell vaccine in the preparation of drugs for the prevention of tumor occurrence and development. In some embodiments, the engineered B-cell vaccine is obtained by culturing immunogenic dead melanoma cell lysates, wherein the tumor is melanoma.

[0024] This invention designs a B-cell vaccine agent stimulated by immunogenic dead tumor cell lysate and aluminum adjuvant for the prevention of tumor (e.g., melanoma) occurrence and development. First, spleens of C57 mice are harvested, lymphocytes are isolated, and CD3 monoclonal antibodies are added to bind to T cells, blocking their activation signal transduction and eliminating T cells to obtain relatively pure B cells. Then, tumor cell lysate is used as a specific antigen, and with the assistance of aluminum adjuvant, B cells are activated to the maximum extent. The activated B cells not only show upregulated expression of activating proteins CD69 and CD138, but also significantly upregulated expression of surface functional proteins (antigen-presenting proteins) such as MHC-II, CD86, and CD80. Activated B cells were used as tumor cell vaccines in an in vitro co-culture system with T cells in equal proportions. This significantly stimulated T cell activation, promoted T cell differentiation into cytotoxic T cells, and upregulated the secretion and expression of perforin and granzyme B. Simultaneously, activated T cells upregulated the expression of CD40, a functional protein on the surface of the antigen-activated B cell vaccine, stimulating the occurrence and development of T cell-dependent humoral immune responses and increasing the production of tumor-specific antibodies. In in vitro studies, the co-culture system of B cell vaccines and T cells significantly inhibited the proliferation of B16 tumor cells and disrupted their normal cell morphology. Using the same method, a 4T1-specific B cell vaccine was prepared and co-cultured with T cells, exhibiting the same in vitro tumor-suppressive activity. Inoculation of C57 mice with the B cell vaccine to establish a mouse melanoma model showed that it also had good in vivo activity in preventing tumor development and progression. Therefore, using in vitro activated B cells as cell vaccines has great potential in preventing tumor occurrence and development. Attached Figure Description

[0025] Figure 1 The purification and activation of B cells in Example 1 are shown. A: Flow cytometry analysis of the expression of CD45R, CD138, and CD3 in purified B cells; B: Flow cytometry analysis of CD69... + B cell expression; C: CD69 expression + Statistical analysis of flow cytometry results of B cells; DI: qRT-PCR analysis of the expression of inflammatory factors IL-6 (D), IL-1β (E), IL-12 (F), TNF-α (G), TGF-β (H), and IL-10 (I); J: Statistical analysis of tumor volume.

[0026] Figure 2Characterization of the cell vaccine in Example 2 is shown. Wherein: A: Representative optical microscopic images of B cells after treatment with different formulations; B: Statistical count of B cell numbers before and after treatment with different formulations; C, E, G, I, K: Flow cytometry gating strategies; D, F, H, J, L: Statistical analysis of the average fluorescence intensity of CD138, CD69, MHC-II, CD80, and CD86.

[0027] Figure 3 The activation of the T cell and B cell co-culture system in Example 3 is shown. A: Flow cytometry detection of CD40. + B cells; B: CD40 + C: Statistical analysis of mean fluorescence intensity of B cells; D: ELISA determination of the content of specific anti-tumor IgG antibodies in different co-culture systems; E: ELISA determination of the content of PE in different co-culture systems; Flow cytometry detection of CD8. + Granzyme B + T cells; F: CD8 + Granzyme B + Statistical analysis of T cells; G: IFN-γ + Relative quantification of T cells; H: IFN-γ + Statistical analysis of T cells.

[0028] Figure 4 The following are examples of the effects of the lymphocyte co-culture system on tumor cells in Example 3 (A), flow cytometry analysis of representative optical microscopic images of B16 cell morphology (B), statistical analysis of B16 proliferation activity (C), flow cytometry analysis of representative optical microscopic images of 4T1 cell morphology (D), and statistical analysis of 4T1 proliferation activity (E).

[0029] Figure 5 The protective effect of the Alum / Lysis vaccine against tumors in Example 4 is illustrated. Wherein: A: Tumor photograph; B: Tumor volume; C: Tumor weight; D: Tumor inhibition efficiency; E: Representative TUNEL stained image of tumor tissue section.

[0030] Figure 6 The in vivo immunostimulatory effect of the Alum / Lysis vaccine in Example 4 is illustrated. Specifically: A: ELISA was used to detect the level of anti-tumor IgG in mouse serum; B: Flow cytometry analysis of spleen CD138... + Plasma cell gating strategy; C: CD138 + Statistical analysis of mean fluorescence intensity of plasma cells; D and F: flow cytometry analysis (D)CD4 + T cells and (F)CD8 +T cell gating strategies in tumor distribution; E and G: (E)CD4 + and (G)CD8 + Statistical analysis of the mean fluorescence intensity of T cells.

[0031] Figure 7 The biosafety assessment in Example 4 is shown. Wherein: A: Mouse weight change; B: Organ coefficient; C: H&E staining of major organs, scale bar = 100 μm; D and E: (D) Biochemical analysis of plasma AST / ALT and (E) ALP.

[0032] Figure 8 The therapeutic effect of the Alum / Lysis vaccine on tumors in Example 5 is illustrated. Wherein: A: Tumor photograph; B: Tumor volume; C: Tumor weight; D: Tumor inhibition efficiency; E: Representative TUNEL stained image of tumor tissue section.

[0033] Figure 9 This illustrates how the Alum / Lysis vaccine enhanced the immune response in tumors, as shown in Example 5. Wherein: A and B: (A) CD19 in tumor tissue sections. + B cells and CD138 + Plasma cells and (B) CD4 + T cells and CD8 + Representative immunofluorescence staining images of T cells, scale bar = 100 μm; CF: ELISA method was used to detect the levels of IFN-γ (C), TNF-α (D), IL-6 (E), and IL-2 (F) in tumor tissue.

[0034] In the image above, P<0.0001, P<0.001, P<0.01, P<0.05, ns, no statistical significance. Detailed Implementation

[0035] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1: Activation and Characterization of B Cells 1. Materials and Instruments 1.1 Materials DOX•HCl (Qingdao Smart Trading Co., Ltd., Shanghai); CD3 monoclonal antibody (GMP Monoclonal Anti-Human CD3 Antibody (OKT3), Beijing Biosciences Biotechnology Co., Ltd., Shanghai); Mouse lymphocyte separation medium (Shanghai Dakowei Biotechnology Co., Ltd., USA); 0.25% trypsin digestion solution (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai); CD69-PacificBlue flow cytometry antibody (BD Biosciences, USA); CD45R-PE flow cytometry antibody (BD Biosciences, USA); CD138-APC-Cy7 flow cytometry antibody (BD Biosciences, USA); CD3-PerCP flow cytometry antibody (BD Biosciences, USA); Total RNA extraction kit (Yisheng Biotechnology Co., Ltd., Shanghai); Hifair® AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR (Cat #11151ES) (Yisheng Biotechnology Co., Ltd., Shanghai); Isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether).

[0039] 1.2 Instruments Electronic balance (BT25S, Sartorius, Germany); Real-time PCR instrument (85-2A, Thermo); Vortex mixer (R8-1, Beijing Wunuos Technology Co., Ltd.); Pure water system (Ultrapure plus-12A, Shanghai Hetai Instrument Co., Ltd.); Flow cytometer (FACS Calibur, BD Pharmingen); Water bath sonicator (SBL-10DT, Ningbo Xinzhi Biotechnology Co., Ltd.); Thermostatic magnetic stirrer (X85-2S, Meiyingpu Instrument Manufacturing Co., Ltd.); Manual pipette (Eppendorf, Germany); Autoclave (SB-5200D, Ningbo Xinzhi Biotechnology Co., Ltd.); Vernier caliper (Guilin Guanglu Digital Measurement and Control Co., Ltd.); Horizontal shaker (X85-2S, Meiyingpu Instrument Manufacturing Co., Ltd.); Culture medium filter (Nalgene, Thermo Scientific, USA).

[0040] 1.3 Experimental Cells and Animals The mouse melanoma cell line (B16) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China); SPF-grade C57 black mice (female, 18–20 g) were purchased from the Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). All mice were housed under standard SPF conditions, with the housing maintained at a constant temperature of 25±2℃, relative humidity of 50%±10%, and a 12-hour light-dark cycle. The animals were kept under sterile conditions with ample access to sterile drinking water and feed, and were allowed free movement. All animal experimental protocols were approved by the Animal Experiment Ethics Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and followed the requirements of the International Association for Laboratory Animal Care (IACAL) Assessment and Accreditation Guidelines.

[0041] 2 Experimental Methods 2.1 Solution Preparation Preparation of recombinant mouse IL-4: Centrifuge at 15,000 rpm for 5 min to ensure a relatively accurate concentration of the cytokine solution, allowing protein powder to settle at the bottom of the tube. Add an appropriate amount of ultrapure water to the tube to prepare a stock solution of IL-4 (500 μg / mL). Gently shake the centrifuge tube until the solution is clear, and carefully pipette several times until the protein powder is fully dissolved. Dilute the stock solution of IL-4 to 100 μg / mL with 5% trehalose solution, then aliquot it into 200 μL centrifuge tubes at a volume of 10 μL each and store at -80℃ for later use.

[0042] Preparation of 1 μM DOX•HCl: The molecular weight of DOX•HCl is 579.98. Weigh 5.8 mg of DOX•HCl powder, dissolve it in 10 mL of DMEM cell culture medium, aliquot 20 μL into 200 μL centrifuge tubes, and store at 4℃ in the dark until use.

[0043] 2.2 Cell Culture Remove the frozen B16 cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath. After the cryopreservation solution thaws, centrifuge at 1500 rpm and 25°C for 3 min. Discard the supernatant and resuspend the cells in DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. Transfer the cells to a cell culture flask and culture at 37°C and 5% CO2. When the cell density reaches 80% of the bottom area of ​​the flask, passage the cells: carefully aspirate and discard the original cell culture medium, carefully wash the cells twice with 2 mL PBS, discard the PBS, carefully add an appropriate amount of cell trypsin digestion solution along the side wall of the culture dish, shake the cell culture flask to allow the trypsin to fully wet the cells at the bottom of the flask, digest for 3 minutes, then aspirate the trypsin digestion solution, carefully add more DMEM culture medium than the volume of trypsin to stop cell digestion, divide the cells in half and passage them into two culture dishes. Transfer the cells to suitable culture flasks or dishes, add sufficient DMEM cell culture medium, and continue culturing after the cells are homogenized by pipetting.

