Engineering cell and application thereof in preparation of whole tumor cell vaccine

Abstract

The invention discloses an engineering cell and application thereof in preparation of a whole-cell tumor vaccine, and belongs to the technical field of biological medicines. According to the engineering cell disclosed by the invention, the form and antigenicity of the tumor cells can be retained through intracellular gelation of the tumor cells, and CD47 blocking and DAMP exposure can enhance the immune recognition capability of the tumor cells and resist immune escape, so that the immunogenicity is improved, and the therapeutic immune response is promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an engineered cell and an application thereof in the preparation of a whole tumor cell vaccine. Background Art

[0002] Vaccines are ideal for treating cancer because they not only target the primary tumor, but also induce long-term anti-tumor immune responses that help prevent recurrence and metastasis. Whole tumor cell vaccines stand out because they contain a full range of tumor-associated antigens from the patient's own tumor cells. This allows whole tumor cell vaccines to be effective even when specific tumor antigens have not been identified.

[0003] Despite their potential, most existing vaccines are preventive rather than therapeutic. Although therapeutic cancer vaccines have been in development for decades, only one—sipuleucel-T—has received approval from the U.S. Food and Drug Administration (FDA), extending survival by four months in prostate cancer patients. Traditional methods of inactivating tumor cells in clinical trials, whether physical, chemical, or biological, often result in the loss and degradation of tumor-associated antigens, which compromises their effectiveness. In addition, whole tumor cell vaccines often have low immunogenicity, which limits their ability to elicit strong anti-tumor responses. To address these challenges, it is critical to maintain antigen integrity and improve immunogenicity.

[0004] In order to improve the immunogenicity of tumor cell vaccines, various in vitro gene modification techniques have been used to introduce target gene fragments (such as MHC-1 molecules and auxiliary stimulatory cytokines (IL-2, IL-12 and GM-CSF)) into tumor cells. However, these methods are often time-consuming and complicated, and cannot be widely used in clinical practice. At the same time, there are currently no relevant studies reporting on how to maintain the antigenic integrity of whole tumor cell vaccines. Therefore, the development of effective therapeutic tumor cell vaccines still faces severe challenges.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an engineered cell and its use in the preparation of a whole tumor cell vaccine. The engineered cell enables the tumor cells to retain their morphology and antigenicity through intracellular gelation of the tumor cells. CD47 blockade and exposure of damage-associated molecular patterns can enhance their immune recognition ability, counteract immune escape, thereby improving immunogenicity and promoting therapeutic immune response.

[0007] In a first aspect, the present invention provides an engineered cell, which is a tumor cell that has hydrogel formation inside the cell and is capable of overexpressing damage-associated molecular patterns and blocking the binding of CD47 to its ligand.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned engineered cells, which comprises: culturing starting cells with a culture medium containing CD47 antibodies and an inducer for inducing overexpression of damage-associated molecular patterns, then mixing the surface-engineered cells with a hydrogel solution, incubating, thawing, and then subjecting the cells to a freeze-thaw cycle, washing, and then irradiating the cells with ultraviolet light to obtain engineered cells.

[0009] In a third aspect, the present invention provides the use of the above-mentioned engineered cells or the engineered cells obtained by the preparation method in the preparation of whole-cell tumor vaccines.

[0010] In a fourth aspect, the present invention provides a whole tumor cell vaccine comprising the above-mentioned engineered cells.

[0011] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned whole tumor cell vaccine.

[0012] In a sixth aspect, the present invention provides the use of the above-mentioned whole tumor cell vaccine or pharmaceutical composition in the preparation of drugs for treating tumors.

[0013] In a seventh aspect, the present invention provides the use of the above-mentioned whole tumor cell vaccine in the preparation of drugs for preventing tumors.

