A hyaluronidase nanogel-loaded whole tumor cell vaccine, and a preparation method and application thereof

Abstract

The application discloses a kind of full tumor cell vaccines loaded with hyaluronidase nanogel knapsack and its preparation method and application.By oxidizing sodium alginate crosslinking hyaluronidase to form nanogel, and further coupling anti-CD47 antibody to construct HAase-patch, it can be stably anchored on the surface of inactivated whole tumor cells.After subcutaneous injection, HAase-patch continuously degrades hyaluronic acid in subcutaneous tissue, produces pro-inflammatory oligomeric hyaluronic acid, promotes dendritic cell (DC) maturation and migration at inoculation site;Surface modified α-CD47 blocks the "don't eat me" signal of TCV, enhances the phagocytosis of DC to vaccine and antigen cross-presentation.The vaccine can stimulate strong and durable anti-tumor immune response without additional traditional immune adjuvant, effectively prevents tumor, inhibits postoperative recurrence and metastasis, and has excellent safety, providing a simple, safe and universal TCV platform for tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy drug technology, specifically to a whole tumor cell vaccine (TCV@HAase-patch) loaded with a hyaluronidase nanogel backpack, its preparation method, and its application in the preparation of anti-tumor drugs. Background Technology

[0002] Tumor vaccines, by activating the patient's own immune system to recognize and eliminate tumor cells, have become one of the most promising cancer treatments after surgery, chemotherapy, and radiotherapy. Whole-cell tumor vaccines (TCVs), containing the full spectrum of tumor-associated antigens and tumor-specific antigens, can induce multivalent anti-tumor immune responses, giving them a unique advantage in personalized immunotherapy. However, inactivated whole-cell tumor cells have low immunogenicity and usually require the combined use of potent adjuvants to elicit an effective anti-tumor immune response. Currently, the types of vaccine adjuvants available clinically are limited. Aluminum salt adjuvants mainly induce Th2 humoral immune responses, with insufficient ability to induce cellular immunity. While novel adjuvants such as TLR agonists (e.g., CpG, poly(I:C)) and MPLA can induce strong cellular immunity, they are often accompanied by dose-dependent systemic inflammatory responses, granuloma formation, cytokine storms, and T-cell exhaustion, severely hindering their clinical translation.

[0003] Recent studies have shown that degradation products of the extracellular matrix (ECM) can act as endogenous "alarms" to activate the innate immune system. Among these, degradation products of hyaluronic acid (HA), especially low-molecular-weight HA and oligomeric HA (sHA), can activate dendritic cells (DCs) through pattern recognition receptors such as TLR2 / TLR4, promoting their maturation and the secretion of pro-inflammatory cytokines. This finding suggests that the controlled degradation of subcutaneous HA via hyaluronidase (HAase) to produce endogenous alarms may be a potential alternative to traditional exogenous adjuvants for vaccine immune enhancement.

[0004] HAase, an enzyme that specifically degrades HA, has been used clinically to enhance drug tissue penetration with a good safety record. However, simply mixing HAase directly with TCV presents a serious pharmacokinetic mismatch: micron-sized TCVs have a long local retention time after subcutaneous injection, while HAase, with a molecular weight of only 55 kDa, is easily diffused and cleared after injection, leading to spatiotemporal separation of the two at the injection site and preventing the synergistic release of endogenous alarm proteins and antigen presentation. Furthermore, the CD47 molecule on the TCV surface interacts with the signal regulatory protein α (SIRPα) on the macrophage surface, transmitting a "don't eat me" signal and inhibiting the phagocytosis of TCV by antigen-presenting cells, which is another key obstacle to TCV immunogenicity. Therefore, developing a TCV modification strategy that can simultaneously achieve spatiotemporal synergistic delivery of HAase and TCV and effectively block the CD47 "don't eat me" signal is of great significance for improving TCV immunogenicity and realizing the preparation of adjuvant-free cancer vaccines. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a whole-cell tumor vaccine (TCV@HAase-patch) loaded with a hyaluronidase (HAase) nanogel backpack, its preparation method, and its application.

