A spleen-targeted mRNA vaccine doped with metal ion adjuvant and a preparation method and application thereof

Abstract

The application discloses a kind of metal ion adjuvant-doped spleen-targeted mRNA vaccine.The mRNA vaccine includes mRNA-encapsulated spleen-targeted lipid nanoparticles and metal ion adjuvant;The mRNA-encapsulated spleen-targeted lipid nanoparticles include spleen-targeted lipid nanoparticles and mRNA;The raw material of the spleen-targeted lipid nanoparticles includes ionizable lipid, auxiliary phospholipid, cholesterol, polyethylene glycol-lipid conjugate and targeting functional lipid (anionic lipid).The application introduces anionic lipid as the fifth component in traditional LNP components, effectively realizes spleen lymphoid organ targeting while using its electrostatic adsorption with adjuvant Mn 2+ Between more efficient encapsulation of adjuvant Mn 2+ , to achieve simultaneous delivery of antigen and adjuvant to antigen-presenting cells in the spleen, improve Mn 2+ Activate the immune efficiency of a specific site, enhance the effectiveness of mRNA vaccine and reduce "off-target" toxicity, and thus achieve the purpose of tumor treatment and prevention.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a spleen-targeting mRNA vaccine doped with metal ion adjuvants, its preparation method, and its application in tumor immunotherapy. Background Technology

[0002] The success of mRNA vaccines in combating SARS-CoV-2 highlights the value of this technology in responding to emerging infectious diseases. In recent years, the development of mRNA anti-tumor vaccines has also attracted increasing attention. Due to their advantages such as short development cycles and the ability to be personalized, numerous preclinical and clinical trials have been conducted, demonstrating positive efficacy against melanoma, non-small cell lung cancer, and colorectal cancer, bringing significant clinical benefits. Nevertheless, there is still considerable room for improvement in the effectiveness of mRNA tumor vaccines. The key points are: 1) how to accurately deliver mRNA to antigen-presenting cells to activate specific immune responses; and 2) how to improve antigen presentation efficiency and immune activation capacity.

[0003] Because mRNA is easily degraded and has difficulty freely crossing cell membranes, developing suitable delivery carriers is crucial. Lipid nanoparticles (LNPs), as a safe and effective delivery carrier, have been successfully applied in the development of mRNA vaccines, most notably the FDA-approved COVID-19 mRNA-LNP vaccine. Furthermore, several anti-tumor mRNA vaccines have shown promising clinical results. Therapeutic anti-tumor mRNA vaccines deliver mRNA containing encoding antigen proteins to antigen-presenting cells, where it is translated to form the corresponding antigen proteins, thereby inducing a specific T-cell response and killing tumor cells. Therefore, targeting immune organs and antigen-presenting cells is an effective way to improve the efficacy of mRNA vaccines. Patent CN112996519A discloses a composition and method for organ-specific delivery of nucleic acids, which, by adding selective organ-targeting lipids to conventional LNP formulations, enables the delivery of mRNA to various extrahepatic organs and tissues. The spleen is the largest peripheral immune organ in the body, and its antigen-presenting cells are adjacent to B cells and T cells, providing an ideal microenvironment for efficiently initiating humoral and cellular immunity. Therefore, the development of spleen-targeted mRNA-LNP vaccines is expected to enhance antigen presentation by antigen-presenting cells and T-cell immune activation, thereby enhancing the anti-tumor immune activity of mRNA-LNP vaccines.

