Chinese herbal medicine vesicle-lipid nanoparticle hybrid tumor mRNA vaccine and application thereof

Abstract

The present application relates to the technical field of biological medicine, and provides a Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine and application thereof.The tumor mRNA vaccine is obtained by hybridizing Chinese herbal medicine nano vesicles and lipid nanoparticles loaded with tumor antigen mRNA.The present application reduces the safety risk of cationic lipid carriers by using the good biocompatibility of Chinese herbal medicine.Combined with the characteristics of Chinese herbal medicine, the nano vesicles are extracted, which promotes the LNP to more effectively target antigen presenting cells, promotes the uptake level of antigen presenting cells, enhances the phagocytosis of mRNA, and can enhance the targeting and transcription expression level of mRNA.Finally, the tumor mRNA vaccine hybridized by Chinese herbal medicine nano vesicles and LNP can strengthen the effect of inhibiting tumor progression, inhibiting tumor recurrence and inhibiting tumor metastasis after vaccination, and opens up a path for its combination with more abundant immunotherapy methods.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a construction strategy of a low-toxicity Chinese herbal medicine nano-vesicle-LNP hybrid tumor mRNA vaccine and biomedical application thereof. BACKGROUND

[0002] With the development of precision medicine, tumor treatment is shifting from "indiscriminate killing" to "individualized targeting", but drug resistance and recurrence are still major challenges, and more efficient and less toxic new strategies are urgently needed. Immunotherapy is a treatment method that uses the human immune system to recognize and kill tumor cells. It does not directly attack cancer cells, but rather activates or enhances the function of immune cells (such as T cells and NK cells) to make them more effective in eliminating tumors. The main types of immunotherapy currently include immune checkpoint inhibitors, chimeric antigen receptor T cell therapy, oncolytic viruses, and cancer vaccines. In recent years, the potential for personalized customization of cancer vaccines has made cancer vaccines a research hotspot in immunotherapy.

[0003] Vaccines have evolved from traditional inactivated / attenuated pathogens to subunit vaccines, protein vaccines, viral vector vaccines, whole cell vaccines, DC vaccines (such as Provenge®), and nucleic acid vaccines (DNA / mRNA). The core goal is to induce immune memory efficiently and safely. In the field of tumors, conventional therapies (surgery, radiotherapy, and chemotherapy) are difficult to eliminate metastatic lesions or drug-resistant cells, while vaccines can activate specific T cell responses and are expected to achieve long-term immune surveillance. However, the heterogeneity of tumor antigens, immune escape mechanisms, and insufficient delivery efficiency have limited the clinical effectiveness of most therapeutic tumor vaccines, and breakthrough technologies are needed to address the three major challenges of antigen selection, delivery, and immune microenvironment regulation.

[0004] Nucleic acid vaccines have rapidly emerged in recent years, mainly influenced by factors such as mRNA sequence optimization and delivery system maturation. Among them, mRNA vaccines encode tumor-associated antigens (TAAs) or neoantigens to activate specific immune responses in the body, and have unique advantages in personalized tumor treatment. Compared with traditional vaccines, mRNA does not need to enter the nucleus, avoiding the risk of genomic integration, and can quickly respond to mutant antigens through sequence design. In recent years, the success of COVID-19 mRNA vaccines has proven their clinical feasibility, but the immunosuppressive properties of the tumor microenvironment (TME) and insufficient delivery efficiency have limited their effectiveness. Therefore, developing efficient and less toxic delivery systems is a core challenge for tumor mRNA vaccines.

[0005] mRNA vaccines rely on delivery vehicles to introduce antigen-encoding mRNA into the cytoplasm, which is then translated by ribosomes, and the antigens are presented by MHC I / II molecules to activate CD8 + / CD4 +T cells. However, naked mRNA is easily degraded by serum RNase, and negatively charged mRNA is difficult to penetrate the cell membrane. Existing delivery systems such as LNP can encapsulate mRNA, but they face problems such as poor targeting, off-target toxicity caused by liver and spleen enrichment, and the like. In addition, the immunogenicity of mRNA can trigger excessive inflammatory response, further restricting its clinical application.

[0006] Currently, the mainstream delivery carrier such as Lipid Nanoparticle (LNP) relies on cationic lipids (such as SM102, DOTAP, etc.) to compress mRNA, but its positive charge leads to binding with serum proteins, accelerated clearance, and cell membrane damage. In addition, although the PEGylation of LNP can prolong the circulation time, it may induce anti-PEG antibodies, reducing the effect of repeated administration. Although viral vectors have high transfection efficiency, they have the risk of insertional mutation and pre-existing immunity. These defects highlight the urgent need for a new delivery platform.

[0007] Mature cationic lipid carriers include various cationic lipid components. Taking SM102 as an example, SM102, as the core component of LNP, has proven its mRNA delivery effectiveness in COVID-19 vaccines, but its cationic properties can cause cell membrane lysis, complement activation, and liver toxicity. Animal experiments show that high-dose SM102 can induce an inflammatory factor storm, and multiple administrations are required for tumor treatment, further amplifying the safety risks. Therefore, it is urgent to shield the toxicity of the carrier through modification, while enhancing its efficient delivery capacity.

[0008] Currently, the LNP carriers (such as SM102, DOTAP, etc.) relied on by mRNA vaccines have significant defects, including potential toxicity risks, induced inflammatory damage or liver damage, which ultimately require restrictions on the administration dose of cationic lipids and mRNA. At the same time, LNP carriers will be non-specifically enriched, and a large number of LNP will be retained at the injection site or captured by the liver and spleen reticuloendothelial system after injection, with a tumor and lymph accumulation rate of less than 5%, which severely limits the efficacy of mRNA. In addition, LNP-mRNA complexes require low-temperature storage and transportation for preservation, increasing the cost of clinical use. SUMMARY

[0009] To solve the above technical problems, the present application provides a low-toxicity Chinese herbal medicine nano-vesicle-LNP hybrid tumor mRNA vaccine. The present application takes advantage of the good biocompatibility of Chinese herbal medicine to reduce the safety risk of cationic lipid carriers. Combined with the characteristics of Chinese herbal medicine, the nano-vesicles are extracted to promote the targeting of LNP to antigen-presenting cells, promote the uptake level of antigen-presenting cells, and enhance the phagocytosis of mRNA. Through the Toll-like receptors on the surface of antigen-presenting cells, the maturation of antigen-presenting cells is promoted, and the targeting and transcription expression level of mRNA are enhanced. Ultimately, the tumor mRNA vaccine hybridized by Chinese herbal medicine nano-vesicles and LNP can strengthen the effect of inhibiting tumor progression, inhibiting tumor recurrence and inhibiting tumor metastasis after tumor mRNA vaccination, and open up a path for its combination with more abundant immunotherapy methods.

