Vaccine pre-sensitization and cpg-conjugated cell preparations, methods of making and using same

Abstract

The application provides a cell preparation based on vaccine pre-sensitization and CpG coupling and a preparation method thereof, the cell preparation comprising immune cells subjected to sensitization treatment by a pre-sensitization vaccine and an immune stimulator CpG coupled to the surface of the immune cells after the sensitization treatment; wherein the pre-sensitization vaccine comprises a whole antigen engineering nanovesicle, the whole antigen engineering nanovesicle carries an agonist of an interferon gene stimulatory receptor inside, and the whole antigen engineering nanovesicle is derived from tumor cells. The CpG-loaded vaccine pre-sensitized T cells (CpG@VT) of the application can stimulate an anti-tumor immune cell promotion type reaction enhancement in a co-stimulation mode of effect T cell targeted delivery, direct killing and CpG-induced immunogenic death (ICD).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a cell preparation based on vaccine presensitization and CpG conjugation, its preparation method, and its application. Background Technology

[0002] Cancer immunotherapy represents a major breakthrough in cancer treatment following surgery, chemotherapy, and radiotherapy. It works by activating or enhancing the body's own immune system to recognize and eliminate cancer cells. Among the many immunotherapy strategies, immune checkpoint inhibitors (ICIs), adoptive cell therapy, and tumor vaccines are particularly noteworthy. These methods share a common mechanism of action: stimulating T cell activation and immune response. Although these single-immune intervention strategies have activated anti-tumor immunity to some extent, their clinical efficacy is often limited by their respective limitations. Their technical bottlenecks mainly lie in the following aspects: Although immune checkpoint blockade therapy relieves immune immobilization by blocking inhibitory signals, its response rate in the tumor microenvironment (TME) is low and difficult to sustain due to the influence of epigenetic changes in tumor cells and the dynamic evolution of immunosuppressive matrix components, and it is very easy to develop acquired drug resistance. Adoptive cell therapy modifies and expands immune cells in vitro and reinfuses them in large quantities into the body to rebuild the functional immune cell lineage. Although it has natural homing ability and direct killing effect, its penetration into solid tumors is poor, and it is often constrained by the high antigenic heterogeneity of solid tumors and the severe immunosuppressive barrier within the TME, causing the reinfused immune cells to quickly enter a state of depletion.

[0003] Although tumor vaccines can cover complete antigenic epitopes and induce long-term immune memory, the affinity between tumor neoantigens and TCRs (T cell receptors) is generally low, making it difficult to induce sufficient amounts of antigen-specific cytotoxic T lymphocytes (CTLs) and resulting in insufficient immune activation efficacy.

[0004] In summary, single-dimensional immunotherapy strategies often fail to overcome the complex and multifaceted immune escape mechanisms of solid tumors. Therefore, developing novel treatment regimens that integrate multiple therapeutic advantages and achieve multidimensional synergistic immune activation to overcome the treatment bottleneck of solid tumors has become a critical technical problem urgently needing to be solved in the current biomedical field. Summary of the Invention

[0005] The purpose of this invention is to provide a cell preparation based on vaccine presensitization and CpG conjugation and its preparation method. CpG-loaded vaccine presensitized T cells (CpG@VT) can stimulate enhanced anti-tumor immune cell pro-type response under the co-stimulation mode of effector T cell targeted delivery direct killing and CpG-induced immunogenic death (ICD).

[0006] According to a first aspect of the present invention, a cell preparation based on vaccine presensitization and CpG conjugation is provided, the cell preparation comprising: Immune cells sensitized by pre-sensitized vaccines; and CpG, an immunostimulant conjugated to the surface of sensitized immune cells; The pre-sensitive vaccine comprises whole antigen-engineered nanovesicles, each containing an agonist of an interferon gene-stimulated receptor, and the whole antigen-engineered nanovesicles are derived from tumor cells.

[0007] As an optional implementation, the CpG includes CpG-ODN 1826, and the 3' end of the CpG is modified with a cholesterol bond.

[0008] As an optional implementation, the particle size of the whole antigen-engineered nanovesicles is 150 nm to 200 nm, and the tumor cells include melanoma cells.

[0009] As an optional implementation, the agonist of the interferon gene-stimulated receptor includes diABZI.

[0010] As an optional implementation, the sensitized immune cells include lymphocytes.

[0011] As an optional implementation, the sensitized immune cells include CD8+. + T cells.