[0006] In a first aspect, the present invention provides a TCV loaded in an HAase nanogel backpack, the vaccine comprising an inactivated TCV and an HAase-patch anchored to the surface of the TCV; the HAase-patch comprising a cross-linked HAase nanogel and an anti-CD47 antibody (α-CD47) coupled to the surface of the nanogel.

[0007] Preferably, the HAase-patch is prepared by the following method: using sodium oxidized alginate (OSA) as a crosslinking agent, it is crosslinked with HAase through a Schiff base reaction to form HAase nanogel; then, using the residual aldehyde groups on the surface of the HAase nanogel as coupling sites, it reacts with the amino groups of α-CD47 to couple α-CD47 to the surface of the nanogel.

[0008] Preferably, the oxidation degree of the OSA is 40%~60%, more preferably 45%~55%, and even more preferably 49%~51%.

[0009] Optionally, the OSA is synthesized as follows: sodium alginate (SA) is dissolved in deionized water, sodium periodate is added for oxidation, and the resulting product is dialyzed and lyophilized to obtain OSA; wherein the molar ratio of sodium periodate to SA is 0.2~0.5:1, and the oxidation reaction is carried out at room temperature in the dark for 2~6 h. The dialysis uses a dialysis membrane with a molecular weight cutoff of 3500 Da, and the dialysis time is 24~72 h.

[0010] Preferably, the HAase nanogel has a particle size of about 200 nm; the HAase-patch has a particle size of about 250 nm.

[0011] Preferably, the HAase-patch is anchored to the TCV surface through the specific binding of α-CD47 to CD47 on the TCV surface.

[0012] Preferably, the TCV is derived from autologous or allogeneic tumor cells, including but not limited to breast cancer cells such as 4T1 and EMT6, melanoma cells such as B16F10, and colon cancer cells such as CT26.

[0013] Preferably, the TCV is inactivated by chemical fixation, preferably using a cell fixation buffer containing 4% paraformaldehyde.

[0014] Optionally, the TCV is prepared by the following method: tumor cells are digested and collected, inactivated by cell fixation buffer, and washed to obtain TCV; wherein the cell fixation buffer is a PBS solution containing 4% paraformaldehyde, and the treatment conditions are 4°C for 30 min.

[0015] Preferably, the α-CD47 blocks the interaction between CD47 on the TCV surface and macrophage signal regulatory protein α (SIRPα), thereby enhancing the phagocytosis of TCV by antigen-presenting cells.

[0016] Preferably, the TCV@HAase-patch is used for subcutaneous injection. The TCV@HAase-patch releases pro-inflammatory sHA as an endogenous alarm factor by degrading subcutaneous HA, thereby promoting the maturation, migration, and antigen cross-presentation of dendritic cells (DCs).

[0017] Secondly, the present invention provides a method for preparing the TCV@HAase-patch, comprising the following steps: (1) Preparation of HAase nanogel: HAase and OSA were dissolved in deionized water respectively, mixed and stirred in an ice bath, and HAase nanogel was obtained by dialysis; (2) Preparation of HAase-patch: α-CD47 was added to the HAase nanogel solution and stirred to couple α-CD47 to the surface of the nanogel. After dialysis and freeze-drying, HAase-patch was obtained. (3) Assembly of TCV@HAase-patch: Mix HAase-patch with TCV and incubate. Centrifuge and wash to remove unbound HAase-patch to obtain TCV@HAase-patch.

[0018] As one of the preferred methods for preparing the above-mentioned TCV@HAase-patch, the mass ratio of HAase to OSA in step (1) is 2~10:1; preferably 5:1.

[0019] As one of the preferred methods for preparing the above-mentioned TCV@HAase-patch, the reaction in step (1) is carried out in an ice bath for 2 to 6 hours.