[0014] The present invention has the following beneficial effects:

[0015] The engineered cells in the present invention are tumor cells that have hydrogel formation in the cells and can overexpress damage-associated molecular patterns and block the binding of CD47 to its ligands. The intracellular gelation of the engineered cells retains the morphology and antigenicity of the tumor cells while making them lose their vitality; CD47 blocking and damage-associated molecular pattern exposure can synergistically enhance the "eat me" signal of tumor cells and inhibit the "don't eat me" signal of tumor cells, thereby significantly improving the immunogenicity of tumor cells. In terms of tumor prevention and existing tumor treatment, the engineered cells can polarize CD4+T cells to TH1 phenotype, reduce regulatory T cells and T cell exhaustion in the inhibitory tumor microenvironment, and stimulate strong tumor antigen-specific T cell responses, thereby completely eliminating tumors. At the same time, the vaccine can develop personalized whole tumor cell vaccines for different tumor types, and has wide applicability. In addition, the engineered cells and autologous tumor cell vaccines provided by the present invention have significant characteristics such as biosafety, no immunotoxicity and easy production, reflecting the potential for clinical anti-tumor applications using surgically removed tumor tissue waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 DAMP exposure and CD47 blockade enhanced the immunogenicity of CD47-DOX-GMC by inducing "eat me signals" and inhibiting "don't eat me signals"; (A) Schematic diagram of the construction of CD47-DOX-GMC; (B) Western blot analysis of CRT and CD47 expression in GMC and CD47-DOX-GMC; (C) Confocal fluorescence imaging: green fluorescent probe labeled CD47 protein, red fluorescent probe labeled CRT protein in GMC and CD47-DOX-GMC; (D) Schematic diagram of improving the recognition ability of macrophages and DCs to CD47-DOX-GMC by inducing CD91-CRT interaction and blocking SIRPα-CD47 interaction;

[0018] Figure 2 CD47-DOX-GMC can effectively prevent tumors and treat early tumors; (A) PBS and CD47-DOX-GMC were intravenously injected into 6-week-old C57BL / 6 male mice, with 10 6 (B) Tumor bioluminescence was monitored using IVIS at different time points (days 0, 3, 7, 11, 15, and 19); (C and D) Recorded melanoma mouse survival curves (C) and semi-quantitative analysis of bioluminescence intensity changes (D); (E) 6-week-old C57BL / 6 male mice were intravenously injected with B16-F10-Luc cells for 3 days and then inoculated with PBS, 10 mg / kg hydrogel, GMC, DOX-GMC, and CD47-DOX-GMC, with each mouse inoculated at a dose of 10 6 (n=5), inoculated twice on day 0 and day 7; (F) Tumor bioluminescence was monitored using IVIS at different time points (days 0, 4, 8, 13, and 19); (G and H) The recorded melanoma mouse survival rate curve (G) and bioluminescence intensity semi-quantitative curve (H);

[0019] Figure 3Immune checkpoint inhibitors promoted CD47-DOX-GMC-induced T cell-mediated tumor killing effect; (A) 6-week-old C57BL / 6 male mice were inoculated with B16-F10-Luc cells for 8 days to establish melanoma mice, and then treated with GMC, DOX-GMC, and CD47-DOX-GMC on days 0 and 7, respectively, at a dose of 10 per mouse. 6 , treated twice in total (n=5); melanoma mice were treated with CD47-DOX-GMC for 3 days (n=5), and then intravenously injected with aPD-L1 at a dose of 4 mg / kg on days 3 and 10, respectively; (B) Tumor bioluminescence was monitored using IVIS at different time points (days 0, 4, 8, 13, and 19); (C and D) Recorded melanoma mouse survival rate curve (C) and semi-quantitative analysis of bioluminescence intensity changes (D);

[0020] Figure 4 The personalized CD47-DOX-GMC vaccine derived from ascites achieved effective anti-tumor efficacy; (A) B16-F10 cells were collected from the ascites of tumor-bearing mice and enriched by filtration capture; (B) 6-week-old C57BL / 6 male mice were injected with B16-F10-Luc cells and 3 days later inoculated with ascites-derived CD47-DOX-GMC at a dose of 10 per mouse. 6 (n=5), injected twice on day 0 and day 7; (C) Tumor bioluminescence was monitored using IVIS at different time points (days 0, 4, 8, 13, and 19); (D and E) Survival curves of melanoma mice recorded; (D) and semi-quantitative analysis of changes in bioluminescence intensity (E);

[0021] Figure 5 The personalized CD47-DOX-GBC vaccine prepared from autologous tumor cells achieved good anti-tumor effects; (A) 4T1 cells were collected from subcutaneous tumor tissue and enriched by filtration capture method; (B) BALB / c male mice were intraperitoneally injected with luciferase-expressing 4T1 cells and then inoculated with tumor-derived CD47-DOX-GBC (10 cells per mouse) 9 days later. 6 (C) Tumor bioluminescence was monitored by IVIS at different time points (days 0, 4, 8, 13, and 19); (D) Semi-quantitative analysis of changes in bioluminescence intensity of tumor-bearing mice.