[0012] According to a second aspect of the present invention, a method for preparing the aforementioned cell preparation based on vaccine presensitization and CpG conjugation is provided, comprising the following steps: Tumor cells were mixed with a buffer solution containing an agonist of the interferon gene stimulation receptor (STING agonist) to obtain a cell resuspension. The cell resuspension was subjected to gradient extrusion and filtered through a microporous membrane to obtain fully antigen-engineered nanovesicles rGBVac; Immune cells were sensitized using rGBVac, and the sensitized immune cells were co-incubated with CpG to obtain the CpG-conjugated cell preparation CpG@VT.

[0013] As an optional implementation, the density of tumor cells in the cell resuspension is 2.0 × 10⁻⁶. 6 cells / mL ~5.0×10 6 The concentration of STING agonist was greater than 0 and less than 2.54 μg / mL, and the CpG loading per unit cell was 1 × 10⁻⁶. 6 0.05 nmol to 0.50 nmol per cell.

[0014] According to a third aspect of the present invention, a melanoma vaccine is provided, the melanoma vaccine comprising whole antigen-engineered nanovesicles, wherein the whole antigen-engineered nanovesicles contain an agonist of an interferon gene-stimulated receptor, the whole antigen-engineered nanovesicles are derived from melanoma cells, and the concentration of the whole antigen-engineered nanovesicles is 200 μg / mL to 400 μg / mL.

[0015] In a third aspect of the present invention, the use of the aforementioned cell preparation based on vaccine presensitization and CpG conjugation, or a cell preparation based on vaccine presensitization and CpG conjugation prepared by the aforementioned method, in the preparation of a medicament for treating melanoma is provided.

[0016] As can be seen from the above technical solutions of the present invention, the cell preparation based on vaccine presensitization and CpG conjugation proposed in this invention, on the one hand, involves CD8 activated by the whole antigen engineered nanovesicle vaccine rGBVac. + T cells (VT) target and hom to the tumor site, exerting direct cytotoxic effects and inducing tumor cell apoptosis. On the other hand, the TLR9 agonist CpG is coupled to the VT surface as a "cell backpack." The local release of CpG, combined with the tumor-killing effect of VT, further induces tumor cells to undergo ICD and release a large number of DAMPs, thereby further promoting the maturation and antigen presentation of tumor-infiltrating dendritic cells, stimulating a stronger adaptive immune response and forming long-term immune memory. It has good biosafety and provides new clinical translational possibilities for synergistic anti-tumor immunotherapy. Attached Figure Description

[0017] Figure 1 This is a morphological image of the whole antigen engineered nanovesicle rGBVac taken under a transmission electron microscope in an embodiment of the present invention (scale bar: 100 nm).

[0018] Figure 2 This is a particle size distribution diagram of the whole antigen engineered nanovesicles rGBVac determined by the DLS method provided in this embodiment of the invention.

[0019] Figure 3 This invention relates to an example of using flow cytometry to evaluate the differentiation effect of PBS buffer and the all-antigen engineered nanovesicle vaccine rGBVac on mouse spleen-derived lymphocytes.

[0020] Figure 4 The results of CpG coupling on the VT surface were captured by confocal microscopy in this embodiment of the invention (scale bar: 20 μm).

[0021] Figure 5 This embodiment of the invention describes the flow cytometry detection of the connection pattern of CpG on the VT surface in CpG@VT.

[0022] Figure 6 This is a graph showing the LDH release concentration from the culture supernatant after co-culturing the cell preparation CpG@VT with melanoma cells in this embodiment of the invention (n=3, Mean±SD; *P<0.05, ****P<0.0001).

[0023] Figure 7 This is a graph showing the ATP release concentration results of the culture supernatant after co-culturing the cell preparation CpG@VT with melanoma cells in the embodiments of the present invention (n=3, Mean±SD; ****P<0.0001).

[0024] Figure 8 This is an example of immunofluorescence staining of B16F10 cells treated with CpG@VT for calreticulin (CRT, red) and high-mobility group box 1 (HMGB1, green) in this invention (scale bar: 20 μm).

[0025] Figure 9 These are the tumor volume (Tumor volume) - time (Time (d)) change curves of tumor-bearing mice in each treatment group of the tumor treatment model in the embodiments of the present invention (n=5, Mean±SD; ***P<0.001).

[0026] Figure 10 The images show representative H&E, Ki67, and TUNEL staining of mouse melanoma tumor tissue in this embodiment of the invention (scale bar: 100 μm).