[0010] In a first aspect of the present application, a Chinese herbal medicine vesicle and LNP hybrid tumor mRNA vaccine is provided, which is obtained by hybridizing Chinese herbal medicine nano-vesicles and lipid nanoparticles loaded with tumor antigen mRNA.

[0011] As an option, the Chinese herbal medicine includes ginseng; and the tumor antigen includes one or more of a combination of model antigens, universal tumor antigens, tumor neoantigens, tumor-associated antigens, or tumor fusion antigens.

[0012] As an option, the operation method of hybridization includes microfluidic method, homogenization method, extruder extrusion method, PEG-mediated membrane fusion method, calcium ion triggering method, charge reversal method, biotin-avidin bridging method, or low-intensity ultrasound method.

[0013] As an option, the preparation method of the lipid nanoparticles loaded with tumor antigen mRNA includes: dissolving LNP raw materials containing cationic lipids and auxiliary lipids in alcohol in proportion to form an alcohol phase; dissolving tumor antigen mRNA in an acid buffer to form an aqueous phase; mixing the alcohol phase with the aqueous phase to obtain a preliminary emulsion; dialyzing the preliminary emulsion and concentrating to obtain the lipid nanoparticles loaded with tumor antigen mRNA.

[0014] As an option, the cationic lipid is SM102, the auxiliary lipid includes DSPC, cholesterol, and DMG-PEG2000; and the proportion is SM102:DSPC:cholesterol:DMG-PEG2000 mass ratio = 50:10:38.5:1.5.

[0015] As an option, the mixing of the alcohol phase with the aqueous phase includes mixing by microfluidic method under the conditions that the molar ratio of nitrogen in SM102 to nucleic acid in mRNA is 6 and the volume ratio of the alcohol phase to the aqueous phase is 3.

[0016] As an alternative, the method of hybridizing the Chinese herbal medicine nanovesicle with the tumor antigen mRNA-loaded lipid nanoparticle comprises: mixing and assembling the Chinese herbal medicine nanovesicle and the tumor antigen mRNA-loaded lipid nanoparticle in a ratio of Chinese herbal medicine nanovesicle protein mass:tumor antigen mRNA mass = 40:1 by using an extruder or a microfluidic method to obtain the tumor mRNA vaccine.

[0017] As an alternative, the cationic lipid comprises a combination of one or more of SM102, DOTAP, DDAB, DOTMA, Dlin-MC3-DMA, ALC-0315, DOSPA, DOGS, DMG-PEG2000, or DSG-PEG2000.

[0018] As an alternative, the helper lipid comprises a combination of one or more of cholesterol, DOPE, DSPC, beta-sitosterol, natural egg lecithin, plant natural lipid components, or DSPE-PEG2000.

[0019] As an alternative, the administration method of the tumor mRNA vaccine comprises intranasal instillation, atomization inhalation, subcutaneous inoculation, intramuscular injection, oral administration, intralymph node injection; and further comprises injecting the activated dendritic cells into the lymph node, vein, or subcutaneously after activating the patient's bone marrow-derived dendritic cells in vitro.

[0020] In a second aspect of the present application, the tumor mRNA vaccine according to any one of the above-mentioned schemes is provided for use in the preparation of a drug for preventing, treating, or inhibiting recurrence or metastasis of a tumor.

[0021] As an alternative, the tumor comprises melanoma, colorectal tumor, breast tumor, lymphocyte tumor, or bladder tumor.

[0022] In a third aspect of the present application, a dendritic cell vaccine is provided, comprising bone marrow-derived dendritic cells activated by the tumor mRNA vaccine according to any one of the above-mentioned schemes.

[0023] In a fourth aspect of the present application, a tumor treatment composition is provided, comprising the tumor mRNA vaccine according to any one of the above-mentioned schemes and a tumor treatment drug, wherein the tumor treatment drug comprises a chemotherapy drug, a radiotherapy drug, siRNA, an immune checkpoint blocker, a T cell, a CAR-T cell, or an oncolytic virus.

[0024] The application establishes a personalized tumor mRNA vaccine by encapsulating a tumor mRNA antigen cationic carrier LNP inside a Chinese herbal medicine nanovesicle or chimerizing the Chinese herbal medicine nanovesicle. The hybridization method such as microfluidics can be used for large-scale production to maintain the biocompatibility of the personalized vaccine and reduce the safety risk of the cationic lipid carrier. At the same time, the Chinese herbal medicine vesicle-LNP hybrid mRNA vaccine can integrate the tumor antigen, adjuvant and delivery carrier, effectively promote the phagocytosis and translation of mRNA by antigen presenting cells, promote the maturation and antigen presenting ability of antigen presenting cells, finally enhance the generation efficiency of specific T lymphocytes and enhance the anti-tumor immune protection of the vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The figure is a schematic diagram of the construction process of the low-toxicity Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine according to an embodiment of the application. Taking the Chinese herbal medicine ginseng nanovesicle G-EVLPs as an example. The tumor antigen mRNA@SM102 vaccine is prepared by microfluidic method. After mixing the differential centrifugation obtained ginseng nanovesicle G-EVLPs with mRNA and mRNA@SM102, extrusion is performed to obtain the mRNA@G-EVLPs (denoted as mRNA@G) tumor vaccine and the mRNA@SM102@G-EVLPs (denoted as SM102@G) tumor vaccine.