[0022] Figure 6 To evaluate the in vivo biosafety and immunotoxicity of CD47-DOX-GMC vaccine; (A) healthy C57BL / 6 mice were given 10 6 / mouse (n=3) were intraperitoneally injected with CD47-DOX-GMC, and the administration was respectively performed on day 0 and day 7; (B) After 14 days of administration, the heart, liver, spleen, lung and kidney were obtained for HE staining. (C) The serum BUN, UA, ALT and AST levels were detected; (D) The TNF-α and IL-6 levels in the ascites were detected by Elisa kit; (E and F) The number of NEU (E), WBC, RBC and PLT (F) of each group of mice was detected by complete blood cell count. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0024] Unless otherwise defined hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the present invention are intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0025] Since tumor cell antigen expression is heterogeneous and genetically unstable, and existing tumor vaccines have the disadvantages of loss or degradation of tumor-associated antigens and low immunogenicity, in order to solve this problem, the present invention first proposes to form a hydrogel in tumor cells to effectively maintain the morphology and structure of the cells, thereby maintaining the integrity and activity of tumor antigens, thereby improving the effectiveness of tumor cell vaccines.

[0026] Under normal physiological conditions, healthy cells maintain a balance between "eat me" and "don't eat me" signals. Tumor cells often upregulate "don't eat me" signals to escape immune surveillance, leading to uncontrolled proliferation. CD47 is a cell surface molecule that is often overexpressed in many tumors. It inhibits the phagocytosis of immune cells by binding to its ligand SIRPα, thereby helping to escape immunity. Monoclonal antibodies targeting CD47 can block this inhibitory signal, enhance the phagocytosis of tumor cells by immune cells, and reduce immune escape. In addition, the surface of apoptotic tumor cells that undergo immunogenic death overexpresses damage-associated molecular patterns (DAMPs). DAMPs act as "eat me" signals by binding to pattern recognition receptors (such as CD91) on phagocytes, promoting recognition and absorption by phagocytes. On the basis of the above-mentioned transformation method, the present invention enhances the immune recognition ability of tumor cell vaccines by combining DAMP exposure and CD47 antibody blocking, counteracts immune escape, thereby improving immunogenicity and promoting therapeutic immune responses.

[0027] According to the above, the present invention can provide a new engineered cell, which constructs a tumor cell with intracellular hydrogel formation through intracellular gelation and cell engineering strategy, and can overexpress DAMP and block the binding of CD47 to its ligand.

[0028] The tumor cells are autologous tumor cells. The engineered cells prepared from autologous tumor cells have a full set of tumor-specific antigens, which can induce specific immune responses, achieve personalized precision treatment, and thus overcome the tumor heterogeneity problem existing in anti-tumor immunotherapy.

[0029] The intracellular hydrogel served as an ideal cytoplasmic skeleton, supported tumor cell morphology, had high stability and biosafety, and maintained the integrity of tumor antigens without any physiological damage. DAMP exposure enhanced the vaccine's "eat me" signal and accelerated DC uptake and maturation. Blocking CD47 inhibited the vaccine's "don't eat me" signal and improved macrophage phagocytosis and inflammatory polarization. Together, these two functions enhanced the immunogenicity of conventional whole tumor cell vaccines and significantly improved the antigen presentation efficiency of T cells, inducing a strong anti-tumor immune response.