[0027] Figure 11 These are H&E staining results of heart, liver, spleen, lung, and kidney tissues from various groups of mice in this invention embodiment (scale bar: 200μm). Detailed Implementation

[0028] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0029] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0030] In one embodiment of the present invention, a cell preparation based on vaccine presensitization and CpG conjugation is provided, the cell preparation comprising: Immune cells sensitized by pre-sensitized vaccines; and CpG, an immunostimulant conjugated to the surface of sensitized immune cells; Among them, the pre-sensitive vaccine includes whole antigen engineered nanovesicles, which contain an agonist of the interferon gene-stimulating receptor. The whole antigen engineered nanovesicles are derived from tumor cells.

[0031] In some embodiments, the 3' end of CpG is modified with a cholesterol bond.

[0032] In some embodiments, CpG includes CpG-ODN 1826.

[0033] In some embodiments, the particle size of the whole antigen-engineered nanovesicles is 150 nm to 200 nm, and the tumor cells include melanoma cells.

[0034] In some embodiments, agonists of interferon gene stimulation receptors include diABZI, which can be used as vaccine adjuvants to induce type I interferon responses and inflammatory cytokines and promote antigen cross-presentation function of dendritic cells (DCs).

[0035] In some embodiments, the sensitized immune cells include lymphocytes.

[0036] In some embodiments, the sensitized immune cells include CD8. + T cells.

[0037] In one preferred example, a cell preparation CpG@VT based on pre-sensitization of the engineered nanovesicle vaccine rGBVac and conjugation of CpG is provided. The engineered nanovesicle vaccine rGBVac is a whole antigen engineered nanovesicle derived from melanoma cells, which carries an agonist of the interferon gene-stimulated receptor. The surface of the pre-sensitized cells is conjugated with CpG-ODN 1826, whose sequence is: TCCATGACGTTCCTGACGTT.

[0038] The CpG sequence is modified with a cholesterol bond at the 3' end. CpG is coupled to the outer membrane surface of presensitized cells via the cholesterol bond, which can induce TLR9-dependent ICD in cancer cells. The induced dead cancer cells will exhibit high immunogenicity and stimulate antigen-presenting cells by secreting DAMPs.

[0039] In another embodiment, a method for preparing the aforementioned cell preparation based on vaccine presensitization and CpG conjugation is provided, comprising the following steps: Tumor cells were mixed with a buffer solution containing an agonist of the interferon gene stimulation receptor (STING agonist) to obtain a cell resuspension. The cell resuspension was subjected to gradient extrusion and filtered through a microporous membrane to obtain fully antigen-engineered nanovesicles rGBVac. Immune cells were sensitized using rGBVac, and the sensitized immune cells were co-incubated with CpG to obtain the CpG-conjugated cell preparation CpG@VT.

[0040] In some embodiments, the density of tumor cells in the cell resuspension is 2.0 × 10⁻⁶. 6 cells / mL ~5.0×10 6 Cells / mL, especially preferably 4.0 × 10⁻⁶. 6 cells / mL ~5.0×10 6 The concentration of the STING agonist is greater than 0 and less than 2.54 μg / mL, particularly preferably 1.20 μg / mL to 1.27 μg / mL.

[0041] In some embodiments, the cell resuspension is passed sequentially through microporous membranes with pore sizes of 2.5 μm to 3.5 μm, 0.4 μm to 0.8 μm, and 0.1 μm to 0.3 μm, and more preferably through microporous membranes with pore sizes of 3.0 μm, 0.6 μm, and 0.2 μm.

[0042] In some embodiments, the CpG loading per unit cell is 1 × 10 6 0.05 nmol to 0.50 nmol per cell, with 0.25 nmol being particularly preferred.

[0043] In some embodiments, the buffer solution used in the preparation process is PBS buffer.