[0026] Figure 2 The figure is the dynamic light scattering (DLS) data of the mRNA@SM102 tumor vaccine. The average particle size of the mRNA@SM102 tumor vaccine is about 142.0 nm, the zeta potential is greater than 0, and the distribution is relatively uniform.

[0027] Figure 3 The figure is the dynamic light scattering (DLS) data of the SM102@G tumor vaccine. The average particle size of the SM102@G tumor vaccine is about 220.0 nm, the zeta potential is less than 0, and the uniformity of the distribution is less than that of the mRNA@SM102.

[0028] Figure 4 The figure is the total protein SDS-PAGE image of the ginseng nanovesicle (G-EVLPs / GdNPs), mRNA, mRNA@SM102, mRNA@G and SM102@G. After extrusion of mRNA@G and SM102@G, the total protein band characteristics of G-EVLPs are still present.

[0029] Figure 5Transmission electron microscopy (TEM) images of mRNA@SM102, G-EVLPs and SM102@G. mRNA@SM102 has the feature of solid spherical structure, and SM102@G still has the feature of double lipid molecule membrane after G-EVLPs encapsulate mRNA@SM102, and the particle size is about 200 nm.

[0030] Figure 6 Flow cytometry analysis of the expression of CD80 and CD86 proteins on the surface of mature BMDCs after co-incubation of mRNA@SM102, G-EVLPs and SM102@G with bone marrow-derived dendritic cells (BMDCs). SM102@G can significantly promote the maturation of BMDCs.

[0031] Figure 7 Flow cytometry analysis of the uptake of mRNA@SM102, G-EVLPs and SM102@G by mature BMDCs after co-incubation with bone marrow-derived dendritic cells (BMDCs). SM102@G can significantly promote the uptake of mRNA@SM102. +

[0032] Figure 8 Tumor volume, mouse weight, complete response rate (CR, mice with no tumor recurrence) and lymph node pictures of the tumor recurrence model and long-term anti-tumor recurrence model after subcutaneous melanoma B16F10-OVA vaccination. The figure shows that the SM102@G tumor vaccine can effectively inhibit the recurrence of B16F10-OVA melanoma and significantly prolong the survival of mice. In long-term anti-tumor protection, it can achieve the effect of less than 20% recurrence rate 60 days after vaccination. At the same time, after SM102@G vaccination, the mouse lymph nodes are significantly larger than the mRNA@SM102 group, indicating a significant enhancement of the vaccine effect.

[0033] Figure 9 Serum pro-inflammatory cytokine levels, including IFN-γ, IL-1β, IL-6, IL-12p70 and TNF-α, of mice after SM102@G tumor vaccine vaccination. The figure shows that the SM102@G tumor vaccine can effectively stimulate the secretion level of anti-tumor cytokines.

[0034] Figure 10 Tumor volume, mouse weight, mouse survival rate and tumor weight of the tumor vaccine treatment model after subcutaneous melanoma B16F10-OVA vaccination. This treatment model is combined with immune checkpoint blocker PD-1 antibody treatment. SM102@G tumor vaccine combined with PD-1 can effectively inhibit the progression of B16F10-OVA melanoma and significantly prolong the survival of mice.

[0035] ​Figure 11 Tumor growth curve and partial remission rate (PR, tumor shrinkage and less than 150 mm 3 ) volume after subcutaneous melanoma B16F10-OVA vaccination treatment model, this treatment model combined with immune checkpoint blocker PD-1 antibody treatment. SM102@G tumor vaccine combined with PD-1 can effectively inhibit the progression of B16F10-OVA melanoma, increase the partial remission rate of mice after treatment.

[0036] Figure 12 Tumor photos of mice in the subcutaneous melanoma B16F10-OVA vaccination treatment model, this treatment model combined with immune checkpoint blocker PD-1 antibody treatment. SM102@G tumor vaccine combined with PD-1 can effectively inhibit the growth of B16F10-OVA melanoma, and some tumors almost completely disappeared.

[0037] Figure 13 Tumor section immunohistochemical staining (PD-L1) pictures of mice in the subcutaneous melanoma B16F10-OVA vaccination treatment model, this treatment model combined with immune checkpoint blocker PD-1 antibody treatment. G-EVLPs can reduce the expression of intratumoral PD-L1, and SM102 can further enhance the expression of intratumoral PD-L1. SM102@G tumor vaccine combined with PD-1 can effectively inhibit the increased expression of PD-L1 caused by SM102, laying a foundation for the combination of PD-1 immunotherapy.

[0038] Figure 14 Tumor immunofluorescence staining (CD8+ T lymphocytes) pictures of mice in the subcutaneous melanoma B16F10-OVA vaccination treatment model, this treatment model combined with immune checkpoint blocker PD-1 antibody treatment. SM102@G tumor vaccine combined with PD-1 can effectively increase the infiltration level of intratumoral CD8+ T lymphocytes in B16F10-OVA melanoma, enhancing the effect of treatment combination.

[0039] Figure 15 Groin lymph node mass of mice in the subcutaneous melanoma B16F10-OVA vaccination treatment model, this treatment model combined with immune checkpoint blocker PD-1 antibody treatment. The lymph nodes of mice in the SM102@G tumor vaccine group were significantly larger than those in the mRNA@SM102 group, indicating a significant enhancement of the vaccine effect.

[0040] Figure 16The figure is the photograph of inguinal lymph nodes of mice in the subcutaneous melanoma B16F10-OVA post-vaccination tumor treatment model, and the treatment model is combined with immune checkpoint blocker PD-1 antibody treatment. The lymph nodes of mice in the SM102@G tumor vaccine group are significantly larger than those in the mRNA@SM102 group, indicating that the vaccine effect is significantly enhanced.

[0041] Figure 17 The figure is the serum pro-inflammatory cytokine level of mice in the SM102@G tumor vaccine post-vaccination treatment model in the subcutaneous B16F10-OVA, including IFN-γ, IL-1β, IL-6, IL-12p70 and TNF-α. The figure shows that the SM102@G tumor vaccine can effectively stimulate the secretion level of anti-tumor cytokines.