[0044] 2.3 Extraction, culture and purification of B cells 2.3.1 Extraction of lymphocytes Before the experiment, ophthalmic scissors, ophthalmic forceps, a 200-mesh filter, and other experimental equipment were soaked in 75% ethanol. Six- to eight-week-old C57 mice were euthanized, and the euthanized mice were immersed in 75% ethanol for 5 minutes. The spleen was isolated, and any adhesions were removed. The spleen was placed in a cell culture dish containing sterile PBS, transferred to a laminar flow hood, and rinsed three times with sterile PBS. An appropriate amount of sterile, room-temperature homogenized mouse lymphocyte separation medium was added to a new 35 mm cell culture dish. Place the mouse spleen above a 200-mesh filter and grind the spleen tissue into a cell suspension using a 5 mL injection plunger. Quickly transfer this cell suspension (splenic cells) to a clean centrifuge tube using a manual pipette. Gently add 1000 μL of RPMI 1640 medium, ensuring a clear liquid level. Centrifuge the tube containing the mixture at 800 ×g on a horizontal rotor at room temperature for 30 min. Set the acceleration (400-600 rpm / min) and deceleration (200-400 rpm / min) to the third setting. After centrifugation, carefully remove the lymphocyte layer, add 10 mL of RPMI 1640 complete medium, invert several times, and centrifuge at 250 ×g at room temperature for 10 min. Collect the cells, decant the supernatant, resuspend the cells in pre-warmed RPMI 1640 cell culture medium, and count the cells.

[0045] 2.3.2 Purification and Culture of B Cells The extracted lymphocytes were processed at a concentration of 6.0 × 10⁻⁶. 9 L -1The concentrations were suspended in RPMI 1640 medium, and IL-4 (20 µg / L) and CD3 monoclonal antibody (5 mg / L) were added separately. The mixture was then incubated at 37°C in a 5% CO2 incubator. During the incubation period, the original medium was discarded every other day, and an equal volume of RPMI 1640 medium and stimulating factors (IL-4 (20 µg / L) and CD3 monoclonal antibody (5 mg / L)) were added. The cells were then cultured for another 4 days to obtain purified B cells.

[0046] 2.3.3 Flow cytometry detection of lymphocytes and their subsets Cells were collected, and the fluorescence intensity of CD3e, CD45R, and CD138 positive cells was detected by flow cytometry.

[0047] 2.4 Preparation of tumor cell lysate particles B16 cells were seeded in 10 cm culture dishes. When the cells reached 50% confluence, the DMEM complete medium was replaced with blank medium containing 1 μM DOX, and the cells were incubated at 37°C with 5% CO2 for 24 h. During this period, the tumor cells underwent immunogenic cell death. The cells were centrifuged at 1500 ×g at room temperature for 15 min, and the resulting cell particles were resuspended in a solution containing 80% DMEM and 20% FBS at 4°C. The suspension was then immersed in liquid nitrogen for 12 h, thawed in a 37°C water bath, and centrifuged at 1500 ×g at room temperature for 15 min to obtain 1 × 10⁻⁶ cells of lysate from the immune-dead tumor cells. 7 indivual.

[0048] 2.5 Activation and flow cytometry characterization of B cells (1) Cell activation and culture B cells were cultured in a 5% CO2 incubator for 36 h at a ratio of B cells to tumor cell lysate particles of 10:1 (cells:particles) to obtain activated B cells. Activated B cells and unactivated B cells were then seeded separately into 6-well plates for further culture.

[0049] (2) Flow cytometry was used to detect the fluorescence intensity of CD69 positive cells. The activated and unactivated B cells cultured above were collected, and the fluorescence intensity of CD69 positive cells was detected by flow cytometry.

[0050] 2.6 Activation of B cells and qPCR verification RNA was extracted from activated and unactivated B cells, and the expression differences of IL-6, IL-1β, IL-12, IL-10, TNF-α, and TGF-β in activated and unactivated B cells were compared.

[0051] 2.7 Confirmation of the activation of B cell antitumor activity in vivo Activated B cells and inactivated B cells were induced using the method described above. C57 mice were randomly assigned to two groups: a B cell group and an activated B cell group, with 5 mice in each group. Mice were then vaccinated at a rate of 5 × 10⁻⁶ cells / mL according to their grouping. 6 Cells were seeded at a specific number per mouse, and different B cells were injected intraperitoneally. One week later, C57 mice were inoculated with melanoma cells. First, B16 cells in the logarithmic growth phase were digested with trypsin and collected in centrifuge tubes. The cells were centrifuged at 1250 rpm for 5 min at 4°C. The cell supernatant was discarded, and an appropriate amount of sterile PBS was added to adjust the cell density to 5 × 10⁻⁶ cells / mouse. 6 Using a sterile 1 mL syringe, draw 100 μL of the above B16 cell suspension (avoiding air bubbles), carefully inject the cell suspension subcutaneously, then carefully turn the syringe needle and slowly remove the syringe. Press the injection site with a clean, dry cotton ball for a moment. Observe the tumor development and progression of each group of mice every two days and perform statistical analysis.

[0052] 3 Results and Discussion Experimental results are as follows Figure 1 As shown.

[0053] The spleen is an important peripheral immune organ, home to mature lymphocytes. B cells account for approximately 60% of the total splenic lymphocytes, while T cells account for about 40%. CD3e, a type I membrane present on the surface of T cells, plays a crucial role in their development. It is a key component in the initial signal transduction phase of T cell activation, and a lack of the CD3e gene can lead to severe immunodeficiency. Therefore, lymphocytes were extracted from the spleen of C57 mice, and then a CD3 monoclonal antibody was introduced into the T cells to inhibit the transmission of activation signals. This process promoted the isolation of T cells, resulting in a purified B cell population.

[0054] Mouse CD45, also known as the leukocyte common antigen (LCA), and CD45R, also known as B220, represent isoform variants of CD45. These are expressed as common antigens on the surface of B cells throughout their development from primitive B cells to mature B cells. Plasma cells are non-dividing terminally differentiated B cells derived from activated germinal center (GC) B cells and possess a unique marker profile. During differentiation of mature B cells into plasma cells, certain surface markers such as CD45R, CD20, and CD19 are downregulated, while plasma cell-specific markers such as CD138 are upregulated. Flow cytometry analysis showed that only 1.47% of mouse spleen lymphocytes were CD3e-positive, 60.65% were CD45R-positive, and 37.78% were CD138-positive. Figure 1 (A). Flow cytometry results showed that spleen T cells (CD3 positive) had been cleared, and B cells (CD45R) had been eliminated. + +CD138 + The purity of the CD3 monoclonal antibody is >90%. Studies have shown that CD3 monoclonal antibodies can effectively inactivate T cells and purify them to obtain relatively pure B cells.

[0055] CD69 is primarily expressed on the surface of activated immune cells, mainly T cells and B cells. After immune cells are stimulated by external factors (such as antigen stimulation), it is rapidly expressed on the cell surface, marking cell activation and response. During the activation of T cells and B cells, CD69 expression can influence cell proliferation and differentiation. Figure 1 In the B cells group, flow cytometry analysis showed that the proportion of CD69-positive B cells was 3.65% in the B cells group and 37.07% in the activated B cells group (tumor cell lysate-activated B cells group). This indicates that the expression of CD69 on the surface of B cells activated by tumor cell lysate was significantly upregulated compared to B cells, suggesting that B cells were successfully activated under the stimulation of tumor cell lysate (activated B cells group). Furthermore, qPCR results showed that the expression of pro-inflammatory factors IL-6, TNF-α, and IL-1β was significantly upregulated in the activated B cells group compared to the B cells group. Figure 1 (DG). Conversely, the expression of anti-inflammatory factors IL-12 and TGF-β was significantly downregulated (DG). Figure 1 (HI). Tumor volume analysis in tumor-bearing mice showed that, compared with the B cells group, B cells inoculated with activated B cells significantly inhibited the occurrence and development of melanoma in the C57 mouse melanoma model. Figure 1 (J), showing certain tumor-preventive activity. In summary, tumor cell lysates can effectively activate B cells and have a certain preventive effect on the occurrence and development of tumors.

[0056] Example 2: Preparation and Characterization of B-cell Vaccine 1 Materials and Instruments 1.1 Materials DMEM cell culture medium (Dalian Meilun Biotechnology Co., Ltd., Dalian); 1640 cell culture medium (Dalian Meilun Biotechnology Co., Ltd., Dalian); fetal bovine serum (Dalian Meilun Biotechnology Co., Ltd., Dalian); 0.25% trypsin (Beyotime Biotechnology Co., Ltd., Shanghai); penicillin-streptomycin dual antibody (Beyotime Biotechnology Co., Ltd., Shanghai); Thermo Scientific TM Imject TM Alum adjuvant (Beijing Newp Biotechnology Co., Ltd., Beijing); CD69-PacificBlue flow cytometry antibody (BD Biosciences, USA); CD45-FITC flow cytometry antibody (BD Biosciences, USA); CD138-APC-Cy7 flow cytometry antibody (BD Biosciences, USA); CD3-PerCP flow cytometry antibody (BD Biosciences, USA); CD19-FITC flow cytometry antibody (BD Biosciences, USA); CD80-PacificBlue flow cytometry antibody (BD Biosciences, USA); CD40-PE flow cytometry antibody (BD Biosciences, USA); CD86-APC flow cytometry antibody (BD Biosciences, USA); MHC-II-BV605 flow cytometry antibody (BD Biosciences, USA); Culture medium filter (Nalgene, Thermo Scientific, USA); BSA bovine serum albumin (Dalian Meilun Biotechnology Co., Ltd., Dalian).