[0044] In one exemplary embodiment, using mice as the subject, the method for preparing a cell preparation based on vaccine presensitization and CpG conjugation includes the following specific steps: Mouse melanoma H4956 cells were mixed with a buffer solution containing diABZI to obtain a cell resuspension, in which the density of tumor cells was 4.0 × 10⁻⁶. 6 cells / mL ~5.0×10 6 The concentration of diABZI was 1.20 μg / mL to 1.27 μg / mL. The cell resuspension was subjected to a gradient extrusion method and filtered through a microporous membrane. The cells were passed sequentially through microporous membranes with pore sizes of 3.0 μm, 0.6 μm, and 0.2 μm, and extruded repeatedly. The number of extrusions was preferably 1 to 10 times to obtain fully antigen-engineered nanovesicles rGBVac. After inoculating mice with rGBVac on days 0, 3, 6, and 9, CD8+ T cells were extracted from sorted mouse spleen lymphocytes and co-incubated with CpG at 35 ℃~37 ℃ (preferably 37 ℃) for 50 min~70 min (preferably 60 min). The CpG loading per cell was 1×10 6 0.25 nmol per cell yielded the cell preparation CpG@VT conjugated with CpG.

[0045] In other examples, a melanoma vaccine is provided, comprising an active ingredient and pharmaceutically acceptable excipients. The active ingredient includes whole-antigen engineered nanovesicles rGBVac, which carry an agonist of an interferon gene-stimulated receptor. The whole-antigen engineered nanovesicles are derived from melanoma cells.

[0046] In some embodiments, the concentration of the whole antigen-engineered nanovesicles in the melanoma vaccine is 200 μg / mL to 400 μg / mL.

[0047] In some embodiments, melanoma vaccine pre-sensitization is performed by subcutaneous injection to immunize the body, preferably with four injections and a three-day interval between two consecutive immunizations.

[0048] In some embodiments, the immunization dose of the melanoma vaccine is based on body weight, preferably a dose of melanoma vaccine greater than 0 and less than 0.67 μg per gram of body weight.

[0049] In other examples, the use of the aforementioned cell preparation based on vaccine presensitization and CpG conjugation, or a cell preparation based on vaccine presensitization and CpG conjugation prepared by the aforementioned method, in the preparation of a drug for treating melanoma is also provided.

[0050] In this invention, vaccine presensitization refers to the pre-sensitization of immune cells with a vaccine.

[0051] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0052] Unless otherwise specified, the following embodiments are all conventional methods.

[0053] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0054] The following examples use mouse melanoma H4956 cells. H4956 cells are engineered cell lines that stably express GM-CSF, generated by transducing B16F10 mouse melanoma cells with a retroviral vector expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF). In clinical formulations, the antigen source can be patient-derived tumor tissue or allogeneic tumor cell membrane material screened from a tissue bank. After mechanical disruption and fractionation to obtain membrane components, GM-CSF is overexpressed, and the membrane components are then co-assembled with a clinically available STING agonist to form rGBVac.

[0055] Example 1 [Preparation of whole antigen engineered nanovesicles rGBVac] (1) Resuscitate melanoma cells and culture them in RPMI-1640 medium (supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1 μg / mL puromycin) at 37 °C and 5% CO2. (2) Collect the melanoma cells from step (1) and resuspend them in phosphate-buffered saline (PBS) containing the STING agonist diABZI, so that the cell density in the cell resuspending solution is 5.0 × 10⁻⁶. 6 The concentration of diABZI in the cell resuspension was 1.27 μg / mL. (3) The cell resuspension in step (2) was extruded sequentially through microporous membranes with pore sizes of 3 μm, 0.6 μm and 0.2 μm using an airtight extruder. This process was repeated three times to obtain fully antigen-engineered nanovesicles rGBVac loaded with adjuvant diABZI.

[0056] Characterization of whole-antigen engineered nanovesicles rGBVac (1) The morphology of rGBVac was identified using transmission electron microscopy.

[0057] Add 10 μL of fresh rGBVac solution to a copper grid with a membrane, allow it to dry at room temperature for 10 min, blot away excess liquid with filter paper, and add 10 μL of 1% phosphotungstic acid buffer to the copper grid containing the sample for negative staining. After 2 min, blot away the staining solution with filter paper, wash the copper grid three times with pure water, and allow it to air dry at room temperature for at least 30 min. After drying, observe and record the results under a transmission electron microscope.

[0058] The results are as follows Figure 1 As shown, the whole antigen engineered nanovesicles rGBVac prepared by this invention have a vacuolar membrane morphology.

[0059] (2) The particle size of the whole antigen engineered nanovesicles rGBVac was determined by dynamic light scattering (DLS).

[0060] The results are as follows Figure 2 As shown, the total antigen-engineered nanovesicles rGBVac prepared by this invention have a particle size of approximately 190 nm and exhibit a normal particle size distribution.