[0030] With respect to the above-mentioned engineered cells, the present invention also provides a method for preparing the above-mentioned engineered cells, comprising the following steps: culturing the starting cells with a culture medium containing CD47 antibodies and an inducer for inducing DAMP overexpression, then mixing the surface engineered cells with a hydrogel solution, incubating, thawing, and then subjecting the cells to a freeze-thaw cycle, washing, and then irradiating the cells with ultraviolet light to obtain the above-mentioned engineered cells.

[0031] In some embodiments, the starting cells are cultured in the culture medium at 37° C. and 5% CO 2 for 24 hours.

[0032] In some embodiments, the incubation condition is: incubation at -80°C for at least 5 min.

[0033] In some embodiments, the ultraviolet irradiation conditions are: 2W ultraviolet irradiation for 10-30 minutes.

[0034] In some embodiments, the inducing agent comprises doxorubicin (DOX); the concentration of doxorubicin in the culture medium is 0.1-1 μM.

[0035] In some embodiments, the hydrogel solution comprises a gelation precursor and a photoinitiator; the gelation precursor comprises polyethylene glycol diacrylate, and the photoinitiator comprises Irgacure 2959. In order to induce the formation of a hydrogel in the cytoplasm, the gel material needs to first penetrate the cell membrane. Since materials with low molecular weight are more likely to penetrate the cell membrane, the gelation precursor selected in the present invention is PEG-DA (polyethylene glycol diacrylate, 700 Da), and the photoinitiator is low molecular weight I2959 (Irgacure 2959).

[0036] In some embodiments, the concentration of polyethylene glycol diacrylate in the hydrogel solution is ≤10 wt %, and the concentration of Irgacure 2959 is 0.1-2 wt %.

[0037] The engineered cells of the present invention can be used to prepare whole tumor cell vaccines and pharmaceutical compositions that can prevent or treat tumors. The prepared whole tumor cell vaccines or pharmaceutical compositions have the active ingredients of the above-mentioned engineered cells, and may also include pharmaceutically acceptable excipients or auxiliary ingredients.

[0038] Experiments have shown that the engineered cells of the present invention or the whole tumor cell vaccine or pharmaceutical composition prepared therefrom have both preventive and therapeutic properties, and can not only prevent tumor occurrence, but also effectively treat early tumors and improve the survival rate of confirmed tumors.

[0039] During the experiment, the inventors found that although the prepared whole tumor cell vaccine has achieved good efficacy in preventing tumors and clearing existing tumors, there is still room for improvement in treating existing tumors. To this end, the inventors combined the vaccine with immune checkpoint inhibitors. The experimental results show that when the whole tumor cell vaccine of the present invention is used in combination with immune checkpoint inhibitors, the efficacy of the vaccine in eradicating confirmed tumors is also enhanced.

[0040] Therefore, the pharmaceutical composition provided by the present invention may also include an immune checkpoint inhibitor. The present invention does not limit the specific type and dosage of the immune checkpoint inhibitor, and those skilled in the art may adjust it according to the specific circumstances.

[0041] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0042] The materials involved in the embodiments of the present invention are as follows:

[0043] Poly(ethylene glycol) diacrylate (PEG-DA, 600 Da) and irgacure 2959 (I2959) were purchased from Aladdin (China).

[0044] 5-isothiocyanate fluorescein (FITC), propidium iodide (PI), 4',6-diamidino-2-phenylindole (DAPI), CFDASE cell proliferation and tracing detection kit (CFSE), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), collagenase II, DNase I and hyaluronidase were provided by Biotech (China).

[0045] Doxorubicin hydrochloride (DOX) was provided by Energy and Chemical Industry (China).

[0046] Anti-CD44 antibody, anti-CD62 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-T-bet antibody, anti-FOXP3 antibody, anti-CTLA-4 antibody, anti-GATA-3 antibody, anti-IL-2 antibody, anti-IFN-γ antibody, anti-TNF-α antibody, anti-granzyme B antibody, anti-PD-1 antibody, anti-CD47 antibody, cadherin antibody, TNF-α enzyme-linked immunosorbent assay kit, and IL-1β enzyme-linked immunosorbent assay kit were all from Proteintech (China).

[0047] AF488 antibody, AF594 antibody, F4 / 80 antibody, CD86 antibody, CD80 antibody, actin tracking probe and tubulin tracking probe were from Biolegend, USA.