[0020] As one of the preferred methods for preparing the above-mentioned TCV@HAase-patch, the dialysis in step (1) uses a dialysis membrane with a molecular weight cutoff of 300 kDa.

[0021] As one of the preferred methods for preparing the above-mentioned TCV@HAase-patch, the mass ratio of α-CD47 to HAase nanogel in step (2) is 1:5~20.

[0022] As one of the preferred methods for preparing the above-mentioned TCV@HAase-patch, the reaction in step (2) is carried out at 2~8°C for 0.5~2 h.

[0023] As one of the preferred embodiments of the above-mentioned TCV@HAase-patch preparation method, in step (3), the feeding ratio of TCV to HAase-patch is 1×10 6 Each TCV corresponds to 5~30 μg HAase; that is, each 1×10 6 For each TCV, the amount of HAase-patch added is 5–30 μg based on the mass of HAase contained therein. More preferably, it is 25 μg of HAase.

[0024] As one of the preferred methods for preparing the above-mentioned TCV@HAase-patch, the incubation conditions in step (3) are 2~8°C for 1~4 h.

[0025] As one of the preferred methods for preparing the above-mentioned TCV@HAase-patch, the centrifugation conditions in step (3) are 300×g centrifugation for 5 min.

[0026] Thirdly, the present invention provides the application of the vaccine in the preparation of antitumor drugs.

[0027] Preferably, the vaccine is used to prevent tumor development and inhibit lung metastasis of tumors, including but not limited to breast cancer, melanoma, and colon cancer.

[0028] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to propose the concept of generating endogenous alarming factors through the controlled degradation of subcutaneous HA to replace traditional exogenous adjuvants. After subcutaneous injection of TCV@HAase-patch, HAase-patch continuously degrades the subcutaneous HA reserve, promoting DC maturation by releasing endogenous alarming factors such as sHA, thereby stimulating a potent anti-tumor immune response without the need for any external adjuvants.

[0029] 2. This invention uses a HAase nanogel "backpack" strategy to stably anchor HAase on the surface of TCV, solving the key problem of pharmacokinetic mismatch between HAase and TCV, and achieving long-term co-retention of HAase and TCV at the injection site and continuous degradation of HA.

[0030] 3. The TCV@HAase-patch constructed in this invention simultaneously blocks the CD47 "don't eat me" signal of TCV through α-CD47 on the surface of HAase-patch, thereby enhancing the phagocytosis of TCV by DCs and antigen cross-presentation; and works synergistically with the endogenous alarms generated by HA degradation to promote DC maturation and enhance DC function.

[0031] 4. This invention utilizes the limited subcutaneous HA reserve as the endogenous alarm factor release threshold, avoiding sustained excessive inflammatory response and T cell depletion compared to traditional exogenous adjuvants. In vitro and in vivo safety evaluations showed that TCV@HAase-patch had no systemic toxicity.

[0032] 5. The TCV@HAase-patch constructed in this invention can provide long-term immune protection. Mice immunized with TCV@HAase-patch showed complete resistance to tumor growth during secondary tumor challenge and survived for more than 90 days.

[0033] 6. TCVs can be derived from different tumor types. The preparation processes of HAase-patch and TCV@HAase-patch are simple, easy to scale up, and have excellent universality, making them promising candidates for development into ready-to-use vaccine platforms. Attached Figure Description