[0061] (3) Evaluation of the differentiation effect of the all-antigen engineered nanovesicle vaccine rGBVac on mouse spleen-derived lymphocytes.

[0062] To detect the differentiation effect of the all-antigen engineered nanovesicle vaccine rGBVac on mouse spleen-derived lymphocytes, flow cytometry was used to detect the expression of CD3, CD4, and CD8 in lymphocytes after vaccine stimulation (the detection index was the proportion of lymphocyte differentiation markers CD3, CD4, and CD8 positive cells), specifically: Lymphocytes were extracted from the spleen of mice and the cells were sorted at a ratio of 1×102 6 Cells were seeded per well in 6-well plates and cultured in RPMI 1640 complete medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin). The whole-antigen engineered nanovesicle vaccine rGBVac and the negative control group (PBS) were added to each well, respectively, to make a culture volume of 2 mL per well.

[0063] After co-incubation for 48 h, lymphocytes were collected from the well plates, centrifuged at 4 °C using a low-temperature centrifuge, and the supernatant was discarded. The cells were then resuspended in 100 μL PBS, and 1 μL each of CD3, CD4, and CD8 flow cytometry antibodies were added. The mixture was thoroughly vortexed and co-incubated at 4 °C in the dark for 30 min.

[0064] After incubation, the cells were centrifuged at 4°C using a low-temperature centrifuge. The supernatant was discarded, and the cells were resuspended in pre-cooled PBS. The cell suspension was then transferred into flow cytometry tubes, with each tube containing 700 μL of cell suspension. The expression of lymphocyte surface markers CD3, CD4, and CD8 was detected by flow cytometry to evaluate the activation of lymphocytes by the whole antigen engineered nanovesicle vaccine rGBVac.

[0065] The results are as follows Figure 3 As shown, based on the flow cytometry results, statistical analysis was performed on the CD3 values ​​of each group. + CD4 in T lymphocyte population + With CD8 + The proportion of T lymphocytes indicated that CD8+ cells were present in the rGBVac group of the whole antigen-engineered nanovesicle vaccine. + The increased proportion of T lymphocytes indicates that rGBVac can significantly induce lymphocytes to convert to CD8. +T lymphocyte differentiation and efficient activation of lymphocyte immune killing function (all the above experiments were conducted in triplicate, and the results are expressed as mean ± standard deviation (Mean ± SD), *P<0.05, ***P<0.001, ****P<0.0001).

[0066] Example 2 [Preparation of CpG@VT, a cell preparation based on pre-sensitization of engineered nanovesicle vaccine rGBVac and conjugation with CpG] (1) After the engineered nanovesicle vaccine rGBVac was injected into mice on days 0, 3, 6 and 9, CD8 cells were extracted and sorted from the spleen lymphocytes of the mice. + T cells were activated and expanded for 5 days using a mouse T cell activator to obtain VT cells presensitized with the engineered nanovesicle vaccine rGBVac; (2) CpG and the above-mentioned cell VT were co-incubated at 37°C for 60 min to obtain the cell preparation CpG@VT based on the pre-sensitized engineered nanovesicle vaccine rGBVac and CpG conjugation.

[0067] [Characteristics of CpG@VT, a cell preparation based on pre-sensitization of engineered nanovesicle vaccine rGBVac and conjugation with CpG] (1) The connection pattern of CpG on the VT surface was evaluated using confocal microscopy.

[0068] Cy5-labeled Cy5-CpG was co-incubated with VT as described above to obtain Cy5-CpG@VT. After washing, Cy5-CpG@VT was fixed in a confocal microscope dish with 4% paraformaldehyde. After removing the fixative, the confocal microscope dish was washed twice with PBS buffer and the cell membrane was stained with DIO staining solution for 2 h. After removing the staining solution, the confocal microscope dish was washed twice with PBS buffer and the cell nucleus was stained with DAPI staining solution for 5 min. After removing the staining solution, the confocal microscope dish was washed twice with PBS buffer. Fluorescence images were obtained by confocal microscopy.

[0069] The results are as follows Figure 4 As shown in the figure, CpG was successfully connected to the surface of VT.

[0070] (2) Flow cytometry was used to assess the connectivity of CpG on the VT surface.

[0071] Cy5-labeled Cy5-CpG was co-incubated with different concentrations and coupled to VT to obtain Cy5-CpG@VT. Cy5-CpG@VT was collected, centrifuged at 4°C using a low-temperature centrifuge, and the supernatant was discarded. The cells were then resuspended in 700 μL PBS. The fluorescence intensity of Cy5 was detected by flow cytometry to evaluate the binding efficiency of CpG on the VT surface.