[0048] 1640 culture medium, fetal bovine serum, D-luciferin monopotassium salt, and anti-PD-L1 antibody were purchased from Thermo Fisher Scientific (USA).

[0049] Mouse skin melanoma cell line (B16-F10) and B16-F10 melanoma cells expressing luciferase (B16-F10-Luc) were purchased from ATCC, USA, and were identified by mycoplasma detection, isozyme detection and DNA fingerprinting.

[0050] Dulbecco's modified Eagle's medium, bison serum and trypsin were from Gibco, USA.

[0051] Six-week-old C57BL / 6 male mice and six-week-old BALB / c male mice were obtained from Shenzhen University. All animal experimental procedures were approved by the Animal Ethics Committee of Shenzhen University (approval number: IACUC-202400065) and were performed in accordance with the Animal Management Regulations of the Ministry of Health of the People's Republic of China.

[0052] The autologous tumor cell isolation method used in the following experiments is as follows:

[0053] For non-solid tumors, 6-week-old C57BL / 6 male mice were intravenously injected with 0.2 mL of B16-F10 cells (1×10 6 ) suspension to construct primary tumors. On day 9, mice were euthanized, ascites was collected, and autologous tumor cells were separated through a filter.

[0054] For solid tumors, 6-week-old BALB / c male mice were subcutaneously injected with 0.1 mL of 4T1 cells to construct a breast cancer model. Then, tumor tissues were collected and cut into 0.5-1 mm 3 The small pieces were soaked in a pre-prepared 100× digestion solution (containing 10 mg / ml collagenase II, 200 U / ml DNase I and 1 mg / ml hyaluronidase) diluted with PBS 10 times. After digestion in a 37°C incubator for 1 hour, the mixture was filtered with a 100 μm cell strainer and the filtrate was collected to obtain a tumor suspension.

[0055] Example 1

[0056] This example is the preparation of engineered cells, and the preparation process is as follows Figure 1 As shown in A, it specifically includes the following steps:

[0057] S1. Melanoma cells were pretreated with 1640 cell culture medium containing 10 μg / ml CD47 antibody and 0.5 μM DOX solution, cultured in an incubator (37°C, 5% CO2) for 24 h, then the culture medium was removed and the cells were rinsed three times with PBS;

[0058] S2. The living tumor cells were incubated with a solution containing 10 wt% PEG-DA and 1 wt% I2959 at -80°C for 10 min and then thawed at room temperature;

[0059] S3. After freeze-thaw cycles, the dissolved PEG-DA solution was quickly washed away;

[0060] S4. Place the tumor cells on ice and irradiate them with 2W ultraviolet light for 15 minutes to obtain CD47-DOX-GMC, i.e., engineered cells.

[0061] Experimental Example 1

[0062] This experiment is to verify the engineering transformation of the intracellular gelling tumor cells obtained in Example 1, as follows:

[0063] To characterize DAMP overexpression and CD47 blockade, the protein levels of CRT and CD47 in CD47-DOX-GMCs were measured by Western blot analysis. In addition, CD47-DOX-GMCs were labeled with CD47 antibodies and CRT antibodies, then incubated with AF488 goat anti-rabbit IgG and AF594 goat anti-rabbit IgG, and fluorescence imaging was performed using CLSM.

[0064] Western blot analysis using rabbit CD47 monoclonal antibody showed that the gray value of CD47 protein was weak ( Figure 1 B), Compared with GMC, the gray value of CRT protein in CD47-DOX-GMC increased. Immunofluorescence staining and flow cytometry analysis showed that the green CD47 fluorescence intensity was low and the red CRT fluorescence intensity was high in CD47-DOX-GMC ( Figure 1 C), further confirming the success of CD47 blockade, as well as overexpression of CRT. Therefore, a combinatorial approach based on promoting “eat me” / suppressing “don’t eat me” signals could be used to overcome the weak immunogenicity of whole tumor cell vaccines ( Figure 1 D).