[0034] Figure 1 Design and characterization of HAase-patch and TCV@HAase-patch. (a) Schematic diagram of OSA synthesis; (b) Schematic diagram of HAase-patch synthesis; (c) TEM and SEM images of HAase-patch (TEM scale bar: 100 nm, SEM scale bar: 200 nm); (d) Hydrodynamic particle size of HAase-nanogel and HAase-patch; (e) Super-resolution fluorescence microscopy image of fluorescently labeled HAase-patch (scale bar: 200 nm); (f) Enzyme activity retention rates of free HAase, HAase-nanogel, and HAase-patch; (g) Schematic diagram of TCV@HAase-patch construction; (h) Proportion of HAase-positive TCV and HAase loading at different feed ratios; (i) Confocal microscopy images of fluorescently labeled TCV and TCV@HAase-patch (scale bar: 5 μm); (j) SEM images of TCV and TCV@HAase-patch (scale bar: 2 μm). (k) Schematic diagram of HAase-patch blocking CD47 on the surface of tumor cells and enhancing DC phagocytosis; (l) Flow cytometry analysis of CD47 surface expression; (m) Flow cytometry quantification of BMDC phagocytosis of TCV@HAase-patch; (n) In vivo distribution experiment timeline; (o) In vivo fluorescence imaging after subcutaneous injection of different formulations; (p) Quantitative analysis of the HAase / TCV fluorescence intensity ratio at the injection site; (q) In vitro fluorescence imaging of major organs and skin at 48 h; (r) Quantitative analysis of the HAase / TCV fluorescence intensity ratio in skin tissue; (s) Zebrafish micro-injection experiment timeline; (t) Fluorescence imaging results of different formulations after micro-injection into the yolk sac of zebrafish (scale bar is 100 μm).

[0035] Figure 2To assess the immune response and tumor prevention effects induced by TCV@HAase-patch. (a) Experimental timeline for evaluating local HA degradation, DC activation, and antigen uptake induced by subcutaneous TCV@HAase-patch; (b) Quantification of HA expression levels in skin tissue; (c) Flow cytometry quantification of mature DCs and CCR7⁺ DCs at the injection site; (d) Schematic diagram of local and systemic immune responses induced by TCV@HAase-patch; (e) Immunofluorescence images of skin and draining lymph node sections (scale bar: 50 μm for skin tissue sections, 100 μm for lymph node tissue sections); (f) Flow cytometry plots of CD11c⁺ DCs carrying TCV fluorescence in skin and lymph nodes; (g) Flow cytometry analysis of CD11c⁺ DCs carrying TCV fluorescence in skin and lymph nodes; (h) Flow cytometry detection of DC antigen cross-presentation in lymph nodes; (i) Immunofluorescence analysis of lymph node sections and in vivo lymph node photographs (scale bar: 50 μm). (j) Flow cytometry quantification of mature DCs, IFN-γ⁺CD8⁺T cells, GC B cells and Tfh cells in lymph nodes; (k) Timeline of 4T1 breast cancer prevention experiment; (l) Bioluminescence imaging of mice in different treatment groups; (m) Tumor growth curve; (n) Proportion of mature DCs and cytotoxic CD8⁺T cells in tumor draining lymph nodes; (o) Survival curve; (p) Bioluminescence imaging of tumor rechallenge in cured and control mice; (q) Proportion of CD44⁺ memory / effect CD8⁺T cells in peripheral blood, lymph nodes and spleen; (r) Survival curve after rechallenge; (s) In vivo bioluminescence imaging of 4T1 lung metastasis prevention experiment; (t) Bioluminescence imaging of ex vivo lung tissue of 4T1 lung metastasis prevention experiment; (u) Number of metastatic nodules on lung tissue surface.

[0036] Figure 3 To demonstrate the preventive effect of TCV@HAase-patch in various tumor models. (a) Representative bioluminescence imaging of mice subcutaneously inoculated with B16F10 melanoma in different vaccine treatment groups (PBS, TCV, TCV+HAase, TCV@HAase-patch); (b) Mean growth curve of B16F10 subcutaneous tumors; (c) Individual growth curve of B16F10 subcutaneous tumors; (d) Survival curve of B16F10 tumor-bearing mice; (e) In vivo bioluminescence imaging of B16F10 lung metastasis model; (f) Bioluminescence imaging of ex vivo lung tissue; (g) Quantitative analysis of lung weight and number of metastatic nodules on lung surface in each group; (h) Survival curve of B16F10 lung metastasis model; (i) Representative bioluminescence imaging of CT26 colon cancer in situ model; (j) Quantitative analysis of bioluminescence intensity of ex vivo gastrointestinal tissue; (k) Quantitative analysis of the number of metastatic nodules in gastrointestinal tract; (l) Representative images of spontaneous metastatic nodules in ex vivo lung tissue; (m) Survival curve of CT26 tumor-bearing mice.