[0072] The results are as follows Figure 5 As shown, the connection efficiency between CpG and VT increases with increasing CpG concentration, reaching the optimal connection efficiency at 0.25 nmol.

[0073] Example 3 [In vitro antitumor effects of cell preparation CpG@VT] Adjust the density of melanoma B16F10 cells to 5 × 10⁻⁶. 5 CpG, VT, and CpG@VT were seeded per well in 6-well plates and co-cultured with B16F10 at different effector-to-target ratios. For the control group, the same volume of PBS buffer was added. After 4 h and 24 h of co-culture, the LDH release level in the supernatant was detected using a lactate dehydrogenase (LDH) assay kit (Beyotime). The LDH content in the culture medium was determined according to the instructions (all experiments were performed in triplicate, and results are expressed as mean ± standard deviation (Mean ± SD). *P < 0.05, ***P < 0.001, ****P < 0.0001).

[0074] The results are as follows Figure 6 As shown, the LDH released during extracellular apoptosis in melanoma cells indicates that the CpG@VT of the present invention has a significant killing effect on melanoma cells, and the killing effect is better when the effector-target ratio is 10:1 than when it is 5:1.

[0075] [Evaluation of the efficacy of CpG@VT in in vitro induction of immunogenic cell death (ICD)] The release or exposure of damage-associated molecular patterns (DAMPs) is a key indicator for assessing the ability of CpG@VT to induce ICD in tumor cells, including adenosine triphosphate (ATP), calreticulin (CRT), and high-mobility group box 1 (HMGB1).

[0076] (1) Adjust the density of melanoma B16F10 cells to 5×10⁻⁶. 5 VT and CpG@VT were seeded per well in 6-well plates, with PBS as the control group. VT and CpG@VT were co-cultured with B16F10. After 24 h of culture, the culture supernatant was collected, and the extracellular ATP content released by ICD was detected using an enhanced ATP assay kit (Beyotime). The luminescence was analyzed by a microplate reader according to the instructions. (All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation (Mean ± SD). *P < 0.05, ***P < 0.001, ****P < 0.0001).

[0077] The results are as follows Figure 7As shown, the ATP released during extracellular immunogenic cell death in melanoma cells indicates that the CpG@VT of the present invention has an ICD-inducing effect on melanoma cells.

[0078] (2) Adjust the density of melanoma B16F10 cells to 5×10⁻⁶. 5 Cells were seeded per well in 6-well plates, with PBS as a control. VT and CpG@VT were co-cultured with B16F10 cells, respectively. After 24 h of culture, the supernatant was discarded, and the cells were fixed with 4% paraformaldehyde after washing and permeabilized with 0.6% Triton X-100 for 30 min. Next, the cells were blocked with 5% BSA and then incubated overnight at 4 °C with primary antibodies (anti-CRT, anti-HMGB1). Finally, unbound antibodies were removed, and the cells were incubated with fluorescently labeled secondary antibodies. After removing unbound secondary antibodies, the cell nuclei were stained with DAPI for 5 min, and immunofluorescence images were obtained using confocal microscopy.

[0079] The results are as follows Figure 8 As shown, melanoma cells B16F10 exhibit significant CRT eversion (…). Figure 8 Part A of the data) and HMGB1 release ( Figure 8 Part B of the paper further verifies that the CpG@VT of the present invention has the effect of inducing ICD in melanoma cells, and at the same time has the potential to further enhance antigen presentation.

[0080] Example 4 [Evaluation of the antitumor efficacy of different groups of cell preparations in in vivo therapeutic experiments on melanoma mice] (1) Healthy 6-week-old female C57BL / 6 mice were selected, and a mouse melanoma model was established by subcutaneously injecting B16F10 cells into the right back of the mice. Each mouse was injected with 1×10 6 Five B16 cells were collected and designated as Day 0. Mice were randomly divided into three groups: PBS, VT, and CpG@VT, with five mice in each group. On days 7 and 15, mice were injected with the corresponding cell preparation at a dose of 2 × 10⁶ cells. 6 Each mouse was euthanized on day 22, with each mouse weighing 14 g to 19 g.