[0004] Unlike preventative vaccines for infectious diseases, therapeutic anti-tumor mRNA vaccines require a stronger immune response to achieve optimal therapeutic effects, often necessitating higher doses and multiple administrations, thus increasing potential safety risks and the risk of immune tolerance. The combined use of metal adjuvants holds promise for addressing the adverse reactions resulting from insufficient immune activation. Numerous studies have demonstrated that activating the interferon gene-stimulating factor (STING) signaling pathway can induce type I interferon (IFN) secretion, thereby enhancing antigen presentation and anti-tumor immunity. In the cGAS-STING signaling pathway, the DNA receptor cGAS recognizes abnormally exposed dsDNA in the cytoplasm and catalyzes the formation of the secondary messenger 2'3'-cyclic-GMP-AMP (cGAMP) from GTP and ATP. cGAMP then binds to the STING homodimer receptor protein located in the endoplasmic reticulum membrane, initiating a conformational change in the STING dimer and causing activation. Recent studies have shown that divalent manganese ions (Mn)... 2+ Mn can directly activate cGAS, significantly enhance the binding affinity of cGAMP and STING, and induce massive secretion of type I IFN, thereby improving the body's antiviral infection and immunomodulatory effects. Therefore, it is necessary to design a method that can effectively encapsulate the adjuvant Mn. 2+ And mRNA-LNP vaccines that deliver Mn to immune cells, improving the immunogenicity of mRNA-LNP vaccines and reducing the side effects of synergistic adjuvants. Currently, therapeutic lymphoid organ-targeted mRNA-LNP vaccines deliver a total of Mn... 2+ Research on adjuvants for tumor immunotherapy has not yet been developed. Summary of the Invention

[0005] To address the problems existing in the prior art and to improve the efficacy of anti-tumor mRNA vaccines while significantly reducing the toxic side effects caused by non-specific delivery and high-dose administration, this invention adopts an "All-in-One" strategy to construct a metal-doped adjuvant Mn 2+ A spleen-targeting mRNA-LNP vaccine. This nanosystem introduces targeted functional lipids into traditional LNPs and co-delivers Mn. 2+ Adjuvants and mRNA promote the interaction of antigens and Mn. 2+ The adjuvant is simultaneously delivered to antigen-presenting cells in the spleen, which helps to increase Mn 2+ It can activate immunity at specific sites with good biosafety, promote the maturation of antigen-presenting cells, and induce antigen-specific immune responses, thereby improving the effectiveness of mRNA vaccines to achieve the purpose of tumor treatment and prevention.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides spleen-targeting lipid nanoparticles loaded with mRNA.

[0008] The spleen-targeting lipid nanoparticles loaded with mRNA provided by the present invention include spleen-targeting lipid nanoparticles and mRNA; the raw materials of the spleen-targeting lipid nanoparticles include ionizable lipids, cofactor phospholipids, steroidal lipids, polyethylene glycol-lipid conjugates, and targeted functional lipids.

[0009] The targeted functional lipid is anionic lipid, which is selected from phospholipids containing at least one of the following anionic heads: phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidic acid (PA).

[0010] Furthermore, the phospholipid containing the phosphatidylglycerol (PG) anionic head is preferably one or more of the following: DMPG (1,2-dimyristoyl-sn-glycerol-3-phosphate-RAC-(1-glycerol) sodium salt), DOPG (1,2-dioleoyl-sn-glycerol-3-phosphate-RAC-glycerol sodium salt), DSPG (1,2-distearyl-tin-glycerol-3-phosphate-rac-(1-glycerol) sodium salt), DLPG (1,2-dilauroyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) sodium salt), DPPG (1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) sodium salt), POPG (1-palmitoyl-2-oleenoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt), etc.; preferably DOPG.

[0011] Furthermore, the phospholipid containing the phosphatidylserine (PS) anionic head is preferably one or more of the following: DOPS (1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine sodium salt), DSPS (1,2-distearate-sn-glycerol-3-phosphate-L-serine sodium salt), 16:0-18:2PS (1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine sodium salt), 18:0-18:1PS (1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine sodium salt), 18:0-18:2PS (1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine sodium salt), and POPS (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine sodium salt); preferably DOPS.