[0065] Experimental Example 2

[0066] This experiment is to verify the effect of CD47-DOX-GMC in preventing tumors. The experimental process is as follows Figure 2 A, as shown below:

[0067] Six-week-old C57BL / 6 male mice were randomly divided into six groups (n=6) and inoculated with 0.2 mL PBS, 10 mg / kg hydrogel, GMC, DOX-GMC, DOX-CD47-GMC (cell number 1×10 6 ). 21 days after inoculation, all mice were intravenously injected with 0.2 mL of B16-F10-Luc cells (1×10 6 ), and the tumor growth of each mouse was imaged by IVIS. Each mouse was intravenously injected with 150 mg / kg of D-luciferin monopotassium solution, and imaging was performed by IVIS 10 minutes after administration. The mice were imaged on days 0, 3, 7, 15, and 19.

[0068] After 19 days of monitoring, the survival rate of CD47-DOX-GMC-treated mice reached 100% ( Figure 2 B and 2C), while the survival rate of mice treated with DOX-GMC was 66%. Almost no fluorescent signal was detected in tumor tissue ( Figure 4D), while only 33% of mice treated with DOX-GMC were tumor-free. Thus, CD47-DOX-GMC showed significant improvement in preventing tumor growth relative to DOX-GMC, suggesting that combined surface engineering via DAMP exposure and CD47 blockade can effectively enhance the in vivo immunogenicity of whole tumor cell vaccines.

[0069] Experimental Example 3

[0070] This experiment uses CD47-DOX-GMC to treat early tumors. The experimental process is as follows Figure 2 E, as shown below:

[0071] Six-week-old C57BL / 6 male mice were intravenously injected with 0.2 mL of B16-F10-Luc cells (1×10 6 ), and were randomly divided into 5 groups (n=5) 3 days after injection. On day 0 and day 7, the tumor-bearing mice were injected with 0.2 mL PBS, 10 mg / kg hydrogel, and 1×10 6 To analyze the effect of treatment on pre-existing tumors, mice were imaged using IVIS on days 0, 4, 8, 13, and 19.

[0072] Consistent with the challenges facing current therapeutic cancer vaccines, GMC had minimal therapeutic effects on pre-existing tumors, similar to those of the PBS and hydrogel groups ( Figure 2 F). However, exposure to DAMPs such as DOX-GMC enhanced the antitumor effect, and all mice survived at 19 days of follow-up ( Figure 2 G). Remarkably, even with this change, only one of the five mice showed little tumor bioluminescence. In contrast, CD47-DOX-GMC completely eliminated peritoneal tumors in four of the five mice ( Figure 2 H). Based on Experimental Examples 3 and 4, CD47-DOX-GMC is constructed by intracellular gelatinized tumor cells, and has both CD47 blockade and DOX-induced DAMP exposure, and has significant ability to prevent tumors and eliminate existing tumors.

[0073] Experimental Example 4

[0074] This experiment verifies the therapeutic effect of CD47-DOX-GMC+aPD-L1 on solid tumors. The verification process is as follows Figure 3 A, as shown below:

[0075] Six-week-old C57BL / 6 male mice were injected with 0.2 mL of B16-F10-Luc cells (1×10 6). Eight days after injection, tumor-bearing mice were constructed and randomly divided into 5 groups (n=5). On day 0 and day 7, tumor-bearing mice were injected with 0.2 mL PBS, GMC, DOX-GMC, and DOX-CD47-GMC, respectively, with a dose of 10 6 After 3 days of CD47-DOX-GMC treatment, tumor-bearing mice were intravenously injected with aPD-L1 at a dose of 4 mg / kg on days 3 and 10, respectively. Mice were imaged using IVIS on days 0, 4, 8, 13, and 19 to analyze the therapeutic effect of the CD47-DOX-GMCe vaccine.

[0076] Since the CD47-DOX-GMC vaccine induces T cell activation, combination therapy with immune checkpoint inhibitors (anti-PD-L1, aPD-L1) is considered to be the best way to enhance the effectiveness of T cells in killing tumor cells. Figure 3 The results of B-3D provide evidence of therapeutic synergy, with a substantial improvement in survival compared to injection of either aPD-L1 or CD47-DOX-GMC vaccine alone, suggesting that aPD-L1 enhances the T cell-mediated tumor killing effect induced by the CD47-DOX-GMC vaccine. Overall, the CD47-DOX-GMC vaccine induced a strong anti-tumor immune response, especially the activation of tumor-associated lymphocytes, while the adjuvant effect of immune checkpoint inhibitors helped prevent T cells from killing cancer cells and avoid an overly strong immune response, resulting in a highly effective therapeutic effect against solid tumors.