[0042] Figure 18 The figure is the serum biochemical index data of mice after vaccination with the SM102@G tumor vaccine, including liver and kidney indexes such as AST, ALT, BUN, CREA, ALP and γ-GT. The figure shows that the SM102@G tumor vaccine has high safety.

[0043] Figure 19 The figure is the blood cell data of mice after vaccination with the SM102@G tumor vaccine, including white blood cell number, lymphocyte number, red blood cell number, platelet number, blood protein level and monocyte percentage. The figure shows that the SM102@G tumor vaccine has high safety. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be described in detail below in combination with the drawings and specific embodiments.

[0045] The present application provides a Chinese herbal medicine vesicle and LNP hybrid tumor mRNA vaccine, which is obtained by hybridizing Chinese herbal medicine nanovesicles and lipid nanoparticles (denoted as mRNA LNP) loaded with tumor antigen mRNA.

[0046] For the Chinese herbal medicine nanovesicles, the Chinese herbal medicine nanovesicles are nanovesicles or exosomes extracted from fresh Chinese herbal medicine plants. The Chinese herbal medicine species include ginseng, etc. The extraction methods of Chinese herbal medicine nanovesicles include differential centrifugation, dialysis, ultracentrifugation, density gradient centrifugation, ultrafiltration, magnetic bead immunosorbent separation, size exclusion and cationic polymer precipitation, etc.

[0047] Chinese herbal medicine or plant-derived exosomes or nanovesicles have natural biocompatibility, low immunogenicity, target organ specificity and the ability to cross biological barriers. These exosomes or nanovesicles have a phospholipid bilayer structure, can effectively encapsulate mRNA or cationic lipid carriers, and the surface proteins can mediate natural targeting. Studies have shown that some Chinese herbal medicine or plant-derived exosomes or nanovesicles can evade immune surveillance, prolong in vivo circulation time, and can be further optimized for targeting ability through genetic engineering or chemical modification. Compared with synthetic carriers, the characteristics of Chinese herbal medicine or plant-derived exosomes or nanovesicles are more in line with the safety requirements of clinical practice.

[0048] For tumor antigen mRNA, including model antigen mRNA, universal tumor antigen mRNA, tumor neoantigen mRNA, tumor-associated antigen mRNA, tumor fusion antigen mRNA, etc.

[0049] For the method of loading LNP with tumor mRNA, including: dissolving LNP raw materials containing cationic lipids and auxiliary lipids in alcohol in proportion to form an alcohol phase; dissolving tumor antigen mRNA in an acidic buffer to form an aqueous phase; mixing the alcohol phase with the aqueous phase to obtain a preliminary emulsion; dialyzing the preliminary emulsion, concentrating to obtain lipid nanoparticles loaded with tumor antigen mRNA. LNP is a complex system, including multiple components, the LNP raw materials, i.e. each component of the LNP complex delivery system, including ionizable cationic lipids (such as SM102) and auxiliary lipids (e.g. DSPC, cholesterol, DMG-PEG2000). As an alternative to the LNP formulation, the cationic lipids can also be a combination of one or more of SM102, DOTAP, DDAB, DOTMA, Dlin-MC3-DMA, ALC-0315, DOSPA, DOGS, DMG-PEG2000 or DSG-PEG2000. The auxiliary lipids can also be a combination of one or more of cholesterol, DOPE, DSPC, beta-sitosterol, natural lecithin, plant natural lipid components or DSPE-PEG2000.

[0050] In a preferred embodiment, the components of the LNP complex system used are SM102, DSPC, cholesterol and DMG-PEG2000, and the mass ratio of each component is 50:10:38.5:1.5 after optimization by multiple experiments. Each component is dissolved in an alcohol such as ethanol to form an alcohol phase. The tumor antigen mRNA to be loaded is dissolved in an acidic buffer such as a sodium citrate buffer at pH 4.0 to form an aqueous phase. Then the alcohol phase and the aqueous phase are mixed to form a crude suspension, i.e. a primary emulsion. More specifically, the ratio of the alcohol phase and the aqueous phase is optimized by multiple experiments, and the mixing is preferably carried out under the conditions of a nitrogen-phosphorus ratio of 6 and an alcohol phase / aqueous phase volume ratio of 3. The mixing can be carried out by microfluidization, i.e. prepared on a microfluidic device, to obtain the primary emulsion (i.e. the suspension). Then the primary emulsion is dialyzed for a certain period of time, for example 2 h, and then concentrated to the desired concentration by ultrafiltration tube to obtain the mRNA-loaded lipid nanoparticles (mRNA LNP). By further optimizing the various ratios and other parameter values described above, the LNP can better load the tumor antigen mRNA, and the subsequent hybridization with the Chinese herbal medicine nanovesicle can form a good effect.

[0051] The hybridization of the Chinese herbal medicine nanovesicle and the mRNA LNP refers to the combination of the Chinese herbal medicine nanovesicle and the tumor antigen mRNA-loaded lipid nanoparticle LNP in the form of fusion, wrapping or chimeric to form a complex structure.