[0012] Furthermore, the phospholipid containing the phosphatidic acid (PA) anionic head is selected from at least one of the following: 17:0PA (sodium salt of 1,2-heptadecanoyl-sn-glycerol-3-phosphate), 18PA (sodium salt of 1,2-dioleoyl-sn-glycerol-3-phosphate), DMPA (sodium salt of 1,2-dimyristoyl-sn-glycerol-3-phosphate), DLPA (sodium salt of 1,2-dilauroyl-sn-glycerol-3-phosphate), DSPA (sodium salt of 1,2-distearate-sn-glycerol-3-phosphate), POPA (sodium salt of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate), etc.; preferably 18PA.

[0013] Preferably, the anionic lipid accounts for 1-50% of the molar percentage of the raw material (total lipids), more preferably 5-30%, further preferably 10-20%, and even more preferably 15%.

[0014] Preferably, the ionizable lipids include, but are not limited to: SM-102 (heptadecane-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)aminooctanoate)), ALC-0315 (((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), DLi n-MC3-DMA (4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester), DLin-KC2-DMA (N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecen-1-yl-1,3-dioxolane-4-ethylamine) and 306Oi10 (tetra(8-methylnonyl)3,3',3",3"'-(((methylazadialkyl)bis(propane-3,1diyl))bis(azatriyl))tetrapropionate);

[0015] Preferably, the auxiliary phospholipids include, but are not limited to: DSPC (1,2-distearyl-sn-glycero-3-phosphatidylcholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), POPE (1-palmitoyl-2-oleoyl-phosphatidylethanolamine), DSPE (1,2-distearyl-sn-glycero-3-phosphatidylethanolamine), and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine).

[0016] Preferably, the steroidal lipids include, but are not limited to: cholesterol, cholesterolanol, 7α-hydroxycholesterol, β-sitosterol, 7β-hydroxycholesterol, cholesterolanone, and cholesterolenone;

[0017] Preferably, the polyethylene glycol-lipid conjugates include, but are not limited to: DMG-PEG2000 (1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000), DSG-PEG2000 (1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol 2000), DSPE-mPEG2000 (distearylphosphatidylacetamide-methoxy polyethylene glycol 2000), and ALC-0159 (methoxy polyethylene glycol bistetradecylacetamide);

[0018] Preferably, the ionizable lipids account for 20-60% of the total lipids in the raw material, more preferably 30-50%, and even more preferably 39.35%.

[0019] Preferably, the auxiliary phospholipid accounts for 1-20% of the molar percentage of the raw material (total lipids), more preferably 2-10%, and even more preferably 7.99%.

[0020] Preferably, the steroidal lipids account for 20-60% of the molar percentage of the raw material (total lipids), more preferably 30-50%, and even more preferably 36.29%.

[0021] Preferably, the molar percentage of the polyethylene glycol-lipid conjugate is 0.1-10%, more preferably 0.5-5%, and even more preferably 1.36%.

[0022] Preferably, the ratio of the total mass of lipid molecules to the mass of nucleic acid in the spleen-targeting lipid nanoparticles loaded with mRNA is (10-50):1, specifically 40:1.

[0023] Preferably, the spleen-targeting lipid nanoparticles carrying mRNA have a particle size of 140–200 nm and a polydispersity index of 0.1–0.2.

[0024] Preferably, the spleen-targeting LNPs that best activate the immune response include any one of 18PALNP, DOPG LNP, and DOPS LNP, and more preferably DOPS LNP.

[0025] In a second aspect, the present invention provides a method for preparing spleen-targeting lipid nanoparticles loaded with mRNA as described in the first aspect.

[0026] The method for preparing the spleen-targeting lipid nanoparticles loaded with mRNA includes the following steps:

[0027] Step (A1): Ionizable cationic lipids, cofactor phospholipids, cholesterol, polyethylene glycol-lipid conjugates, and targeted functional lipids are dissolved in an organic solvent at a specific molar percentage to obtain a lipid organic phase;

[0028] Step (A2): Dissolve the mRNA in an appropriate buffer solution to obtain an mRNA solution;

[0029] Step (A3): Mix the lipid organic phase from step (A1) and the mRNA solution from step (A2) and incubate to obtain a solution of spleen-targeting lipid nanoparticles loaded with mRNA.