[0081] (2) Weigh the mice every 3 days and measure the long and short diameters of the tumors to calculate the tumor volume. Results are as follows: Figure 9 As shown, the tumors in the CpG@VT group mice were significantly smaller than those in other groups, indicating that the CpG@VT of the present invention has a good anti-tumor effect on mouse melanoma (*P<0.05, ***P<0.001, ****P<0.0001).

[0082] (3) Take tumor tissues from each group of mice for H&E and Ki67 immunohistochemical staining and TUNEL immunofluorescence staining. H&E is used to assess the morphology of surviving tumors and necrotic tumors. Ki67 is a nuclear proliferation antigen of cells, which reflects the proliferation of cells. TUNEL method is a method to detect cell apoptosis.

[0083] The results are as follows Figure 10 As shown, the trends of H&E, Ki67, and TUNEL staining results are basically consistent, indicating that the proportion of apoptotic tumor cells further decreased after CpG@VT treatment, further demonstrating that the cell preparation of the present invention can effectively inhibit tumor growth and promote tumor tissue apoptosis, and has a good anti-tumor effect on mouse melanoma.

[0084] Example 5 [Safety Evaluation of Cellular Preparations Based on Vaccine Presensitization and CpG Conjugation for the Treatment of Melanoma in Mice] Heart, liver, spleen, lung, and kidney sections from mice in each group were subjected to H&E staining. The results are as follows: Figure 11 As shown in the figure, the histological structures of the major organs are intact, and no differential adverse pathological manifestations are observed, proving that the melanoma vaccine of the present invention has good safety in treating melanoma mice.

[0085] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A cell preparation based on vaccine presensitization and CpG conjugation, characterized in that, This cell preparation includes: Immune cells sensitized by pre-sensitized vaccines; and CpG, an immunostimulant conjugated to the surface of sensitized immune cells; The pre-sensitive vaccine comprises whole antigen-engineered nanovesicles, each containing an agonist of an interferon gene-stimulated receptor, and the whole antigen-engineered nanovesicles are derived from tumor cells.

2. The cell preparation based on vaccine presensitization and CpG conjugation according to claim 1, characterized in that, The CpG includes CpG-ODN 1826, and the 3' end of the CpG is modified with a cholesterol bond.

3. The cell preparation based on vaccine presensitization and CpG conjugation according to claim 1, characterized in that, The particle size of the whole antigen-engineered nanovesicles is 150 nm to 200 nm, and the tumor cells include melanoma cells.

4. The cell preparation based on vaccine presensitization and CpG conjugation according to claim 1, characterized in that, The interferon gene-stimulating receptor agonists include diABZI.

5. The cell preparation based on vaccine presensitization and CpG conjugation according to claim 1, characterized in that, The sensitized immune cells include lymphocytes.

6. The cell preparation based on vaccine presensitization and CpG conjugation according to claim 1 or 6, characterized in that, The sensitized immune cells include CD8. + T cells.

7. A method for preparing a cell preparation based on vaccine presensitization and CpG conjugation as described in any one of claims 1-6, characterized in that, Includes the following steps: Tumor cells were mixed with a buffer solution containing an agonist of the interferon gene stimulation receptor (STING agonist) to obtain a cell resuspension. The cell resuspension was subjected to gradient extrusion and filtered through a microporous membrane to obtain fully antigen-engineered nanovesicles rGBVac; Immune cells were sensitized using rGBVac, and the sensitized immune cells were co-incubated with CpG to obtain the CpG-conjugated cell preparation CpG@VT.

8. The method for preparing a cell preparation based on vaccine presensitization and CpG conjugation according to claim 7, characterized in that, The density of tumor cells in the cell resuspension was 2.0 × 10⁻⁶. 6 cells / mL ~5.0×10 6 The concentration of STING agonist was greater than 0 and less than 2.54 μg / mL, and the CpG loading per unit cell was 1 × 10⁻⁶. 6 0.05 nmol to 0.50 nmol per cell.

9. A melanoma vaccine, characterized in that, The melanoma vaccine comprises whole antigen-engineered nanovesicles, each containing an agonist of the interferon gene-stimulated receptor. The whole antigen-engineered nanovesicles are derived from melanoma cells, and the concentration of the whole antigen-engineered nanovesicles is 200 μg / mL to 400 μg / mL.

10. The use of a cell preparation based on vaccine presensitization and CpG conjugation as described in any one of claims 1-6, or a cell preparation based on vaccine presensitization and CpG conjugation prepared by the method described in any one of claims 7-8, in the preparation of a medicament for treating melanoma.

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