[0057] 1.2 Instruments Pure water system (Ultra Pure Plus-12A, Shanghai Hetai Instrument Co., Ltd., China); Electronic balance (BT25S, Sartorius, Germany); Vortex mixer (R8-1, Beijing Wunuos Technology Co., Ltd.); Pure water system (Ultrapure Plus-12A, Shanghai Hetai Instrument Co., Ltd.); Flow cytometer (FACS Calibur, BD Pharmingen); Water bath sonicator (SBL-10DT, Ningbo Xinzhi Biotechnology Co., Ltd.); Automated cell counter (ThermoScientific, USA); Optical microscope (XD-202, Jiangnan, China).

[0058] 1.3 Experimental Cells Same as Example 1.

[0059] 2 Experimental Methods 2.1 Solution Preparation (1) Preparation of sodium carbonate buffer (pH 7.4): Weigh 1.59 g of sodium carbonate powder and 2.93 g of sodium bicarbonate powder and dissolve them in 1 L of distilled water. Adjust the pH to 7.4 with sodium hydroxide.

[0060] (2) Preparation of coating dilution buffer for ELISA detection: For the BCA protein quantification method to detect the protein content of tumor cell lysate, take the above neutral sodium carbonate buffer and dilute the tumor cell lysate to the appropriate plating concentration.

[0061] (3) Preparation of PBST: Measure 1 L of PBS solution, use a 1000 μL pipette to transfer 500 μL of Tween 20, and sonicate in a water bath to mix the two solutions evenly to obtain PBST solution. Store at room temperature for later use.

[0062] 2.2 Preparation of B-cell vaccine co-stimulated by aluminum adjuvant and tumor cell lysate Prepare aluminum adjuvant / RPMI 1640 culture medium; take aluminum adjuvant (Thermo Scientific) TM Imject TM Alum adjuvant was used to dilute the aluminum adjuvant / RPMI 1640 culture medium to a concentration of 5 μg / mL. Preparation of B16 cell lysate / RPMI 1640 culture medium: B16 cell lysate particles were dissolved in the aforementioned aluminum adjuvant / RPMI 1640 culture medium to obtain B16 cell lysate + aluminum adjuvant / RPMI 1640 culture medium. The B cells purified by the method in 2.3.2 of the above examples were divided into four groups: B cells group, Alum group (B cells + aluminum adjuvant, aluminum adjuvant: 5 μg / mL), Lysis group (B cells + tumor cell lysate particles, cell number: tumor cell lysate particles ratio of 10:1), and Alum / Lysis group (B cells + aluminum adjuvant + tumor cell lysate particles, aluminum adjuvant: 5 μg / mL; cell number: tumor cell lysate particles ratio of 10:1). They were cultured in a cell culture incubator for 36 h, and then centrifuged to remove B16 cell lysate / culture medium, resulting in four groups of B cell vaccines.

[0063] 2.3 Morphological and quantitative changes of B-cell vaccines under different stimuli under an optical microscope Appropriate amounts of cell suspensions from the B cells group, Alum group, Lysis group, and Alum / Lysis group were taken and observed under an optical microscope at the same magnification to observe the growth and cell morphology of the four groups of B cells.

[0064] 2.4 Characterization of B-cell vaccines Cells were collected, and each sample was aliquoted into two clean centrifuge tubes labeled "1" and "2". Flow cytometry was used to detect the fluorescence intensity of CD45, CD3e, CD69, and CD138 positive cells in sample tube "1" and the fluorescence intensity of CD19, MHC-II, CD80, and CD86 positive cells in sample tube "2".

[0065] 3 Results and Discussion Experimental results are as follows Figure 2 As shown.

[0066] Compared with the other three groups, the number of B cells was significantly upregulated under the synergistic effect of tumor cell lysis buffer and aluminum adjuvant (Alum / Lysis cell group). Figure 2 (AB). Microscopic observation showed that it had exhibited clumping growth. Figure 2 Therefore, aluminum adjuvants and tumor cell lysates can maximize the stimulation of B cell proliferation.

[0067] The number of B cells in the Lysis group also increased to some extent, and cell aggregation was also observed. Figure 2 As shown in Figure A), tumor cell lysates can also effectively stimulate B cell proliferation. The cell numbers in the Alum group and the B cells group were not significantly upregulated, and no cell clumping was observed under a microscope, indicating that aluminum adjuvant alone cannot effectively stimulate B cell proliferation. Flow cytometry results showed that, under stimulation by tumor cell lysates or aluminum adjuvant alone, CD69 levels in the Lysis and Alum groups increased. + B cells and CD138 + The number of plasma cells increased to some extent. Figure 2 (CF). Alum / Lysis group CD69 + B cells and CD138 + Plasma cells have the highest number ( Figure 2 (CF). In summary, both aluminum adjuvants and tumor cell lysates can activate B cells to some extent, and their synergistic use maximizes B cell activation.

[0068] Previous studies have shown that B cells alone cannot effectively inhibit tumor growth under in vitro culture conditions. However, in the in vivo anti-tumor immune response environment, B cells can act as antigen-presenting cells, interacting with T cell antigen receptors by upregulating the expression of major histocompatibility complex II (MHC-II), thereby initiating activation signal 1. B cells bind to corresponding receptor molecules on T cells through their surface protein B7 molecules (CD86, CD80), activating co-stimulatory signals (activation signal 2), leading to complete T cell activation. Activated T cells expressing CD40L can interact with CD40 on activated B cells, promoting T cell-dependent humoral immune responses. Flow cytometry analysis showed that aluminum adjuvant or tumor cell lysates alone could enhance the expression of CD86 on the surface of B cells (…). Figure 2 (KL). Furthermore, the synergistic use of aluminum adjuvant and tumor cell lysates significantly increased the expression of B cell surface antigen-presenting related proteins MHC-II, CD86, and CD80. Figure 2 The study (GL) further confirmed that the combined action of aluminum adjuvant and tumor lysate enhanced the B-cell antigen presentation capacity.

[0069] Example 3: In vitro study of B-cell vaccine 1 Materials and Instruments 1.1 Materials Thermo Scientific TM Imject TMAlum adjuvant (Beijing Newp Biotechnology Co., Ltd., Beijing); Horseradish peroxidase-labeled goat anti-mouse IgG (Beyotime Biotechnology Co., Ltd., Shanghai); Horseradish peroxidase-labeled secondary antibody IgG (Beyotime Biotechnology Co., Ltd., Shanghai); TMB chromogenic solution (Beyotime Biotechnology Co., Ltd., Shanghai); TMB chromogenic stop solution (Beyotime Biotechnology Co., Ltd., Shanghai); CD19-FITC flow cytometry antibody (BD Biosciences, USA); CD138-APC-Cy7 flow cytometry antibody (BD Biosciences, USA); CD3-PerCP-Cy5.5 flow cytometry antibody (BD Biosciences, USA); CD80-PacificBlue flow cytometry antibody (BD Biosciences, USA); MHC-II-BV605 flow cytometry antibody (BD Biosciences, USA); CD86-APC flow cytometry antibody (BD Biosciences, USA); CD40-PE flow cytometry antibody (BD Biosciences, USA); CD45-FITC flow cytometry antibody (BD Biosciences, USA). BD Biosciences, USA); CD8-APC flow cytometry antibody (BD Biosciences, USA); Granzyme B-PE flow cytometry antibody (BD Biosciences, USA); IFN-γ-APC-Cy7 flow cytometry antibody (BD Biosciences, USA); BSA bovine serum albumin (Dalian Meilun Biotechnology Co., Ltd., Dalian); Mouse perforin ELISA kit (Guangzhou Aorida Biotechnology Co., Ltd., Guangzhou); EdU-488 cell proliferation assay kit (Shanghai Saiyi Biotechnology Co., Ltd., Shanghai). 1.2 Instruments Pure water system (Ultra Pure Plus-12A, Shanghai Hetai Instrument Co., Ltd., China); Flow cytometer (FACSCalibur, BD Pharmingen); Microplate reader (MULTISKAN GO, Thermo Scientific); Optical microscope (XD-202, Jiangnan, China); pH meter (LAB 850, Schott); Water bath ultrasonic analyzer (SBL-10DT, Ningbo Xinzhi Biotechnology Co., Ltd., China); Horizontal shaker (X85-2S, Meiyingpu Instrument Manufacturing Co., Ltd.); High-speed micro-volume temperature-controlled centrifuge (Eppendorf, USA). 1.3 Experimental Cells Same as Example 1.

[0070] 2 Experimental Methods 2.1 Cell Culture B16 cells: Remove the frozen B16 cells from the liquid nitrogen container and immediately place them in a 37°C constant temperature water bath to thaw the cell cryopreservation solution. Centrifuge at 1500 rpm for 3 min at 25°C, discard the supernatant, resuspend the cells in DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and transfer them to a cell culture flask. Incubate at 37°C and 5% CO2. When the cell density reaches 80% of the bottom area of ​​the flask, passage the cells: carefully aspirate and discard the original cell culture medium, and use 2 mL of... Carefully rinse the cells twice with PBS, discard the PBS, and carefully add an appropriate amount of cell trypsin digestion solution along the side wall of the culture dish. Shake the cell culture flask to allow the trypsin to fully wet the cells at the bottom of the flask. After digestion for 3 minutes, aspirate the trypsin digestion solution. Carefully add more DMEM culture medium than the volume of trypsin to stop cell digestion. Divide the cells into two portions and passage them into two culture dishes. Transfer the cells to suitable culture flasks or culture dishes, add sufficient DMEM cell culture medium, and gently pipette the cells to ensure even distribution before continuing culture.

[0071] 4T1 cells: Remove the cryopreserved 4T1 cells from the liquid nitrogen container and immediately place them in a 37°C constant temperature water bath to thaw the cell cryopreservation solution. Centrifuge at 1500 rpm for 3 min at 25°C, discard the supernatant, resuspend the cells in RPMI 1640 medium supplemented with fetal bovine serum and penicillin-streptomycin antibiotic solution, and transfer them to a cell culture flask. Incubate at 37°C and 5% CO2. When the cell density reaches 80% of the bottom area of ​​the flask, passage the cells: Carefully aspirate and discard the original cell culture medium, carefully wash the cells twice with 2 mL PBS, discard the PBS, carefully add an appropriate amount of cell trypsin digestion solution along the side wall of the culture dish, shake the cell culture flask to ensure the trypsin fully wets the cells at the bottom of the flask, digest for 3 minutes, then aspirate the trypsin digestion solution, carefully add more RPMI 1640 culture medium than the volume of trypsin to stop cell digestion, divide the cells in half and passage them into two culture dishes, then transfer them to suitable culture flasks or dishes, and replenish with sufficient RPMI. Use 1640 cell culture medium to mix the cells thoroughly and continue culturing.