[0030] Preferably, the organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, tetrahydrofuran, and diethyl ether.

[0031] Preferably, the pH value of the buffer solution is 2.0-8.0, more preferably 4.0. Further, the buffer solution is a citrate buffer or an acetate buffer, more preferably a citrate buffer.

[0032] Preferably, the mass ratio of the total mass of lipid molecules in the lipid organic phase to the mass of mRNA in the mRNA solution is (10-50):1, and more preferably, the mass ratio is 40:1.

[0033] Preferably, the volume ratio of the lipid organic phase to the mRNA solution is 1:(1-5), and more preferably, the volume ratio is 1:3.

[0034] Preferably, the incubation conditions are static incubation at room temperature for 10-15 minutes.

[0035] The method further includes a step of dialysis of the spleen-targeting lipid nanoparticle solution containing mRNA obtained in step (A3).

[0036] The dialysis was performed in Pur-A-Lyzer Maxi 3500 dialysis tubes and with a large volume of 0.01M PBS for at least 2 hours to remove ethanol.

[0037] This invention optimizes the molar ratio of anionic lipids in LNPs to achieve higher spleen-targeting efficiency. The spleen-targeting LNPs involved in this invention can be effectively taken up by antigen-presenting cells (APCs) both in vitro and in vivo, thereby activating strong humoral immunity and antigen-specific T-cell responses, and enhancing the delivery efficiency and efficacy of mRNA vaccines.

[0038] Thirdly, the present invention provides spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions.

[0039] The spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions provided by the present invention include the spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions as described in the first aspect of the present invention; the metal ions include, but are not limited to, one or more of manganese ions, copper ions, zinc ions, aluminum ions, iron ions, ferrous ions and cobalt ions, preferably manganese ions.

[0040] When the adjuvant metal ion is an adjuvant metal ion, the anionic lipids in the spleen-targeting lipid nanoparticles carrying mRNA react with the adjuvant metal ion (such as Mn). 2+ The mass ratio of the two components is 1:(0-10), more preferably 1:(1-5), and most preferably 1:1.

[0041] Preferably, the adjuvant metal ion is a divalent manganese salt, including any one of manganese chloride, manganese nitrate, or manganese sulfate, and preferably manganese chloride.

[0042] Fourthly, the present invention provides a method for preparing spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions as described in the third aspect of the present invention.

[0043] The method for preparing spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions according to the present invention includes the following steps:

[0044] Step (B1): Ionizable cationic lipids, cofactor phospholipids, cholesterol, polyethylene glycol-lipid conjugates, and targeted functional lipids are dissolved in an organic solvent at a specific molar percentage to obtain a lipid organic phase;

[0045] Step (B2): Dissolve the adjuvant metal ions and mRNA in an appropriate buffer solution to obtain an aqueous solution;

[0046] Step (B3): The lipid organic phase from step (B1) and the aqueous phase solution from step (B2) are mixed and incubated to obtain a solution of spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions.

[0047] Preferably, the organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, tetrahydrofuran, and diethyl ether.

[0048] Preferably, the pH value of the buffer solution is 2.0-8.0, more preferably 4.0. Further, the buffer solution is a citrate buffer or an acetate buffer, more preferably a citrate buffer.

[0049] Preferably, the mass ratio of anionic lipids to adjuvant metal ions in the lipid organic phase is 1:(0-10), more preferably 1:(1-5), and most preferably 1:1.

[0050] Preferably, the mass ratio of the total mass of lipid molecules in the lipid organic phase to the mass of mRNA in the mRNA solution is (10-50):1, and more preferably, the mass ratio is 40:1.

[0051] Preferably, the volume ratio of the lipid organic phase to the mRNA solution is 1:(1-5), and more preferably, the volume ratio is 1:3.

[0052] Preferably, the incubation conditions are static incubation at room temperature for 10-15 minutes.