[0052] More specific hybridization methods include microfluidization, homogenization, extrusion instrument extrusion, PEG-mediated membrane fusion, calcium ion triggering, charge reversal, biotin-avidin bridging or low-intensity ultrasound, etc. Among them, the microfluidization refers to the use of a microfluidic chip (such as a laminar flow focusing method) to control the precise mixing of LNP and vesicles. By adjusting the flow rate ratio (aqueous phase: oil phase) and channel size, a uniform wrapping structure (particle size controllable at 80-150 nm) is formed. The homogenization refers to mixing LNP and plant vesicles, then adding the above mixed liquid into a homogenizer, and repeatedly circulating under a pressure of 200-800 Pa for 20-25 times to obtain the product. A condensing device is used to ensure that the temperature of the mixed liquid is at 4-8℃. The extrusion instrument extrusion refers to mixing LNP and plant vesicles, and then extruding multiple times through a polycarbonate membrane (such as a 100-200 nm pore size) to recombine the vesicle membrane and wrap the LNP. The PEG-mediated membrane fusion refers to modifying short-chain PEG (such as DSPE-PEG2000) on the surfaces of LNP and vesicles, respectively, to promote membrane fusion through the dehydration effect of PEG. The calcium ion triggering refers to using Ca 2+The neutralization of the negative charge of the vesicle and the LNP membrane phospholipid induces temporary membrane fusion, and the free calcium ions can be removed by EDTA subsequently. The charge reversal method is to mix LNP (positively charged) with negatively charged plant vesicles (such as soybean vesicles) to form a loose complex by electrostatic adsorption, and then to promote the internalization of LNP by vesicles through mild ultrasound or pH adjustment (such as pH 5.0). The biotin-avidin bridging method is to modify biotin and streptavidin on the surface of LNP and vesicles, respectively, to achieve directional wrapping by using high-affinity binding. This method has high wrapping efficiency (> 90%). The low-intensity ultrasound method is to mix LNP with plant vesicles, and then to place the mixture under an ultrasonic probe for ultrasonic treatment under ice bath conditions, using an ultrasonic intensity of 125-250 W for 5 min, with 3 s of ultrasonic treatment and 2 s of rest for each cycle. For the specific schemes and implementation effects of hybridization, for example, in an embodiment of the present application (such as the specific construction method of the tumor mRNA vaccine of the scheme shown in Figure 1 The LNP-wrapped mRNA (i.e., lipid nanoparticles loaded with tumor antigen mRNA) is hybridized with plant-derived nanovesicles to form a structure similar to a "shell-core" structure. The inner layer SM102 of the hybrid particle is used to compress the tumor antigen mRNA, and the outer layer is used to provide a biocompatible barrier and a targeting barrier by using Chinese herbal medicine vesicles. This design uses Chinese herbal medicine vesicles to neutralize the surface charge of LNP, reducing non-specific adsorption; its natural membrane proteins and natural sterol compounds can promote enrichment at the tumor site, while the pH-responsive phospholipid of LNP can trigger mRNA release in the acidic environment of the lysosome of antigen-presenting cells (APCs). This structure combines the efficiency of synthetic materials with the safety of natural carriers, breaking through the bottleneck of existing technologies.

[0053] The tumor mRNA vaccine of the present application constructed based on the above schemes has a dosage form including a liquid preparation, and the final inoculation method includes but is not limited to: nasal instillation, aerosol inhalation, subcutaneous inoculation, intramuscular injection, oral administration, intralymph node injection, intravenous injection after in vitro activation of patient-derived dendritic cells, subcutaneous injection after in vitro activation of patient-derived dendritic cells, etc. Accordingly, the present application also provides a dendritic cell vaccine, which is the bone marrow-derived dendritic cells activated by the tumor mRNA vaccine of the present application.

[0054] Therefore, the tumor mRNA vaccine of the present application can also be used as a main component or a combined component of a tumor treatment drug, and applied to tumor treatment; more specifically, the present application also provides the use of the tumor mRNA vaccine in the preparation of a tumor disease prevention, treatment drug or a drug for inhibiting tumor recurrence or metastasis. In some schemes, the tumor includes melanoma, colorectal tumor, breast tumor, lymphocyte tumor, or bladder tumor, etc.

[0055] In addition, the Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine of the present application can also be used in combination with conventional and immunotherapy, including chemotherapy, radiotherapy, siRNA therapy, PD-1 / PD-L1 immune checkpoint blockade therapy, T cell adoptive transfer therapy, CAR-T therapy, and oncolytic virus therapy, or CRISPR technology or small molecule drugs, etc.

[0056] That is, the present application also provides a tumor treatment composition, which comprises the tumor mRNA vaccine and a tumor treatment drug, wherein the tumor treatment drug comprises a chemotherapeutic drug, a radiotherapeutic drug, an siRNA drug, an immune checkpoint inhibitor, a T cell, a CAR-T cell, or an oncolytic virus, etc.

[0057] Further, the tumor mRNA vaccine of the present application can also be combined with a genetically editable oncolytic virus therapy to establish a universal Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine, and reduce the antigen screening step.

[0058] In addition, as a vaccine, the present application also provides a storage method, which comprises liquid storage at 4°C, lyophilized powder storage, storage by combining a gel matrix, storage by adding a preservative, or storage by adding a co-emulsifier and the like.

[0059] During the process, the activity verification method of the prepared tumor mRNA vaccine comprises, but is not limited to, in vitro bone marrow-derived dendritic cell uptake and activation verification, in vivo lymph node accumulation verification, and in vivo lymph node dendritic cell activation verification, etc.

[0060] Taking ginseng nanovesicles (G-EVLPs) as an example, and taking SM102 as an example of the cationic lipid of LNP, in an embodiment, the establishment of the low-toxicity Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine (SM102@G) mainly comprises three steps:

[0061] The first step is to obtain Chinese herbal medicine nanovesicles by density gradient centrifugation, which is an integrated platform for adjuvant and carrier of tumor antigen antigen delivery.

[0062] The second step is to establish tumor antigen mRNA-loaded cationic lipid particles LNP using microfluidic technology, and establish tumor antigen mRNA-loaded LNP (mRNA@SM102).

[0063] The third step is to hybridize the above-mentioned Chinese herbal medicine nanovesicles and mRNA LNP to establish a Chinese herbal medicine nanovesicle-LNP hybrid tumor mRNA vaccine (SM102@G), which can deliver tumor antigen mRNA, promote the translation of mRNA by antigen-presenting cells, enhance the maturation of dendritic cells and the expression of MHC-I-antigen peptide complexes.

[0064] Compared with the prior art, the present application has obvious innovation. Compared with a single carrier, the present composite system has three innovations: (1) the Chinese herbal medicine nano vesicle reduces the systemic toxicity and non-specific adsorption and targeting of SM102, and reduces the safety risk of SM102; (2) through the surface protein and glycolipid characteristics of the Chinese herbal medicine nano vesicle itself, the purpose of active targeting is achieved, and the delivery efficiency of SM102 in lymph nodes is improved; (3) the Chinese herbal medicine nano vesicle itself has the characteristics of synergistic immune response activation, which can enhance the antigen peptide presentation level of SM102 to dendritic cells. Experiments have proved that the carrier significantly enhances the antigen presentation ability of antigen presenting cells in a mouse model, and significantly reverses the expression level of PD-L1 and the like, and improves the whole blood index and serum biochemical index.