[0077] Experimental Example 5

[0078] This experiment verifies the therapeutic effect of ascites-derived CD47-DOX-GMC on solid tumors, as follows:

[0079] CD47-DOX-GMC was prepared using ascites-derived melanoma cells according to the above preparation method. Then, 0.2 mL of B16-F10-Luc cells (1×10 6 ), and were randomly divided into three groups (n=5) 3 days after injection. On day 0 and day 7, tumor-bearing mice were intravenously injected with 0.2 mL CD47-DOX-GMC (1×10 per mouse). 6 cells), and aPD-L1 was intravenously injected 3 days after CD47-DOX-GMC treatment, with a dose of 4 mg / kg on days 3 and 10. PBS-treated mice and free CD47-DOX-GMC-treated mice served as controls. To analyze tumor growth, mice were imaged using IVIS on days 0, 4, 8, 13, and 19.

[0080] In this study, B16-F10 cells were collected from the ascites of tumor-bearing mice, and flow cytometry was used to determine that 40.8% of the B16-F10 cells in the ascites were Figure 4 A). After enrichment with a reversible filter, the concentration of melanoma cells reached 63.7%, and these cells were used to construct the ascites-derived CD47-DOX-GMC vaccine. Subsequently, 6-week-old C57BL / 6 male mice were injected with B16-F10-Luc cells for 3 days and then vaccinated with ascites-derived CD47-DOX-GMC at a dose of 10 per mouse. 6 (n=5), two doses in total, each dose was given on day 0 and day 7 respectively ( Figure 4 B).

[0081] like Figure 4 As shown in C, ascites-derived CD47-DOX-GMC effectively inhibited tumor growth, which was similar to the antitumor effect of CD47-DOX-GMC developed using cancer cell lines. All mice treated with ascites-derived CD47-DOX-GMC survived the 19-day follow-up, and tumors were completely cleared in two of the five mice ( Figure 4 D and 4E). Therefore, it is feasible to make personalized whole tumor cell vaccines using existing tumor tissues through subsequent CD47 blockade, DOX treatment to induce -DAMP exposure and intracellular gelation. In clinical practice, cancer vaccines can be made from surgically resected tumor tissues or biopsy samples for eradication of residual tumors and / or prevention of tumor recurrence.

[0082] Experimental Example 6

[0083] This experiment was further extended to different cancer cell lines to develop whole-cell vaccines, specifically to verify the therapeutic effect of CD47-DOX-GBC on breast cancer cells.

[0084] Six-week-old BALB / c male mice were injected with luciferase-expressing 4T1 cells for 9 days and then blindly randomized into three groups (n=5). Tumor-derived CD47-DOX-GBCs were inoculated into mice at a dose of 106 (n=5) per mouse, twice on days 0 and 7, and co-inoculated with aPD-L1 on days 3 and 10. PBS and free CD47-DOX-GBCs served as controls. Mice were imaged using IVIS on days 0, 4, 8, 13, and 19 to analyze tumor growth.

[0085] Autologous 4T1 cells were obtained from subcutaneous tumor tissue, and the proportion of 4T1 cells reached 69.9% ( Figure 5A). After engineering modifications such as CD47 blocking, DAMP exposure, and intracellular gelation, CD47-DOX-GBC was constructed and used for antitumor immunotherapy of subcutaneous breast cancer ( Figure 5 B) Figure 5 As shown in C, tumor tissue-derived CD47-DOX-GBC showed a moderate anti-tumor effect. Two of the five mice showed no luminescence at day 19, while the tumors of the other three mice increased in size. After the addition of aPD-L1, the luminescence of three of the five mice disappeared, and the tumor size of the remaining two mice was slightly increased ( Figure 5 D), indicating that aPD-L1 also enhanced the anti-tumor immunotherapy effect of the autologous CD47-DOX-GBC vaccine. This result indicates that the surface engineering strategy of CD47 blockade and intracellular gelation after DAMP exposure is indeed a platform technology suitable for the preparation of a variety of tumor vaccines with intact tumor antigens and better immunogenicity for the immunotherapy of different types of tumors.