[0037] Figure 4 To assess the biosafety of TCV@HAase-patch. (a) Results of hematological parameters (RBC, WBC, HGB) and serum biochemical parameters (ALT, AST, UREA, CREA, LDH) in peripheral blood of mice at different time points after TCV@HAase-patch immunization; (b) H&E stained histological images of major organs on day 14 after repeated TCV@HAase-patch inoculation (scale bar is 100 μm). Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be described in detail below with reference to specific examples. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Example 1

[0039] Synthesis of Oxidized Sodium Alginate (OSA): The preparation process is as follows Figure 1 As shown in (a), specifically, sodium alginate (SA, molecular weight approximately 80–120 kDa, 2.01 g) was dissolved in 200 ml of deionized water to prepare a 1.0 wt% solution. Sodium periodate (NaIO4, 1.08 g, 5.05 mmol) was added to the SA solution, and the mixture was stirred at room temperature in the dark for 4 h. The reaction was terminated by adding 1.5 ml of ethylene glycol. The resulting OSA solution was dialyzed against deionized water for 48 h using a dialysis membrane with a molecular weight cutoff of 3500 Da, with the water changed every 8 h. The purified OSA was lyophilized to obtain a white powder. The aldehyde content of OSA was determined to be 5.1 μmol / mg by hydroxylamine hydrochloride titration, corresponding to an oxidation degree of 50.5%.

[0040] Preparation of HAase nanogels and HAase-patch: The preparation process is as follows Figure 1As shown in (b), specifically, HAase (derived from bovine testis, 3000 U / mg) was dissolved in deionized water to obtain a 4 mg / mL HAase solution, and OSA was dissolved in deionized water to obtain a 20 mg / mL OSA solution. 2 mL of the HAase solution was mixed with 80 μL of the OSA solution and stirred in an ice bath for 5 h to allow HAase and OSA to crosslink via Schiff base reaction, forming a HAase nanogel. Unreacted HAase and OSA were removed by dialysis with PBS (pH 7.4) using a dialysis membrane with a molecular weight cutoff of 300 kDa, yielding purified HAase nanogel (HAase-nanogel). α-CD47 (10 mg / mL aqueous solution, 0.1 mL) was added to the above HAase nanogel solution and stirred at 4°C for 1 h to couple α-CD47 via its amino group to the residual aldehyde group on the surface of the HAase nanogel. Uncoupled antibodies were removed by dialysis with PBS using a dialysis membrane with a molecular weight cutoff of 300 kDa, and the resulting HAase-patch was obtained by lyophilization.

[0041] Figure 1 Characterization of HAase-patch: Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that HAase-patch had a spherical morphology (c), and dynamic light scattering (DLS) measured a particle size of approximately 256.3 ± 8.0 nm, while HAase-nanogel had a particle size of approximately 212.4 ± 4.4 nm (d), and a Zeta potential of approximately -30.81 ± 1.42 mV. Super-resolution fluorescence microscopy showed that FITC-labeled OSA, Cy3-labeled HAase, and Cy5-labeled α-CD47 were co-localized in HAase-patch, confirming the successful integration of each component (e). Enzyme activity retention experiments showed that after incubation at 37°C for 12 h, the retention rate of free HAase decreased to approximately 20%, while the retention rates of HAase-nanogel and HAase-patch remained above 80%, indicating that nanogel encapsulation significantly improved the stability of HAase (f). Example 2

[0042] Preparation of TCV: Tumor cells in logarithmic growth phase (such as 4T1 breast cancer cells, B16F10 melanoma cells, CT26 colon cancer cells, or patient-derived tumor cells) were collected by trypsin digestion and washed twice with sterile PBS. The cell pellet was resuspended in cell fixation buffer (PBS containing 4% paraformaldehyde) at a density of 1×10⁻⁶. 7Cells / mL, treated at 4°C for 30 min. After fixation, wash three times with PBS to remove residual fixative. Inactivated TCVs were resuspended in sterile PBS to the desired concentration and stored at 4°C for later use. Live / Dead co-staining reagent was used to confirm the absence of viable cells after fixation.