[0065] In the following more specific examples 1-10, the model antigen OVA mRNA is used as the research object to verify the universality of the above platform. The cationic lipid and the Chinese herbal medicine nano vesicle are respectively verified by using SM102 and ginseng nano vesicle.

[0066] Specifically, the above prepared ginseng nano vesicle-LNP hybrid tumor mRNA vaccine (SM102@G) is verified for its effectiveness at the cell level and the animal level respectively, and the therapeutic effect of the tumor vaccine SM102@G is verified based on the established tumor recurrence, tumor progression, in situ tumor and tumor metastasis models. The therapeutic effect is repeatedly verified by establishing tumor models of different sources, and the safety is evaluated.

[0067] The abbreviations possibly involved in the following specific examples are explained and described as follows:

[0068] Control group (Control), complete blank without any experiment;

[0069] SM102 carrier group of model antigen OVA mRNA (OVA mRNA@SM102, mRNA@SM102), only using cationic lipid SM102 to wrap model antigen OVA mRNA for stimulation, that is, only tumor model antigen OVA mRNA stimulation and carrier stimulation;

[0070] Ginseng nano vesicle group (G-EVLPs), only using ginseng nano vesicle for stimulation, that is, only the effect of adjuvant, without tumor model antigen;

[0071] Ginseng nano vesicle-LNP hybrid group (SM102@G), using microfluidic and the like to wrap G-EVLPs outside SM102 to form a "shell-core" structure, that is, tumor model antigen OVA mRNA and immune adjuvant are delivered at the same time. Among them, the immune checkpoint blocker PD-1 is used for treatment synergy.

[0072] Example 1: Isolation and extraction of ginseng nanovesicles, the process is as follows:

[0073] ① Obtain crude fresh ginseng extract: Take fresh ginseng (9-10 months of genuine medicinal materials), wash with double distilled water, add the juice of fresh ginseng extracted by the wall-breaking machine, and centrifuge at 200 rpm for 15 min using a high-speed centrifuge; centrifuge at 2000 rpm for 30 min; then centrifuge at 10000 rpm for 60 min, repeat this step 3-5 times.

[0074] ② Extraction of fresh ginseng nanovesicles: collect the supernatant after centrifugation, centrifuge at 100000 rpm for 60 min using an ultracentrifuge, collect the precipitate, add 1xPBS solution, and mix gently using an injection. Determine the concentration of fresh ginseng nanovesicles using a BCA protein quantification kit. Store the obtained fresh ginseng nanovesicles at -80°C for long-term preservation.

[0075] Example 2: Establishment of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine (as shown in Figure 1 ), the specific process is as follows:

[0076] ① Preparation of mRNA@SM102: SM102, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at a ratio of 50:10:38.5:1.5 to form an alcohol phase. mRNA was dissolved in a pH 4.0 sodium citrate buffer to form an aqueous phase. Under the conditions of a nitrogen-phosphorus ratio (molar ratio of nitrogen in SM102 to nucleic acid in mRNA) of 6 and an alcohol phase / aqueous phase volume ratio of 3, mRNA@SM102 was prepared on a microfluidic device to obtain a primary emulsion. The primary emulsion was dialyzed for 2 h and concentrated to the desired concentration by ultrafiltration tube.

[0077] ② Preparation of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine: Mix the above G-EVLPs and mRNA@SM102 according to the concentration (G-EVLPs protein amount:mRNA mass=40:1 ratio), and use an extruder / microfluidic device to prepare SM102@G.

[0078] Example 3: Characterization of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine, the specific process is as follows:

[0079] ① Particle size distribution characterization: Collect the above mRNA@SM102 and SM102@G solutions, and use dynamic light scattering and nanoparticle size determination instrument to characterize the particle size distribution of the above two solutions (Zetasizer Nano ZS, Malvern Instruments, UK) and (Nanotrac UPAs, Microtrac, USA). Figure 2 Figure 3 ).

[0080] ​② Component characterization: SDS-PAGE, proteomics and non-target lipidomics techniques were used to characterize the G-EVLPs and SM102@G solutions obtained above Figure 4 ).

[0081] ③ Particle morphology characterization: The G-EVLPs, mRNA@SM102, and SM102@G solutions were collected, and transmission electron microscopy was used to characterize the particle morphology of the three solutions Figure 5 ).

[0082] As shown in the figure, the average particle size of SM102@G is about 220.0 nm, and the zeta potential is less than 0 mV. The SM102@G protein electrophoresis strip has obvious characteristics, and the SDS-PAGE banding is similar to that of ginseng nanocapsule G-EVLPs. G-EVLPs contain various lipids, proteins, nucleic acids, and organic acid components, and have a lipid bilayer structure, which is a significant exosome feature.

[0083] Example 4: Fresh ginseng nanocapsule-LNP hybrid tumor mRNA vaccine in vitro effect on dendritic cell uptake and maturation, the specific process is as follows:

[0084] ① Activation of SM102@G on bone marrow-derived dendritic cells (BMDCs): Extract, induce and culture BMDCs, and co-incubate the above G-EVLPs, mRNA@SM102 and SM102@G with BMDCs for 24 hours (mRNA at a dose of 1 μg / mL, G-EVLPs at a dose of 10 μg / mL), and analyze the levels of CD80 and CD86 activation markers of BMDCs using flow cytometry Figure 6 ).

[0085] ② Uptake of SM102@G by BMDCs: Extract, induce and culture BMDCs, and use ICG to encapsulate SM102 and DiD to label G-EVLPs. Co-incubate the above fluorescently labeled G-EVLPs, mRNA@SM102 and SM102@G with BMDCs for 24 hours (mRNA at a dose of 1 μg / mL, G-EVLPs at a dose of 10 μg / mL), and analyze the uptake of G-EVLPs and mRNA@SM102 by BMDCs using flow cytometry Figure 7 ).