[0072] 2.2 Solution Preparation Preparation of fixative: Weigh 2 g of paraformaldehyde powder, dissolve it in 50 mL of neutral PBS, transfer it to a magnetically stirred container, add a few drops of sodium hydroxide solution, heat it to 60°C in a fume hood to dissolve it, cool it to room temperature, adjust its pH to 7.4 with hydrochloric acid solution, and store it at 4°C for later use.

[0073] Preparation of washing solution: Place BSA powder at room temperature until it returns to room temperature, weigh 1.5 g of BSA powder, dissolve it in 50 mL of PBS under vortex conditions, and store at 4℃ for later use.

[0074] Preparation of permeation solution: Measure 20 mL of PBS into a 50 mL centrifuge tube, add 100 µL of Triton X-100 to the PBS, vortex to dissolve, and store at room temperature for later use.

[0075] 2.3 Activation of B cell vaccine under T cell co-culture T cells were divided into four groups according to cell density. Each group was co-cultured with different B cell vaccines at a 1:1 ratio for 24 h with B cells, Alum, Lysis, and Alum / Lysis groups (the same as in Example 2). B cells alone (B cells group) were used as a control. Flow cytometry was used to detect the fluorescence intensity of CD80, CD86, MHC-II, CD40, and CD138 in B cells co-cultured with T cells and with different B cell vaccines.

[0076] 2.4 ELISA detection of tumor-specific IgG content in the supernatant of different B-cell vaccines co-cultured with T cells (1) ELISA plate Tumor cell lysates were diluted with PBS to a protein concentration of 100 μg / ml. 100 μL of tumor cell lysate / phosphate buffer was carefully added to a 96-well adsorption plate using a 100 μL pipette, sealed with a sealing film, and incubated overnight at 4°C for 12 h.

[0077] (2) Collect cell culture supernatant a. Using a 5 mL pipette, thoroughly mix the five groups of lymphocyte co-culture systems, and take 0.5 mL into a 1.5 mL centrifuge tube. Take 3 sample tubes from each group.

[0078] b. Centrifuge at 1500 rpm for 5 min at 4℃.

[0079] c. Transfer the supernatant to a 1.5 mL clean centrifuge tube and label it with the group and number.

[0080] (3) ELISA detection of tumor-specific IgG content in cell culture supernatant: a. Washing the plate: Take out the 96-well plate that has been incubated overnight at 4°C, pour out the tumor cell lysate / carbonate buffer, fill each well with PBST washing buffer, let stand for 1 min, shake off the washing buffer, pat dry on absorbent paper, and repeat this process three times.

[0081] b. Blocking: Add 100 μL of 1% BSA PBS solution to each well for blocking. Blocking conditions: Incubate at 37℃ for 1.5 h.

[0082] c. Wash the plate: Repeat the above plate washing operation.

[0083] d. Antibody incubation: Add 100 μL of cell culture supernatant to each well and incubate at 37°C for 1 h.

[0084] e. Wash the plate: Repeat the above plate washing operation.

[0085] f. Incubation with secondary antibody: Dilute IgG 1000 times with PBS, add 100 μL of the diluted secondary antibody solution to each well, and incubate at 37°C for 1 h.

[0086] g. Washing the plate: Repeat the above plate washing operation.

[0087] h. Color development: Add 100 μL of TMB color development solution to each well and incubate at 37°C for 10 min.

[0088] i. Termination: Add 100 μL of TMB stop solution to each well.

[0089] j. Detection: OD value of each well was measured at a wavelength of 450 nm.

[0090] 2.5 Flow cytometry investigation of the effect of B cell vaccine on the differentiation capacity of cytotoxic T cells T cells were divided into five groups according to cell density. They were co-cultured with RPMI-1640 medium (T cells group), B cells group, Alum group, Lysis group, and Alum / Lysis group of B cell vaccines for 24 h. The fluorescence intensity of CD45, CD4, CD8a, Granzyme B, and IFN-γ positive cells was detected by flow cytometry to analyze the effect of different B cell vaccines on cytotoxic T cell differentiation.

[0091] 2.6 ELISA detection of the effect of B cell vaccine on perforin secretion by T cells After co-incubating the above four groups of different B-cell vaccines with T cells for 24 h, and using T cells alone (T cells group) as a control, the effects of the five groups of T cells on perforin (PE) secretion were investigated by ELISA. The following experiments were all conducted under these light-protected incubation conditions: (1) Take out the slats that have been refrigerated at 4°C, equilibrate them at room temperature for 25 minutes, put the remaining slats back into the aluminum foil bag and seal them, and keep them in the refrigerator at 4°C for further storage.

[0092] (2) Set up the sample wells and standard wells in advance according to the number of experimental samples and the instructions. Use a manual pipette to add the standard of different concentrations into the standard wells, 50 μL per well.

[0093] (3) Take 10 μL of each group of test samples and add them to the sample wells. Then add 40 μL of sample diluent to each well. Note that blank wells should not be added.

[0094] (4) Use a pipette to add 100 μL of horseradish peroxidase (HRP) labeled antibody to the standard wells and sample wells. Note that no antibody should be added to the blank wells. Seal the standard wells and sample wells tightly with sealing film and incubate on a shaker (150 rpm, 37℃) for 60 min.

[0095] (5) Discard the above reaction solution, pat the excess liquid in the plate thoroughly dry on the filter paper, add 300 μL of washing solution to each well, soak for 1 min, shake off the washing solution in the well, pat thoroughly dry on the dry and clean filter paper, and repeat the above steps 5 times.

[0096] (6) Substrate A and substrate B were added to each well in quick succession, 50 μL per well, and incubated at 37°C in the dark for 15 min.

[0097] (7) Add stop solution quickly to each well, 50 μL per well. After reacting for 10 min, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value of each well at a wavelength of 450 nm and perform statistical analysis.

[0098] 2.7 Effects of different B-cell vaccine and T-cell co-culture systems on tumor cell growth and morphology B16 cells were fed at a concentration of 1.0 × 10⁶ 5 The cells were seeded at the specified density in 24-well plates and cultured overnight until they recovered to normal morphology and state. T cells were then co-cultured with B cell vaccines of equal proportions in the B cells group, Alum group, Lysis group, and Alum / Lysis group, with a total lymphocyte to B16 cell ratio of 2:1. After co-culturing at 37°C and 5% CO2 for 24 h, the lymphocytes in the 24-well plates were aspirated and discarded, and the suspended cells in the wells were rinsed with PBS. The growth of B16 cells under lymphocyte stimulation in the five groups and the differences in B16 cell growth and cell morphology under blank culture conditions were observed under an optical microscope at the same magnification.

[0099] 2.8 Effects of different B-cell vaccine and T-cell co-culture systems on the proliferation activity of B16 / 4T1 cells (1) B16 cells were treated at a concentration of 1.0 × 10⁻⁶. 5 Cells were seeded at the specified density in 24-well plates and cultured overnight until they recovered to their normal morphology and state. Then, the five different lymphocyte co-culture systems were co-cultured with B16 cells for 24 h.

[0100] (2) Prepare 1×EdU working solution: Dilute 10 mM EdU working solution with DMEM complete medium to the working concentration of 1×EdU.

[0101] (3) Before the experiment, the above 1×EdU working solution was preheated in a constant temperature water bath at 37℃. The lymphocytes in the 24-well plate were aspirated and discarded. The suspended cells in the well plate were rinsed clean with PBS. An equal volume of 1×EdU working solution was added to the 24-well plate.

[0102] (4) Continue incubating the cells for an appropriate time.

[0103] (5) After EdU labeling, remove the culture medium, digest the cells with trypsin, centrifuge at 500 ×g at room temperature for 5 min, and collect the cells in a 1.5 mL centrifuge tube.

[0104] (6) Add 0.5 mL of fixative to each tube and fix at room temperature for 20 min.

[0105] (7) Discard the fixative and add 0.5 mL of washing solution to each tube to wash the cells 3 times, each time for 5 min.

[0106] (8) Discard the washing solution, add 0.5 mL of permeation solution to each tube, and incubate at room temperature for 15 min.

[0107] (9) Discard the permeation solution and add 0.5 mL of washing solution to each tube to wash the cells twice, 5 min each time.

[0108] (10) Prepare the Click reaction solution according to the number of cells.

[0109] (11) Discard the washing solution. Add 100 μL of the above Click reaction solution to each tube under light-protected conditions, mix well to ensure full contact between the cells and the reaction solution.

[0110] (12) Incubate the above system for 30 min at room temperature in the dark.

[0111] (13) Discard the Click reaction solution and add 0.5 mL of washing solution to each tube to wash the cells thoroughly 3 times, 5 min each time.

[0112] (14) Resuspend the cells in 150 μL of washing buffer in each tube, and then analyze the proliferation activity of B16 cells using a flow cytometer. (5 × 10⁶ cells) 4 Four groups of 4T1 cells were seeded in 24-well plates and cultured overnight until they recovered to normal morphology and state. Four groups of B cell preparations with the same formulation were obtained by stimulating 4T1 tumor cell lysate with aluminum adjuvant. These preparations were then co-cultured with T cells in equal proportions. The above five different lymphocyte co-culture systems were co-cultured with 4T1 cells for 24 h. The proliferation activity of 4T1 cells under the five culture conditions was measured using the same cell proliferation activity assay (EdU) method.

[0113] 3 Results and Discussion Experimental results are as follows Figure 3 and Figure 4 As shown.

[0114] Flow cytometry analysis showed that when the Lysis group was co-cultured with T cells, the expression of CD40 on B cells was upregulated. Figure 3 (AB). Furthermore, the combined use of aluminum adjuvant and tumor cell lysates significantly enhanced the expression of CD40 on the surface of B cells (AB). Figure 3 (AB). At the same time, the interaction with T cells maximizes the activation of the humoral immune response of B cells.