[0043] Assembly of TCV@HAase-patch: such as Figure 1 As shown in (g), different amounts of HAase-patch (corresponding to 0~30 μg HAase) were mixed with 1×10 6 One TCV was incubated in 200 μL PBS at 4°C for 2 h. The mixture was centrifuged at 300×g for 5 min to remove unbound HAase-patch. The precipitate was washed twice with PBS and resuspended in 200 μL PBS. The supernatant was collected, and the content of unbound HAase was determined using a BCA protein quantification kit. See [link to BCA protein quantification kit]. Figure 1 The results showed that when the feed ratio was 25 μg HAase / 10 6 During TCV, approximately 100% of the TCV was labeled with HAase-patch, with a drug loading of 10.41 ± 0.78 μg HAase per 10. 6 TCV(h). Confocal microscopy and SEM confirmed the successful anchoring of the HAase-patch on the TCV surface (i, j). Example 3

[0044] The principle of CD47 blocking and DC phagocytosis enhancement in TCV@HAase-patch is as follows: Figure 1 As shown in (k). Flow cytometry analysis revealed that TCVs derived from 4T1, EMT6, B16F10, and CT26 all highly expressed CD47. HAase-patch treatment significantly reduced the fluorescence intensity of CD47 on the TCV surface, while HAase-nanogel treatment had no such effect (l). After co-incubating CFSE-labeled TCV@HAase-patch with CellTracker Deep Red-labeled BMDCs for 24 h, flow cytometry analysis showed that the DC phagocytosis rate of the TCV@HAase-patch group was significantly higher than that of the TCV group and the TCV+HAase-nanogel physical mixture group (m). Example 4

[0045] Investigate the in vivo distribution and retention of TCV@HAase-patch: such as Figure 1As shown in Figure (n), TCV was labeled with DiR and HAase with Cy5 to prepare three formulations: (1) a physical mixture of TCV and HAase; (2) a physical mixture of TCV and HAase-nanogel; and (3) a TCV@HAase-patch. Each formulation was subcutaneously injected into BALB / c mice, and the fluorescence signal was monitored by IVIS in vivo imaging. The results are shown in Figure (n). Figure 1 As shown in (o, p), the HAase / TCV fluorescence ratio in the TCV+HAase group decreased sharply within 6 h; the decrease was slower but continued to decline in the TCV+HAase-nanogel group; while the HAase / TCV ratio in the TCV@HAase-patch group remained stable within 48 h. Ex vivo imaging at 48 h showed that TCV and HAase signals in the TCV@HAase-patch group were mainly co-localized in the skin at the injection site, with no significant accumulation in other organs (q, r). See also Figure 1 In the zebrafish model, good co-localization of TCV and HAase in the TCV@HAase-patch group was also observed, suggesting that the backpack structure of TCV is conducive to realizing spatiotemporal co-delivery of TCV and HAase. Example 5