[0086] ③ Specific anti-tumor immune response of SM102@G: Extract BMDCs, co-incubate SM102@G with BMDCs, and obtain T lymphocytes from the spleen above the BMDCs. Analyze CD45 + CD3 + CD8+ The proportion of T cells; cells in the supernatant were collected and added to a 24-well plate coated with tumor cells (B16F10-OVA), and the content of LDH in the supernatant (a marker of tumor cell death) was determined using a kit, and the release level of IFN-γ in the supernatant was determined using an ELISA kit, which was the activation level of specific anti-tumor immune response.

[0087] As shown in Figure 6 , Figure 7 , SM102@G can significantly enhance the maturation level of BMDCs, and the expression amount of surface CD80 and CD86 molecular markers is significantly increased. At the same time, SM102@G hybridized with G-EVLPs can significantly improve the phagocytosis level of BMDCs for SM102 (and the mRNA inside).

[0088] Example 5: Evaluation of the in vivo specific anti-tumor immune response activation of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine, using model antigen OVA mRNA as an example, the specific determination process is as follows:

[0089] ① Lymph node targeting of SM102@G vaccine: using fluorescent molecule DiD to label G-EVLPs, and using ICG to encapsulate mRNA@SM102, a fluorescently labeled SM102@G vaccine was prepared, and the above obtained mRNA vaccine was subcutaneously inoculated, and small animal imaging was used to analyze the targeting situation, and the fluorescence intensity of each tissue organ (especially the lymph node) was counted. Lymph node single cells were isolated, and flow cytometry was used to analyze the phagocytosis efficiency of CD11c + cells for the above, that is, the lymph node targeting efficiency of SM102@G.

[0090] ② SM102@G vaccine forms immune memory: subcutaneously injecting SM102@G vaccine, and then removing the spleen after vaccination and preparing a single cell suspension. Flow cytometry was used to analyze the proportion of CD45 + CD3 + CD8 + CD44 high CD62L low memory T cells, that is, the formation strength of immune memory.

[0091] The results show that SM102@G can significantly improve the targeting of mRNA to the lymph node and DC cells in the lymph node, and enhance the generation of central memory T cells in the spleen.

[0092] Example 6: Evaluation of the in vivo anti-tumor recurrence efficacy of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine, using model antigen OVA mRNA as an example, the specific determination process is as follows:

[0093] ①Establishment of melanoma recurrence model: 3x10 5 Figure 8 ).

[0094] ②Evaluation of long-term immune protection: The above mice were completely resected at 24 days, and 3x10 5 Figure 8 ) at 30 days. The mice were euthanized at 40 days, and the serum inflammatory cytokine levels were determined.

[0095] The results are shown in Figure 8 , tumor volume data show that SM102@G vaccine can significantly inhibit the recurrence of B16F10-OVA tumor and form long-term anti-tumor protection. At the same time, the complete response rate (complete inhibition rate) of B16F10-OVA tumor after SM102@G vaccination can be increased to 80%, effectively prolonging the survival of mice, and the survival rate is about 100%. There was no significant change in body weight before and after injection of vaccine and inoculation of tumor, indicating that SM102@G has high biocompatibility. As shown in Figure 9 , SM102@G vaccine can significantly increase the level of pro-inflammatory cytokines in serum after tumor inoculation.

[0096] Example 7: Fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine in vivo efficacy study for inhibiting acute tumor metastasis, using model antigen OVA mRNA as an example, the specific determination process is as follows:

[0097] Establishment of melanoma B16F10-OVA tumor acute metastasis model: subcutaneous inoculation of vaccine (G-EVLPs, mRNA@SM102 and SM102@G) on days 1, 4 and 8, 5 days later, tumor cells (B16F10-OVA, 1x10 5

[0098] Example 8: Fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine in vivo efficacy study for treating tumors, using model antigen OVA mRNA as an example, the specific determination process is as follows: ​​​

[0099] Establishment of melanoma progression model: First, subcutaneously administer OVA-expressing melanoma B16F10 cells 3x10 5 individuals, subcutaneously inoculate the vaccine at 1 day, 4 days, and 8 days after tumor inoculation, interval administration of immune checkpoint blocker PD-1, and then continuously detect mouse body weight, tumor volume, tumor weight, lymph node mass, survival rate, intratumoral PD-L1 expression, and intratumoral CD8 + T cell infiltration.

[0100] The results, as shown in Figures 9-16 , tumor data show that SM102@G vaccine + PD-1 can significantly inhibit the progression of B16F10-OVA tumors and form long-term anti-tumor protection. At the same time, the partial response rate (tumor regression and final volume less than 100 mm 3 ) of B16F10-OVA after SM102@G vaccine + PD-1 inoculation can be increased to 100%, effectively prolonging the survival of mice, and the survival rate of mice in the SM102@G vaccine + PD-1 group is about 100%. The body weight of mice did not change significantly before and after injection of the vaccine and inoculation of the tumor, indicating that SM102@G combined with PD-1 has high biocompatibility, but can enhance the vaccine activity of SM012 in the lymph nodes, increase the volume and mass of the lymph nodes. SM102@G can significantly enhance the level of intratumoral CD8+ T cell infiltration. More importantly, G-EVLPs can significantly reverse the high expression of intratumoral PD-L1 caused by mRNA@SM102 injection, reduce the immunosuppressive microenvironment, and enhance the effect of immunotherapy. As shown in Figure 17 , SM102@G vaccine + PD-1 can significantly increase the level of pro-inflammatory cytokines in serum after tumor inoculation

[0101] Example 9: Pharmacodynamic study of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine and oncolytic virus combination, using model antigen OVA mRNA as an example, the specific determination process is as follows:

[0102] ① Pharmacodynamic evaluation of mixed tumor cells: mix B16F10 and B16F10-OVA cell lines in proportions of 10:0, 9:1, 7:3, 5:5, and 0:10. Subcutaneously administer the mixed melanoma B16F10 cells 3x10 5 individuals, subcutaneously inoculate the vaccine at 1 day, 4 days, and 8 days after tumor inoculation, interval administration of immune checkpoint blocker PD-1, and then continuously detect mouse body weight, tumor volume, survival rate.