[0115] Subsequently, the levels of specific antitumor IgG antibodies in the cell culture supernatants of the five groups were detected by ELISA. The results showed that the levels of specific antitumor IgG antibodies in the cell culture supernatants of the Lysis group and the Alum / Lysis group were significantly higher than those of the other three groups. Figure 3 (C). The Alum / Lysis group produced the highest levels of specific anti-tumor IgG antibodies, but there was no significant difference in the levels of specific anti-tumor IgG antibodies between the Alum / Lysis group and the Lysis group. Figure 3 (C). The above results indicate that when tumor cell lysates or their combination with aluminum adjuvant stimulate B cells, co-culturing them with T cells can enhance the humoral immune response of B cells, thereby increasing the production of specific anti-tumor IgG antibodies.

[0116] When B cell vaccines are co-cultured with T cells, B cells also have a certain stimulatory effect on T cells. B cells in the Alum / Lysis group showed induction of CD8+. + GranzymeB + T cells and IFN-γ + The ability of T cells to proliferate and differentiate ( Figure 3 In Alum / Lysis, the combination of tumor cell lysate and aluminum adjuvant significantly upregulated the number of cytotoxic T cells. The results indicated that the combined use of tumor cell lysate and aluminum adjuvant maximally activated B cells, promoted cytotoxic T cell differentiation, and thus enhanced the anti-tumor immune response. ELISA results showed differences in perforin secretion among the five T cell groups, consistent with flow cytometry results; B cells in Alum / Lysis effectively activated T cells, increasing their Granzyme B (Granzyme B) secretion. Figure 3 EF) and PE Figure 3 Increased expression and secretion of PE (diethylstilbestrol) enhances T cell cytotoxicity against tumors. Although B cells in the Lysis group can also induce T cells to secrete PE (diethylstilbestrol). Figure 3 While B cells in the Alum / Lysis group showed good activity (D), their induction ability was weaker compared to those in the Alum / Lysis group. Conversely, B cells in both the Alum and B cell groups failed to effectively activate cytotoxic T cells or induce T cells to secrete PE (diethyltoxin). Figure 3 (D).

[0117] After co-culturing with five groups of lymphocytes for 24 h, there was no significant difference in cell density and morphology of B16 cells between the T cells group and the B16 group. Figure 4 (A). This indicates that T cells alone do not affect the growth of B16 cells. Cell density gradually decreased in both the T cells + B cells group and the Alum group, while the normal morphology of tumor cells remained unchanged. Figure 4 (A). This indicates that co-culturing T cells and B cells can affect the growth of B16 cells, and this effect is enhanced in the presence of aluminum adjuvant. The tumor cell density in the Lysis and Alum / Lysis groups was significantly lower than that in the B16 group, and the spindle cell morphology of B16 cells was essentially lost. Notably, under a light microscope, the number of B16 cells in the Alum / Lysis group was reduced to the lowest, accompanied by significant changes in tumor cell morphology (A). Figure 4 (A). The above results indicate that B-cell vaccines stimulated by tumor cell lysates, in conjunction with T cells, can significantly affect the morphology and proliferative activity of tumor cells. Furthermore, B-cell vaccines obtained through the combined stimulation of aluminum adjuvant and tumor cell lysates, in conjunction with T cells, can maximally disrupt tumor cell morphology and inhibit their proliferative activity.

[0118] To further investigate the effects of B-cell vaccine and T-cell co-culture system on tumor cell proliferation, the inventors co-cultured T-cells, T-cells+B-cells, Alum, Lysis, and Alum / Lysis groups with B16 cells for 20 hours. The proliferation activity of different groups of B16 cells was assessed using the EdU cell proliferation assay kit. EdU, linked to the fluorescent probe Alexa Fluor 488 Azide, can replace thymidine and be incorporated into newly synthesized DNA during the S phase of the cell cycle; the newly synthesized DNA is then labeled with green fluorescence. Flow cytometry analysis showed that the Alum / Lysis co-culture system exhibited the strongest inhibitory effect on B16 cell proliferation. Figure 4 (B16). Furthermore, the Alum co-culture system also showed a certain degree of inhibition on B16 cell proliferation, indicating that aluminum adjuvant may activate T cells to some extent and exert a specific anti-tumor effect. B cells were activated using 4T1 cell lysates in the same manner, and identical experimental groups were established under the same conditions. After co-culturing with 4T1 tumor cells for 20 hours, the proliferation activity of 4T1 cells in each group was assessed using the EdU cell proliferation activity assay kit. Consistent experimental results were observed. Figure 4 (DE), and obtained the same results as the above experiment.

[0119] Example 4: In vivo investigation of the effect of B-cell vaccine in preventing tumor growth 1 Materials and Instruments 1.1 Materials Thermo Scientific TM Imject TM Alum adjuvant (Beijing Newp Biotechnology Co., Ltd., Beijing); Horseradish peroxidase-labeled goat anti-mouse IgG (Beyotime Biotechnology Co., Ltd., Shanghai); Horseradish peroxidase-labeled secondary antibody IgG (Beyotime Biotechnology Co., Ltd., Shanghai); TMB chromogenic solution (Beyotime Biotechnology Co., Ltd., Shanghai); TMB chromogenic stop solution (Beyotime Biotechnology Co., Ltd., Shanghai); CD19-FITC flow cytometry antibody (BD Biosciences, USA); CD138-APC-Cy7 flow cytometry antibody (BD Biosciences, USA); CD3-PerCP-Cy5.5 flow cytometry antibody (BD Biosciences, USA); CD45-FITC flow cytometry antibody (BD Biosciences, USA); CD45R-PE flow cytometry antibody (BD Biosciences, USA); CD45-APC-Cy7 flow cytometry antibody (BD Biosciences, USA); CD3-BB700 flow cytometry antibody (BD Biosciences, USA); CD4-BV605 flow cytometry antibody (BD Biosciences, USA). Biosciences, USA); CD8-APC flow cytometry antibody (BDBiosciences, USA); BSA bovine serum albumin (Dalian Meilun Biotechnology Co., Ltd., Dalian); xylene (Sinopharm Chemical Reagent, Shanghai); citrate buffer (Beijing Zhongshan Jinqiao Biotechnology, Beijing).

[0120] 1.2 Instruments Pure water system (Ultra Pure Plus-12A, Shanghai Hetai Instrument Co., Ltd., China); Flow cytometer (FACSCalibur, BD Pharmingen); Microplate reader (MULTISKAN GO, Thermo Scientific); Tissue dehydrator (Excelsior TMES, Thermo, USA); Tissue embedding machine (EC350, Thermo Scientific, USA); Horizontal shaker (X85-2S, Mei Yingpu Instrument Manufacturing Co., Ltd.); High-speed micro-volume temperature-controlled centrifuge (Eppendorf, USA); Small animal anesthesia device (EZ-ANESTHESIA, USA).

[0121] 1.3 Experimental Cells and Animals Same as Example 1.

[0122] 2 Experimental Methods 2.1 Solution preparation: (1) Preparation of pH 7.4 sodium carbonate buffer: Weigh 1.59 g of sodium carbonate powder and 2.93 g of sodium bicarbonate powder and dissolve them in 1 L of distilled water. Add an appropriate amount of flake sodium hydroxide and adjust the pH to 7.4.

[0123] (2) Preparation of coating dilution buffer for ELISA detection: For the BCA protein quantification method to detect the protein content of tumor cell lysate, take the above-prepared neutral sodium carbonate buffer and dilute the tumor cell lysate to a suitable plating concentration.

[0124] (3) Preparation of PBST: Measure 1 L of pH 7.4 PBS solution, use a 1000 μL pipette to transfer 500 μL of Tween 20, and sonicate in a water bath to mix the two solutions evenly to obtain PBST solution. Store at room temperature for later use.

[0125] (4) 0.5% BSA solution: Equilibrate the BSA powder to room temperature, weigh 2.5 g of BSA powder into 500 mL of PBS, vortex to dissolve, and store at 4°C for later use.

[0126] (5) 4% Paraformaldehyde: Weigh 40 g of paraformaldehyde powder and dissolve it in an appropriate amount of PBS solution. Add a small amount of sodium hydroxide solution until the paraformaldehyde powder is completely dissolved. After it is dissolved, transfer it to a 1 L glass bottle, add PBS solution to make up to 1 L, adjust its pH value to 7.4 with hydrochloric acid solution in a fume hood, filter to remove insoluble matter, and store at 4℃ for later use.

[0127] (6) Permeabilization solution: Measure 5 mL of 4×Fixation / Permeabilization solution and 15 mL of Dilution buffer and vortex mix to obtain the permeabilization solution.

[0128] (7) Washing buffer: Measure 5 mL of 5×Perm / wash buffer and mix it with 20 mL of distilled water by vortexing to obtain washing buffer.

[0129] (8) Tissue digestion solution: Weigh 76 mg each of hyaluronidase and type IV collagenase, and 22.8 mg of DNase, add 76 mL of serum-free culture medium to dissolve (i.e., the final concentration of hyaluronidase and type IV collagenase is 1 mg / mL, and the final concentration of DNase is 0.3 mg / mL), and place on ice for later use.

[0130] 2.2 Establishment of a mouse B16 tumor model The B-cell vaccine was induced according to the method described in Example 2 above. Mice were randomly assigned to five groups: PBS group; B-cells group; Alum group; Lysis group; and Alum / Lysis group. Mice were administered the vaccine at a dose of 5 × 10⁻⁷ mmol / L according to their group for 7 days. 6 The number of cells / mouse for cell vaccination was determined, and different single B-cell vaccines / PBS solutions were administered via intraperitoneal injection. On day 0, a mouse orthotopic melanoma model was established. First, B16 cells in the logarithmic growth phase were digested with trypsin and collected in centrifuge tubes. The cells were centrifuged at 1250 rpm for 5 min at 4°C. The cell supernatant was discarded, and an appropriate amount of sterile PBS solution was added to adjust the cell density to 5 × 10⁶ cells / mouse. 6 Cells / mL, the cell suspension was placed on ice at 4℃ to maintain cell viability. C57 mice were fully anesthetized in an isoflurane gas anesthesia machine in the biosafety cabinet of the animal room. Hair on the right hind leg of the mouse was thoroughly removed using a shaver and depilatory cream. The skin at the tumor site was gently wiped with a 75% alcohol swab. The cell suspension was then thoroughly dispersed using a manual pipette. 100 μL of the B16 cell suspension was drawn up with a sterile 1 mL syringe (avoiding air bubbles) and carefully injected subcutaneously. The syringe needle was then carefully reversed and slowly removed. A clean, dry cotton ball was pressed against the injection site for a moment. Tumor development and weight changes in each group of mice were observed and statistically analyzed every two days.