[0086] Experimental Example 7

[0087] In order to translate into clinical application, this experiment is to evaluate the safety of CD47-DOX-GMC in healthy mice, as follows:

[0088] Six-week-old healthy C57BL / 6 male mice were intravenously injected with CD47-DOX-GMC at a dose of 10 6 (n=3), injected twice on day 0 and day 7. Blood and organs (heart, liver, spleen, lung and kidney) were collected after 14 days of treatment for further evaluation. HE staining was performed on sections of heart, liver, spleen, lung and kidney to study organ damage ( Figure 6 A). The levels of BUN, UA, ALT and AST in serum were determined by biochemical analysis, and the levels of TNF-α and IL-6 in ascites were determined by Elisa kits. The number of NEU, WBC, RBC and PLT in the test mice was detected by complete blood cell count.

[0089] The vaccinated mice did not suffer from hair loss, diarrhea, or weight loss. Histological analysis showed negligible damage to the heart, liver, spleen, lungs, and kidneys of the vaccinated mice 14 days after vaccination ( Figure 6 B). Serum levels of blood urea nitrogen (BUN), uric acid (UA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were similar to those of healthy mice ( Figure 6 C), indicating that the liver and kidney functions of the mice were healthy. The levels of inflammation represented by TNF-α and IL-6 in the ascites of the vaccinated mice remained normal ( Figure 6 D). The number of neutrophils (NEU) in vaccinated mice increased moderately ( Figure 6 E), but no purulent peritonitis symptoms were observed. In addition, the differences in the numbers of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) between the vaccinated mice were negligible ( Figure 6 F) In conclusion, whole tumor cell vaccines treated with surface engineering and intracellular gelation showed negligible immunotoxicity in inducing antitumor immune responses.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered cell, characterized in that: The engineered cells are tumor cells that have hydrogel formation in the cells and are capable of overexpressing damage-related molecular patterns and blocking the binding of CD47 to its ligand.

2. The method for preparing an engineered cell according to claim 1, characterized in that: include: The starting cells are cultured with a culture medium containing CD47 antibodies and an inducer for inducing overexpression of damage-associated molecular patterns, and then the surface-engineered cells are mixed with a hydrogel solution, incubated, thawed, and subjected to freeze-thaw cycles, washed, and then irradiated with ultraviolet light to obtain the engineered cells; Preferably, the starting cells are cultured in the culture medium at 37° C. and 5% CO 2 for 24 h; Preferably, the incubation conditions are: incubation at -80°C for at least 5 min; Preferably, the ultraviolet irradiation conditions are: 2W ultraviolet irradiation for 10-30min.

3. The preparation method according to claim 2, characterized in that: The inducing agent includes doxorubicin; Preferably, the concentration of doxorubicin in the culture medium is 0.1-1 μM.

4. The preparation method according to claim 2, characterized in that: The hydrogel solution comprises a gelation precursor and a photoinitiator; Preferably, the gelling precursor comprises polyethylene glycol diacrylate; Preferably, the photoinitiator comprises Irgacure 2959; Preferably, the concentration of polyethylene glycol diacrylate in the hydrogel solution is at least 10 wt %, and the concentration of Irgacure 2959 is 0.1-2 wt %.

5. Use of the engineered cell according to claim 1 or the engineered cell obtained by the preparation method according to any one of claims 2 to 4 in the preparation of a whole tumor cell vaccine.

6. A whole tumor cell vaccine, characterized in that: Comprising the engineered cell according to claim 1; Preferably, the whole tumor cell vaccine also includes pharmaceutically acceptable excipients or auxiliary ingredients.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the whole tumor cell vaccine.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also includes an immune checkpoint inhibitor.

9. Use of the whole tumor cell vaccine according to claim 6 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug for treating tumors.

10. Use of the whole tumor cell vaccine according to claim 6 in the preparation of drugs for preventing tumors.

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