[0046] See Figure 2 In the experimental procedure shown in (a), BALB / c mice were subcutaneously injected with PBS, TCV, HAase-patch, or TCV@HAase-patch. Skin samples from the injection sites were collected at 4 h and 48 h for HA immunofluorescence staining and DC flow cytometry analysis. This was to verify the local HA degradation and immune response induced by TCV@HAase-patch. Results showed that the TCV@HAase-patch group maintained significant HA degradation at both 4 h and 48 h (b); flow cytometry analysis showed that the TCV@HAase-patch group still maintained a high proportion of mature DCs and CCR7 at 48 h. + DCs in the TCV+HAase and TCV+HAase-nanogel groups returned to baseline levels after 48 h (c). TCV@HAase-patch promoted DC recruitment, maturation, and antigen uptake by continuously degrading HA to produce endogenous alarmins (d). Immunofluorescence showed that a large number of CD11c⁺ DCs in the skin and lymph nodes of the TCV@HAase-patch group carried TCV fluorescence signals (e), and flow cytometry further confirmed the presence of CD11c in the lymph nodes of this group. +DCs showed the highest TCV uptake rate (f, g). OVA expression of TCV and detection of anti-SIINFEKL-H-2Kb antibody revealed the strongest antigen cross-presentation in lymph node DCs in the TCV@HAase-patch group (h). Immunofluorescence and photographs of lymph node sections showed significant enlargement of lymph nodes in the TCV@HAase-patch group, and CD8+... + T cells and CD11c + DC infiltration was significantly increased (i). Flow cytometry analysis showed that mature DCs and IFN-γ were present in the lymph nodes of the TCV@HAase-patch group. + CD8 + The proportions of T cells, GC B cells, and Tfh cells were all significantly higher than in other groups (j). Example 6

[0047] See Figure 2 In the middle ku, the prophylactic antitumor effect and long-term immune memory of TCV@HAase-patch were further verified. The experimental procedure is as follows: Figure 2 As shown in (k), BALB / c mice were subcutaneously immunized with PBS, TCV, TCV+HAase, TCV+HAase-nanogel, or TCV@HAase-patch on days -10, -5, and 0 (n=5 per group, 1×10⁻⁶). 6 TCV per mouse), subcutaneous injection of 4 T1-luc cells (2 × 10⁻⁶) on day 0. 5 Bioluminescence imaging showed that all mice in the TCV@HAase-patch group (5 / 5) showed no tumor growth, while the TCV+HAase and TCV+HAase-nanogel groups showed only partial inhibition. Tumors progressed rapidly in the PBS and TCV groups (l, m). The TCV@HAase-patch group had the highest proportion of mature dendritic cells (DCs) and cytotoxic CD8 T cells in the tumor-draining lymph nodes (n). Mice in the TCV@HAase-patch group had a 100% 60-day survival rate, while all mice in other groups died within 40 days (o).

[0048] To assess the persistence of immune memory, cured mice in the TCV@HAase-patch group were subcutaneously inoculated with 4T1-luc cells at four different sites on day 60, with blank mice serving as a control. Results showed that no tumor growth occurred at any injection site in the cured mice, while progressive tumors appeared in the blank mice (p). CD44 levels were found in the peripheral blood, lymph nodes, and spleen of the cured mice. + Memory / Effect CD8 + The proportion of T cells was significantly higher than that of the blank control group (q), and all cured mice survived during the 90-day observation period (r). In the 4T1 lung metastasis prevention model, mice were immunized with the same protocol and then intravenously injected with 1×106 4T1-luc. Bioluminescence imaging showed almost no metastatic signal in the lung tissue of the TCV@HAase-patch group, while other groups showed extensive lung metastases (s, t). Ex vivo lung tissue metastatic nodule counting confirmed that the number of metastatic nodules on the lung surface in the TCV@HAase-patch group was close to zero, significantly less than in other groups (u). Example 7

[0049] To verify the universality of TCV@HAase-patch across different tumor types, its preventive effect was evaluated in B16F10 melanoma and CT26 colon cancer models. See [link to relevant documentation]. Figure 3 In the B16F10 melanoma model (a), C57BL / 6 mice were subcutaneously inoculated with B16F10-luc after immunization using the same protocol. Tumor growth was significantly inhibited in the TCV@HAase-patch group (b, c), and survival was significantly prolonged (d). In the B16F10 lung metastasis model (e, f), the TCV@HAase-patch group had very few lung metastatic nodules, significantly lower lung weight than other groups (g), and significantly prolonged survival (h). In the CT26 colon cancer orthotopic model (i), BALB / c mice were inoculated with CT26-luc in the cecal wall after immunization. The TCV@HAase-patch group had the lowest bioluminescence intensity in the isolated gastrointestinal tract tissue (j), the fewest metastatic nodules (k), and no spontaneous metastatic lesions were observed in the isolated lung tissue (l), and significantly prolonged survival (m). These results indicate that TCV@HAase-patch can induce a broad-spectrum and highly effective prophylactic antitumor immune response in different tumor types. Example 8