[0131] 2.3 ELISA detection of serum tumor-specific IgG levels (1) ELISA plate Measure 100 μL of tumor cell lysate / carbonate buffer using a 100 μL pipette, carefully add it to a 96-well adsorption plate, cover it with a sealing film, and incubate it overnight at 4°C for 12 h.

[0132] (2) Blood collection from tumor-bearing mice a. Seven days after the establishment of the mouse B16 tumor model, mice were anesthetized with isoflurane.

[0133] b. Blood was collected from the eye sockets of mice via capillary tubes. 500 μL of blood was collected from each mouse and placed into a 1.5 mL centrifuge tube.

[0134] c. Let stand at room temperature for 2 hours.

[0135] Centrifuge at 4500 rpm and 4℃ for 15 min.

[0136] e. Take 200 μL of supernatant from each sample tube and centrifuge again under the same conditions.

[0137] f. Take the supernatant from each sample tube into a 1.5 mL clean centrifuge tube, and label the group and number.

[0138] g. Take 20 μL of sample from each tube, add 80 μL of 0.01% BSA PBS solution, dilute five times, and place at 4℃ for testing. Store the remaining sample at -80℃.

[0139] (3) ELISA detection of serum tumor-specific IgG levels: a. Washing the plate: Take out the 96-well plate that has been incubated overnight at 4°C, pour out the tumor cell lysate / carbonate buffer, fill each well with PBST washing buffer, let stand for 1 min, shake off the washing buffer, pat dry on absorbent paper, and repeat this process three times.

[0140] b. Blocking: After platelet application, add 100 μL of 1% BSA PBS solution to each well for blocking. Blocking conditions: Incubate at 37℃ for 1.5 h.

[0141] c. Wash the plate: Repeat the above plate washing operation.

[0142] d. Antibody incubation: Add 100 μL of diluted serum to each well and incubate at 37°C for 1 h.

[0143] e. Wash the plate: Repeat the above plate washing operation.

[0144] f. Incubation with secondary antibody: Dilute IgG 1000 times with PBS, add 100 μL of the diluted secondary antibody solution to each well, and incubate at 37°C for 1 h.

[0145] g. Washing the plate: Repeat the above plate washing operation.

[0146] h. Color development: Add 100 μL of TMB color development solution to each well and incubate at 37°C for 10 min.

[0147] i. Termination: Add 100 μL of TMB stop solution to each well.

[0148] j. Detection: OD value of each well was measured at a wavelength of 450 nm.

[0149] 2.4 Evaluation of the preventive efficacy of different B-cell vaccines in a melanoma model -7 days later, C57 mice were randomly divided into PBS group, B cell group, Alum group, Lysis group, and Alum / Lysis group, ensuring 5 mice in each group and assigning each mouse a number. C57 mice were intraperitoneally injected with different groups of B cell vaccines. 0 days later, a mouse melanoma model was established according to the groups. Every other day, the weight changes of the experimental mice in each group were measured using an electronic scale. Simultaneously, the maximum and minimum diameters of the tumors in each tumor-bearing mouse were measured using electronic calipers. The tumor volumes of all tumor-bearing mice were calculated and statistically analyzed. The entire experimental period was 21 days. At the end of the experiment, all experimental mice were euthanized in a CO2 asphyxiation apparatus. Melanoma tissue was carefully removed subcutaneously from each group of experimental mice using ophthalmic scissors and forceps. An appropriate amount of neutral PBS solution was placed in a cell culture dish, and the tumor tissue from each group was rinsed thoroughly. Then, the tissue was repeatedly pressed onto filter paper to fully drain excess water. Place a clean white cardstock horizontally on the table. Arrange the melanomas from each group on the cardstock according to their group numbers, and take photos for record-keeping. Finally, weigh and record the weight of the melanoma tissue from each group of mice to calculate the average tumor weight of each group and perform statistical analysis to calculate the tumor prevention efficiency of different B-cell vaccines.

[0150] 2.5 Evaluation of tumor cell apoptosis using the TUNEL assay After the experimental endpoint, melanoma tissues were obtained from mice in each group. A portion of the tumor tissue was carefully excised and immersed in 4% paraformaldehyde fixative. After fixation at room temperature for 48 h, the TUNEL assay was used to detect the degree of apoptosis in the tumor cells of each group of tumor-bearing mice under the intervention of different B-cell vaccines. The procedure is as follows: (1) Place the paraffin slices in an oven and dry them at 60°C for 2 hours; dewax the slices in xylene for 5 minutes, and then dewax the slices again in fresh xylene for 5 minutes. (2) Soak the sections in ethanol at concentration gradients of 100%, 95% and 85% twice for 10 min each time; then soak the sections in distilled water three times for 10 min each time; finally soak them in neutral PBS three times for 5 min each time. (3) Place the slides in citrate buffer (0.1 M) at pH 6, and place them in a high-temperature and high-pressure environment for 15 min after high heat for 2 min and low heat for 8 min to repair the antigen. Then wash the slides three times with PBS for 5 min each time. (4) Perform TUNEL reaction: Place the slides in Protease K and incubate them at 37°C for 30 min; take TdT Equilibration Buffer and treat the slides at room temperature for 30 min; treat the slides with Labeling Solution at 37°C in the dark for 1 h. (5) Mounting: Counterstain the slides with DAPI at room temperature for 5 min and mount them with an anti-fluorescence quencher; (6) Observe the apoptosis of tumor cells in each group under an inverted fluorescence microscope.

[0151] 2.6 Effect of B-cell vaccine on plasma cell enrichment in the spleen of tumor-bearing mice Preparation and flow cytometry processing of single-cell suspensions from spleen tissue (cell group only) (1) Preparation of single-cell suspension of spleen tissue: At the end of the experiment, the experimental mice were euthanized by CO2 asphyxiation instrument, the tumor-bearing mice were dissected, the spleen tissue of the tumor-bearing mice was taken out, washed in PBS, and excess water was drained on clean filter paper. The spleens of each group of mice were placed in 12-well plates, 1 mL of PBS was added, and the spleen tissue was carefully ground into a single-cell suspension using a 2 mL syringe plunger. The single-cell suspension was blown evenly with a 1 mL pipette and transferred to a 1.5 mL clean centrifuge tube. The tube was centrifuged at 3000 rpm for 6 min at 4℃. The supernatant was discarded, and 1 mL of red blood cell lysis buffer that had been brought to room temperature was added to each tube to resuspend it. The tube was placed at room temperature for 5 min. (2) Blocking: Centrifuge at 3000 rpm and 4℃ for 5 min, discard the supernatant, add 300 μL of blocking solution to each sample tube, incubate on ice for 30 min, and gently shake the sample tube every ten minutes to ensure that the sample is in full contact with the blocking solution. (3) Staining: After blocking, centrifuge at 3000 rpm and 4℃ for 5 min, discard the supernatant, add 100 μL of the prepared flow cytometry surface antibody CD45, CD45R and CD138 to each tube, incubate on ice at 4℃ in the dark for 30 min, and gently shake the sample tube every 10 minutes to ensure that the sample is in full contact with the flow cytometry antibody dilution solution. (4) Fixation: Add 100 μL of 4% paraformaldehyde to each sample tube, fix on ice for 30 min, centrifuge at 3000 rpm and 4℃ for 5 min, discard the supernatant, add 100 μL of 0.5% BSA solution to each sample tube for resuspending, and store on ice. (5) Flow cytometry: Flow cytometry was used to detect the fluorescence intensity of CD45, CD45R and CD138 positive cells.

[0152] 2.7 Effect of B-cell vaccine on T-cell enrichment in tumor tissues of tumor-bearing mice Tumor tissue flow cytometry (1) Preparation of tumor single-cell suspension: At the end of the experiment, the experimental mice were euthanized by CO2 asphyxiation instrument. The tumor-bearing mice were dissected, and the subcutaneous melanoma tissue was taken out. The tissue was washed in PBS, drained on clean filter paper, weighed and photographed. 100 mg of melanoma tissue was weighed from each mouse into a 4 mL clean centrifuge tube. The tissue was cut into small pieces with surgical scissors. 2 mL of digestion solution was added to each sample tube. Each sample tube was sealed with sealing film and arranged in a test tube rack according to the group number. The test tube rack was wrapped tightly with plastic wrap and then placed on a shaker for 1.5 h (shaking conditions: 37℃, 220 rpm) until the tissue digestion solution became turbid. The sample tubes were taken out, and the samples were repeatedly blown with a 1 mL pipette. The undigested tissue clumps in the tumor digestion solution were filtered out with a 200-mesh filter. (2) Blocking: Centrifuge at 3000 rpm and 4℃ for 5 min, discard the supernatant, add 300 μL of blocking solution to each sample tube, incubate on ice for 30 min, and gently shake the sample tube every 10 min to mix the blocking solution with the sample evenly. (3) Staining: After blocking, centrifuge at 3000 rpm and 4℃ for 5 min, discard the supernatant, add 100 μL of the prepared flow cytometry antibody CD45, CD3e, CD4 and CD8 to each sample tube, incubate on ice for 30 min, and gently shake the sample tube every 10 min to mix the flow cytometry antibody with the sample evenly. (4) Flow cytometry: Flow cytometry was used to detect the fluorescence intensity of CD45, CD3e, CD4 and CD8 positive cells.