[0103] ② Oncolytic virus pharmacodynamics and OVA editing activity: establish an oncolytic virus strain with OVA editing activity. Subcutaneously administer the mixed melanoma B16F10 cells 3x105 Mice were inoculated with the aforementioned oncolytic virus intratumorally 10 and 20 days after tumor inoculation. Body weight, tumor volume, survival rate, and OVA-positive expression rate in B16F10 cells were continuously monitored.

[0104] ③ Efficacy of combined treatment with SM102@G and oncolytic virus: 3×10-10 melanoma B16F10 cells were administered subcutaneously as described above. 5 Mice were injected intratumorally with oncolytic virus 5 and 10 days after tumor inoculation. SM102@G vaccine was subcutaneously administered 3, 8, and 15 days after tumor inoculation, with the immune checkpoint inhibitor PD-1 administered at intervals. Subsequently, mouse body weight, tumor volume, and survival rate were continuously monitored.

[0105] The results showed that SM102@G could significantly inhibit the volume of mixed tumors in mice; oncolytic viruses could inhibit tumor volume while promoting the expression of heterologous antigens not encoded by the tumor itself; SM102 combined with oncolytic viruses could serve as a universal tumor vaccine strategy to inhibit or reverse tumor growth.

[0106] Example 10: In vivo biocompatibility study of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine, using OVA mRNA as a model antigen, the specific measurement process is as follows:

[0107] ① Pathological examination of major organs after administration of SM102@G vaccine: The heart, liver, spleen, lungs, kidneys, brain and other major organs of the mice after vaccination were taken and subjected to pathological (H&E) staining.

[0108] ② Detection of blood and major serum biochemical indicators after administration of SM102@G vaccine: Blood was collected from mice after vaccination. A portion of the blood was directly measured for blood cell indicators. The other portion was coagulated at room temperature for 30 min, centrifuged at 2500 rpm for 5 min, and the resulting serum was then used to detect biochemical indicators (AST, ALT, BUN, CREA, ALP and γ-GT).

[0109] The results are as follows Figure 18 , Figure 19 As shown, the SM102@G vaccine did not cause significant morphological damage to major tissues and organs after administration, demonstrating good biocompatibility. The results are as follows... Figure 18 , Figure 19 As shown, the serum biochemical indicators such as AST, ALT, BUN, CREA, ALP and γ-GT did not change significantly after administration of SM102@G vaccine, indicating that SM102@G vaccine has good biocompatibility and safety.

Claims

1. A tumor mRNA vaccine hybridized with LNP and Chinese herbal vesicle, characterized in that, The tumor mRNA vaccine is prepared by hybridizing Chinese herbal medicine nanovesicles and lipid nanoparticles loaded with tumor antigen mRNA. The Chinese herbal medicine nanovesicles are ginseng nanovesicles; and the structure of the vaccine is a shell-core structure formed by ginseng nanovesicles wrapped outside the LNP. The preparation method of the lipid nanoparticles loaded with tumor antigen mRNA comprises the following steps: dissolving SM102, DSPC, cholesterol and DMG-PEG2000 in alcohol in a certain proportion to form an alcohol phase; dissolving tumor antigen mRNA in an acid buffer to form an aqueous phase; mixing the alcohol phase and the aqueous phase to obtain a preliminary emulsion; and dialyzing and concentrating the preliminary emulsion to obtain the lipid nanoparticles loaded with tumor antigen mRNA.

2. The tumor mRNA vaccine of claim 1, wherein, The tumor antigen comprises one or more of a combination of model antigens, universal tumor antigens, tumor neoantigens, tumor-associated antigens or tumor fusion antigens.

3. The tumor mRNA vaccine of claim 1, wherein, The mass ratio of SM102:DSPC:cholesterol:DMG-PEG2000 is 50:10:38.5:1.

5.

4. The tumor mRNA vaccine of claim 3, wherein, The mixing of the alcohol phase and the aqueous phase comprises mixing by microfluidization under the conditions that the molar ratio of nitrogen in SM102 to nucleic acid in tumor antigen mRNA is 6 and the volume ratio of the alcohol phase to the aqueous phase is 3.

5. The tumor mRNA vaccine of claim 4, wherein, The hybridization comprises: mixing and assembling the ginseng nanovesicles and the lipid nanoparticles loaded with tumor antigen mRNA by extrusion instrument extrusion method or microfluidization method at a ratio of ginseng nanovesicle protein mass:tumor antigen mRNA mass=40:1 to obtain the tumor mRNA vaccine.

6. The tumor mRNA vaccine according to any one of claims 1 to 5, characterized in that, The administration method of the tumor mRNA vaccine comprises intranasal instillation, atomization inhalation, subcutaneous inoculation, intramuscular injection, oral administration, intralymph node injection; and further comprises injecting the activated dendritic cells into the lymph node, vein or subcutaneously after the tumor vaccine activates the patient's bone marrow-derived dendritic cells in vitro.

7. The tumor mRNA vaccine of any one of claims 1-5 in the preparation of a melanoma prevention, treatment drug or a drug for inhibiting melanoma recurrence or metastasis; the tumor antigen of the tumor mRNA vaccine is a model antigen OVA.

8. A dendritic cell vaccine, characterized in that, The bone marrow-derived dendritic cells activated by the tumor mRNA vaccine of any one of claims 1-5.

9. A tumor therapeutic composition, characterized by, The tumor mRNA vaccine of any one of claims 1-5 and a tumor treatment drug, wherein the tumor treatment drug comprises a chemotherapy drug, a radiotherapy drug, an siRNA, an immune checkpoint blocker, a T cell or an oncolytic virus.

10. The neoplasm treatment composition according to claim 9, characterized by The T cell comprises a CAR-T cell.

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