[0050] To assess the in vivo safety of TCV@HAase-patch, BALB / c mice were subcutaneously inoculated with either TCV@HAase-patch or PBS control according to the prophylactic regimen. Peripheral blood was collected on days 1, 7, and 14 for hematological and serum biochemical analyses. See [link to relevant documentation] Figure 4The results showed that, compared with the PBS control group, hematological indicators such as red blood cell count (RBC), white blood cell count (WBC), and hemoglobin (HGB) in the TCV@HAase-patch group were all within the normal physiological range at all time points; serum biochemical indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), creatinine (CREA), and lactate dehydrogenase (LDH), showed no abnormal elevation (a). On day 14 after repeated vaccination, H&E staining histological analysis of major organs such as the heart, liver, spleen, lungs, and kidneys showed that, compared with the PBS control group, no obvious pathological abnormalities, tissue necrosis, or inflammatory cell infiltration were observed in any organ in the TCV@HAase-patch group (b). These results indicate that the TCV@HAase-patch vaccine has good biocompatibility and low systemic toxicity at both prophylactic and therapeutic doses, providing strong safety support for its clinical application.

[0051] The TCV@HAase-patch preparation process provided by this invention is simple, low-cost, and easy to scale up. HAase and α-CD47 are both biomolecules with existing clinical experience, and TCV can be derived from surgically removed tumor tissue, providing a good foundation for clinical translation. This vaccine platform can be used for postoperative recurrence prevention, cancer prevention in high-risk populations, and treatment of advanced tumors, demonstrating broad clinical application prospects and economic value.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A whole-cell tumor vaccine loaded with a hyaluronidase nanogel backpack, characterized in that, The vaccine comprises inactivated whole tumor cells and HAase-patch anchored on the surface of the whole tumor cells; the HAase-patch is a hyaluronidase nanogel with surface-conjugated anti-CD47 antibody.

2. The whole-tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to claim 1, characterized in that, The hyaluronidase nanogel is obtained by cross-linking sodium alginate with hyaluronidase.

3. The whole-tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to claim 2, characterized in that, The oxidation degree of the oxidized sodium alginate is 40%~60%.

4. The whole-tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to claim 1, characterized in that, The tumor cells are derived from autologous or allogeneic tumor cells.

5. The whole-tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to claim 1, characterized in that, The vaccine is administered via subcutaneous injection.

6. The method for preparing the whole tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Dissolve HAase and sodium oxidized alginate separately in deionized water, mix them, stir and react in an ice bath, and obtain HAase nanogel by dialysis; (2) α-CD47 was added to the HAase nanogel solution and stirred to couple α-CD47 to the surface of the nanogel. The HAase-patch was obtained by dialysis and freeze-drying. (3) Mix HAase-patch with TCV and incubate. Centrifuge and wash to remove unbound HAase-patch to obtain TCV@HAase-patch.

7. The method for preparing a whole-tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to claim 6, characterized in that, In step (1), the mass ratio of HAase to sodium oxidized alginate is 2~10:

1.

8. The method for preparing a whole-tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to claim 6, characterized in that, In step (2), the mass ratio of α-CD47 to HAase nanogel is 1:5 to 1:

20.

9. The method for preparing a whole-tumor cell vaccine loaded with a hyaluronidase nanogel backpack according to claim 6, characterized in that, In step (3), the feed ratio of TCV to HAase-patch is 1 × 10 6 Each TCV feed should contain 5-30 μg of HAase.

10. The use of the whole tumor cell vaccine loaded with hyaluronidase nanogel backpack according to any one of claims 1-5 in the preparation of antitumor drugs.

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