[0153] 2.8 Safety evaluation of different B-cell vaccines Since the establishment of the melanoma model, the body weight of each group of tumor-bearing mice was recorded every 1 day, and the mean value was used to plot a body weight-time curve. Upon reaching the treatment endpoint, all experimental mice were euthanized using a CO2 asphyxiation device. The mice in each group were immediately dissected to obtain the five major organs: heart, liver, spleen, lungs, and kidneys. These major organs were carefully cleaned in PBS to remove any residual adhesions and blood from their surfaces. Excess moisture was thoroughly drained from the organ surfaces using clean filter paper, and the weight of the major organs in each group was quickly weighed and recorded. The organ coefficients of each group were calculated, and a bar chart of the organ coefficients for each group was plotted based on their mean values. Subsequently, one mouse from each experimental group was selected according to the same number, and its five major organs were placed in centrifuge tubes. The tubes were then fixed by immersion in 4% paraformaldehyde for 48 hours at room temperature. The fixed organs were then embedded in paraffin, sectioned, and the sections were stained with H&E. The morphology of the major organs before and after different B-cell vaccines was observed using a pathological section fluorescence scanner to evaluate the safety of different B-cell vaccines. At the end of the experiment, 0.5 mL of blood was collected from each mouse via the orbital vein and placed in a clean 1.5 mL centrifuge tube. After standing at room temperature for 2 hours, the tubes were centrifuged at 4500 rpm for 15 minutes at 4°C. 300 μL of the supernatant was collected, and the tubes were centrifuged again under the same conditions. The supernatant was then transferred to a clean 1.5 mL centrifuge tube, labeled with the group and number. Serum was used to detect liver function indicators such as AST, ALT, and ALP.

[0154] 3 Results and Discussion Experimental results are as follows Figures 5 to 7 As shown.

[0155] like Figure 5 As shown in Figures A and B, both the PBS group and the B cells group showed rapid tumor development and progression, with comparable tumor growth rates to the Alum group, and neither group exhibited ideal tumor prevention activity. Figure 5 As shown in Figure C, after the experimental endpoint, there were no significant differences in tumor tissue weight among the above groups of mice; the Lysis group vaccine had a certain tumor prevention effect; the Alum / Lysis group vaccine showed a better tumor prevention effect; as shown in Figure C. Figure 5 As shown in Figure C, after the experimental endpoint, the weight of the two groups of tumor tissues also showed the same trend; Figure 5The results showed that after vaccination with different B-cell vaccines, the tumor growth inhibition rate in the Alum / Lysis group was 97.80%; the tumor growth inhibition rate in the Lysis group was 63.26%; and the tumor growth inhibition rates in the B-cell group and the Alum group were 28.76% and 16.61%, respectively. These results indicated that the B-cell vaccines in the B-cell group and the Alum group had no significant tumor prevention effect; the Alum / Lysis group showed the strongest anti-tumor activity. The principle of the TUNEL assay for evaluating tumor cell apoptosis: During apoptosis, a large number of intracellular DNA endonucleases are activated, cutting the DNA double strand of chromosomes and producing a large number of 3'-OH sticky ends. Under the action of deoxynucleotidyl transferase (TdT), these sticky ends can connect to fluorescein-labeled dUTPs, and finally specifically bind to HRP-labeled fluorescein antibodies. GFP fluorescence is observed using a fluorescent inverted microscope to display bright green apoptotic cells, accurately and specifically locating apoptotic cells in tumor tissue to examine the apoptosis situation in tumor tissue. TUNEL results (e.g.) Figure 5 The results showed that very few cells in the tumor tissues of the PBS group, B cells group, and Alum group underwent apoptosis; a large number of cells in the Lysis group underwent apoptosis, indicating that B cells stimulated by tumor cell lysates have certain tumor prevention activity; the Alum / Lysis group had the highest number of apoptotic cells. The experimental results were consistent with the in vitro study results, indicating that the B cell vaccine stimulated by aluminum adjuvant and tumor cell lysates has good tumor prevention activity.

[0156] Fourteen days after mice were vaccinated with a B-cell vaccine (when the humoral immune response is strongest), orbital venous blood samples were collected. The levels of specific anti-tumor IgG in the serum of five groups were measured using ELISA. Figure 6 As shown in Figure C, the serum levels of specific antitumor IgG antibodies in the Lysis and Alum / Lysis groups were significantly higher than those in the other three groups. Furthermore, the Alum / Lysis group had the highest serum levels of specific antitumor IgG antibodies, but no significant difference was observed between the Lysis and Alum / Lysis groups. These results are consistent with in vitro experimental results, indicating that stimulation of B-cell vaccines with tumor cell lysates or in combination with aluminum adjuvants can induce humoral immunity in mice and promote the production of specific antitumor IgG antibodies.

[0157] Flow cytometry analysis showed that CD138 levels in the spleens of mice in different groups were significantly increased after vaccination with different B-cell vaccines. + Changes in plasma cells, such as Figure 6 As shown in Figures A and B. In tumor-bearing mice, CD138 levels in the spleen were increased after vaccination with the Alum / Lysis vaccine. +The percentage of plasma cells was 41.64%, which was 1.6, 1.5, 1.5, and 1.2 times higher than that in the PBS group, B cells group, Alum group, and Lysis group, respectively. The results indicate that CD138 in the spleen of mice in the Lysis group and Alum / Lysis group... + Plasma levels were significantly increased. However, similar to the results regarding IgG levels in cell supernatants, no significant difference in IgG levels was observed between the Lysis and Alum / Lysis groups. Overall, the Alum / Lysis vaccine enhanced CD138 levels in the spleen of tumor-bearing mice. + Plasma cell differentiation increases the production of tumor-specific antibodies and enhances anti-tumor activity. Furthermore, T cells are crucial for tumor immunity; this study also investigated the effects of different B-cell vaccines on T cells, using CD4 as an indicator of helper T cell activity. Figure 6 The results shown in D and E indicate that CD4 in the Alum / Lysis group + The number of T cells increased significantly, being 8.5-fold, 4.9-fold, 3.2-fold, and 2.3-fold higher than those in the PBS, B cells, Alum, and Lysis groups, respectively. CD8 was used as a marker of cytotoxic T cell activity. Figure 6 As shown in F and G, CD8 in tumor tissue of Alum / Lysis group mice + The number of T cells increased significantly, by 11.5 times, 6.2 times, 3.4 times, and 2.3 times respectively compared to the PBS group, B cells group, Alum group, and Lysis group. These results indicate that the Alum / Lysis vaccine effectively stimulates an anti-tumor immune response in vivo.

[0158] In pharmacodynamic studies evaluating cell vaccines, monitoring the weight changes of experimental animals throughout the process is a common method for assessing vaccine biosafety. For example... Figure 7 Figure A shows the trend of body weight change in the experimental animals during the experiment. There was no significant difference in body weight between the mice in the different B-cell vaccine groups and the PBS group; all groups showed an increasing trend. This indicates that B-cell vaccines do not cause safety issues such as loss of appetite or emaciation in mice. At the end of the experiment, the body weight of each group of mice was weighed and recorded. The major organs of each group of mice were dissected, washed, drained, and weighed. The organ coefficients of each group of mice were calculated. Figure 7 As shown in Figure B, no significant difference was observed in organ coefficients between mice vaccinated with each group of B-cell vaccines and the PBS control group, preliminarily verifying that all groups of B-cell vaccines have good safety. Pathological sections of major organs are shown below. Figure 7As shown in Figure C, no abnormalities were observed in the morphology of the major organs and tissues of mice in each group. After the experimental endpoint, serum samples from mice in each B-cell vaccine-inoculated group were collected to measure relevant liver function biochemical indicators, including AST, ALT, and ALP, to evaluate the degree of liver damage caused by different B-cell vaccines in the experimental animals. The experimental results are as follows: Figure 7 As shown in Figures D and E, the serum biochemical indicators of mice in each group remained within the normal range and showed no significant differences, indicating that the B-cell vaccines in each group had good biosafety.

[0159] Example 5: Evaluation of the in vivo tumor treatment efficacy of B-cell vaccine 1 Materials and Instruments 1.1 Materials Thermo Scientific TM Imject TM Alum adjuvant (Beijing Newp Biotechnology Co., Ltd., Beijing); Horseradish peroxidase-labeled goat anti-mouse IgG (Beyotime Biotechnology Co., Ltd., Shanghai); Horseradish peroxidase-labeled secondary antibody IgG (Beyotime Biotechnology Co., Ltd., Shanghai); TMB chromogenic solution (Beyotime Biotechnology Co., Ltd., Shanghai); TMB chromogenic stop solution (Beyotime Biotechnology Co., Ltd., Shanghai); Mouse TNF-α ELISA kit (Shanghai Dakwei Biotechnology Co., Ltd., Shanghai); Mouse IFN-γ ELISA kit (Shanghai Dakwei Biotechnology Co., Ltd., Shanghai); Mouse IL-2 ELISA kit (Shanghai Dakwei Biotechnology Co., Ltd., Shanghai); Mouse IL-6 ELISA kit (Shanghai Dakwei Biotechnology Co., Ltd., Shanghai); Xylene (Sinopharm Chemical Reagent, Shanghai); Citrate buffer (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing).

[0160] 1.2 Instruments Pure water system (Ultra Pure Plus-12A, Shanghai Hetai Instrument Co., Ltd., China); Microplate reader (MULTISKAN GO, Thermo Scientific); Inverted fluorescence microscope (Nikon Instruments (Shanghai) Co., Ltd., China); Tissue dehydrator (Excelsior TMES, Thermo, USA); Tissue embedding machine (EC350, Thermo Scientific, USA); Horizontal shaker (X85-2S, Mei Yingpu Instrument Manufacturing Co., Ltd.); High-speed micro-volume temperature-controlled centrifuge (Eppendorf, USA); Small animal anesthesia device (EZ-ANESTHESIA, USA).

[0161] 1.3 Experimental Cells and Animals Same as Example 1.

[0162] 2 Experimental Methods 2.1 Evaluation of the preventive efficacy of different B-cell vaccines in a melanoma model Four groups of B-cell vaccines were induced according to the method in Example 2. Using the same method (except that B cells were replaced with DC cells), dendritic cells (DCs) were stimulated with tumor cell lysates and aluminum adjuvant to obtain DC cell vaccines. Mice were randomly assigned to six groups: PBS group; B-cell group; Alum group; Lysis group; Alum / Lysis group; and Alum / Lysis@DCs group. On day 0, a mouse orthotopic melanoma model was established. On day 1, mice were vaccinated according to their groups at 5 × 10⁻⁶ mg / L. 6 The number of cells / mouse vaccinated with different cell vaccines was determined by intraperitoneal injection. Tumor development and weight changes in each group of mice were observed and statistically analyzed every two days. Simultaneously, the maximum and minimum diameters of the tumors in each tumor-bearing mouse were measured using electronic calipers to calculate the total tumor volume and perform statistical analysis. The entire experiment lasted 21 days. At the end of the experiment, all mice were euthanized using a CO2 asphyxiation apparatus. Melanoma tissue was carefully removed subcutaneously from each group of mice using ophthalmic scissors and forceps. A suitable amount of neutral PBS solution was added to a cell culture dish, and the tumor tissues were rinsed thoroughly. Afterward, the tissues were repeatedly pressed onto filter paper to drain excess moisture. A clean white card was placed horizontally on a table, and the melanomas from each group were arranged according to their group numbers. Photos were taken and recorded. Finally, the weight of the melanoma tissues from each group of mice was weighed and recorded to calculate the average tumor weight and statistically analyze the tumor treatment efficiency of different B-cell vaccines.

[0163] 2.2 Evaluation of tumor cell apoptosis using the TUNEL assay After the experimental endpoint, melanoma tissues were obtained from mice in each group. A portion of the tumor tissue was carefully excised and immersed in 4% paraformaldehyde fixative. After fixation at room temperature for 48 h, the TUNEL assay was used to detect the degree of apoptosis in the tumor cells of each group of tumor-bearing mice under the intervention of different B-cell vaccines. The procedure is as follows: (1) Place the paraffin slices in an oven and dry them at 60°C for 2 hours; dewax the slices in xylene for 5 minutes, and then dewax the slices again in fresh xylene for 5 minutes. (2) Soak the sections in ethanol at concentration gradients of 100%, 95% and 85% twice for 10 min each time; then soak the sections in distilled water three times for 10 min each time; finally soak them in neutral PBS three times for 5 min each time. (3) Place the slides in citrate buffer (0.1 M) at pH 6, and place them in a high-temperature and high-pressure environment for 15 min after high heat for 2 min and low heat for 8 min to repair the antigen. Then wash the slides three times with PBS for 5 min each time. (4) Perform TUNEL reaction: Place the slides in Protease K and incubate them at 37°C for 30 min; take TdT Equilibration Buffer and treat the slides at room temperature for 30 min; treat the slides with Labeling Solution at 37°C in the dark for 1 h. (5) Mounting: Counterstain the slides with DAPI at room temperature for 5 min and mount them with an anti-fluorescence quencher; (6) Observe the apoptosis of tumor cells in each group under an inverted fluorescence microscope.

[0164] 2.3 Investigation of lymphocyte infiltration within the tumor after cell vaccine administration To investigate the infiltration of T cells and B cells in tumors after inoculation, paraffin-embedded tumor sections were dewaxed with xylene, graded and hydrated with ethanol, and placed in 0.01 M citrate buffer at 98°C for 30 minutes for antigen recovery. Tumor tissue samples were blocked with 5% bovine serum albumin and stained with TYR-570Plus-CD4 (Biolegend), TYR-520Plus-CD8 (Biolegend), TYR-570Plus-CD19 (Biolegend), and TYR-520Plus-CD138 (Biolegend) at 4°C. The infiltration of T cells and B cells within the tumor was observed under a confocal microscope.

[0165] 2.4 ELISA was used to evaluate the levels of intratumoral inflammatory factors after cell vaccine administration. Mouse tumor tissue samples were homogenized with PBS to extract total protein. After determining the total protein concentration, the levels of cytokines in the tumor tissue homogenate were quantified using an ELISA kit, with all procedures performed according to the manufacturer's specifications.

[0166] 6.3 Results and Discussion Experimental results are as follows Figure 8 and Figure 9 As shown.

[0167] In addition to the four groups of B-cell vaccines mentioned above, this study used the same method to co-stimulate DCs with tumor cell lysates and aluminum adjuvant to obtain the Alum / Lysis@DCs vaccine. After establishing a mouse melanoma model, mice were inoculated with five different cell vaccines. The results showed that the tumors in the PBS blank control group grew rapidly, while no significant tumor treatment effect was observed in the B-cell group and the Alum group. Figure 8 In the middle AD group, the tumor growth rate was comparable to that in the PBS group. Lysis and Alum / Lysis@DCs vaccines showed some tumor treatment effects, with the Alum / Lysis vaccine exhibiting the best tumor treatment effect. Tumor tissue weight also showed a similar trend. Figure 8 (C) For example Figure 8 The results showed that after vaccination with different cell vaccines, the tumor growth inhibition rates of the Alum / Lysis, Lysis, Alum / Lysis@DCs, Alum, and B cells groups were 85.32%, 62.39%, 57.62%, 15.29%, and 8.66%, respectively. These results indicate that B cells and Alum cell vaccines had no significant tumor therapeutic effect, while Lysis and Alum / Lysis@DCs vaccines showed some tumor therapeutic activity. The Alum / Lysis vaccine exhibited the best tumor suppression efficacy. Figure 8 TUNEL assays in mice showed that in the PBS control group, mice vaccinated with B cells, and mice vaccinated with Alum vaccines, fewer cells in the tumor tissue underwent apoptosis. Vaccination with Lysis or Alum / Lysis@DCs vaccines induced a certain degree of apoptosis in the tumor tissue, with Alum / Lysis vaccine inducing the most significant apoptosis. Figure 9 As shown in Figures A and B, immunofluorescence results indicate that Alum / Lysis vaccine administration can induce CD4+. + T cells, CD8 + T cells, CD19 + B cells and CD138 + Plasma cells maximally infiltrate tumor tissue. Inoculation with Lysis or Alum / Lysis@DCs vaccines can also promote the infiltration of these immune lymphocytes into tumors, but their activity is significantly lower than that of the Alum / Lysis vaccine. ELISA results show that Alum / Lysis vaccine inoculation can induce the enrichment of inflammatory factors such as IFN-γ, TNF-α, IL-6, and IL-2 in tumor tissue. Figure 9 The aluminum adjuvant (CF) activates the anti-tumor immune response. The experimental results are consistent with the tumor prevention effect of B-cell vaccines, indicating that B-cell vaccine agents stimulated by aluminum adjuvant and tumor cell lysates have good tumor therapeutic activity. However, DC vaccines and B-cell vaccines stimulated by tumor cell lysates under the same conditions, although they have certain tumor therapeutic activity, cannot be regarded as the optimal prescription for tumor vaccines.

Claims

1. A method for preparing an engineered B cell vaccine, comprising: S1, preparation of B cells: culturing isolated lymphocytes with interleukin-4 (IL-4) and CD3 monoclonal antibody to remove T cells to obtain purified cultured B cells; S2, preparation of immunogenic death tumor cell lysate particles: subjecting cultured tumor cells to immunogenic death treatment to obtain immunogenic death tumor cell lysate particles; S3, activation of B cells: co-culturing the B cells obtained in S1 with the tumor cell lysate particles obtained in S2 at a ratio of 5:1 to 50:1 of cell number: particle number to obtain activated B cells, thereby obtaining an engineered B cell vaccine.

2. The method of producing an engineered B cell vaccine according to claim 1, wherein, In S1, the isolated lymphocytes are lymphocytes isolated from blood or lymphocytes isolated from spleen.

3. The method of producing an engineered B cell vaccine according to claim 1 or 2, wherein, In S1, the isolated lymphocytes are suspended in a culture medium (e.g., RPMI 1640 medium) at a concentration of 1.0 x 10 9 cells / L to 1.0 x 10 10 cells / L, preferably 6.0 x 10 9 cells / L, and 10-30 μg / L, preferably 20 μg / L, of IL-4 and 3-10 mg / L, preferably 5 mg / L, of CD3 monoclonal antibody are added, respectively, for culture. During the culture, the original culture medium is discarded and an equal volume of the culture medium and the IL-4 and CD3 monoclonal antibody are added every other day for continued culture, and the purified cultured B cells are obtained after the culture.

4. The method of producing an engineered B cell vaccine according to any one of claims 1-3, wherein, In S1, the culturing time is 3-5 days, preferably 4 days.

5. The method for preparing an engineered B cell vaccine according to any one of claims 1-4, wherein: In S2, the tumor cells are cells with proliferative ability isolated from melanoma tissue, or the tumor cells are commercial melanoma cells; and / or In S2, the immunogenic death treatment is performed using a drug capable of inducing immunogenic death of tumor (e.g., a chemotherapeutic drug, such as doxorubicin), preferably, the amount of the drug capable of inducing immunogenic death of tumor is 0.5-1.5 μM; and / or In S2, the tumor cell lysate particles are obtained by freeze-thaw method.

6. The method of producing an engineered B cell vaccine according to any one of claims 1-5, wherein, In S3, the B cells obtained in S1 are co-cultured with the tumor cell lysate particles obtained in S2 at a ratio of 10:1 of cell number: particle number; and / or In S3, the co-culturing time is 24-48 hours, preferably 36 hours.

7. The method of producing an engineered B cell vaccine according to any one of claims 1-6, wherein, In S3, further an aluminum adjuvant is used to obtain activated B cells upon co-stimulation of immunogenic dead tumor cell lysate particles with an aluminum adjuvant, preferably, as aluminum adjuvant, a alum adjuvant is used, in particular Thermo Scientific Alhydrogel® TM Imject TM The amount of alum adjuvant, also preferably aluminum adjuvant, is preferably 3-10 μg / mL, preferably 5 μg / mL.

8. An engineered B cell vaccine prepared by the method for preparing an engineered B cell vaccine according to any one of claims 1-7.

9. Use of the engineered B cell vaccine according to claim 8 in the preparation of a medicament for preventing the occurrence and development of tumors.

10. Use according to claim 9, wherein, The engineered B cell vaccine is obtained by culturing immunogenic death melanoma tumor cell lysate particles, and the tumor is melanoma.

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