[0053] The method further includes a step of dialysis of the spleen-targeting lipid nanoparticle solution containing mRNA and adjuvant metal ions obtained in step (B3).

[0054] The dialysis was performed in Pur-A-Lyzer Maxi 3500 dialysis tubes, with a large volume of 0.01M PBS used for at least 2 hours to remove unencapsulated adjuvant Mn. 2+ .

[0055] This invention utilizes anionic lipids and adjuvant metal ions (such as Mn) 2+ Electrostatic adsorption between the two enables LNPs to efficiently encapsulate adjuvant metal ions. Combined with the method described in the second aspect, the anionic lipid is preferably DOPS, which encapsulates the adjuvant Mn. 2+ The mRNA encoding the tumor antigen was dissolved in citrate buffer, then rapidly mixed into the lipid solution. The solution was incubated at room temperature for 10–15 min, and the resulting solution was dialyzed for at least 2 hours to remove unencapsulated adjuvant Mn. 2+ Ultimately, mRNA and the metal ion adjuvant Mn were co-encapsulated. 2+ DOPS LNP, when the adjuvant metal ion is Mn 2+ It is abbreviated as Mn@mRNA-LNP.

[0056] In a preferred embodiment of the present invention, the anionic lipid DOPS and the adjuvant Mn 2+ The mass ratio of Mn@mRNA-LNPs was 1:(0-10). Under this ratio, LNPs achieved high encapsulation efficiency, uniform dispersion, and improved mRNA transfection efficiency. The optimized Mn@mRNA-LNP enhanced the cGAS-STING pathway in antigen-presenting cells, promoted the activation of antigen-presenting cells, and further triggered a stronger OVA-specific CTL response than DOPS LNPs.

[0057] Fifthly, the present invention provides the application of spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions in the preparation of vaccines for tumor immunotherapy.

[0058] The tumors include, but are not limited to, one or more of the following: melanoma, colon cancer, liver cancer, breast cancer, glioma, ovarian cancer, stomach cancer, prostate cancer, and lung cancer.

[0059] In this invention, Mn is doped 2+ The adjuvanted spleen-targeting mRNA-LNP vaccine effectively inhibited the progression of established and postoperative B16-OVA tumors and prolonged the overall survival of mice after vaccination.

[0060] In a sixth aspect, the present invention provides a pharmaceutical composition for treating tumors.

[0061] The pharmaceutical composition for treating tumors provided by this invention includes the mRNA-encapsulated component and adjuvant metal ions (such as Mn) as described in the third aspect of this invention. 2+ Spleen-targeting lipid nanoparticles and immune checkpoint inhibitors.

[0062] Preferably, the immune checkpoint inhibitors include, but are not limited to, PD-1 antibodies, PD-L1 antibodies, or CTLA-4 antibodies.

[0063] In a seventh aspect, the present invention also provides a method for combined anticancer treatment with a spleen-targeted mRNA-LNP vaccine based on a metal ion adjuvant.

[0064] The method includes administering the pharmaceutical composition for treating tumors described in the sixth aspect above to a subject in need.

[0065] In this invention, the mRNA is any mRNA molecule that can encode one or more tumor antigens. Examples show that the tumor antigen mRNA can encode seven MC38 neoantigens, which have been confirmed in previous studies to be effectively presented by MHC I and induce CD8+ T cell responses.

[0066] In this invention, by doping Mn 2+ An adjuvanted spleen-targeted mRNA-LNP vaccine was prepared to deliver mRNA encoding the MC38 neoantigen. The neoantigen mRNA-LNP vaccine effectively alleviated the progression of MC38 tumors. Notably, the combination with anti-PD-1 antibodies further increased the percentage of complete tumor regression and prolonged overall survival in mice.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] (1) The preparation method of the present invention is simple, and the required equipment is all conventional equipment, which can realize large-scale production and has good prospects for clinical translation.

[0069] (2) This invention uses lipid nanoparticles as mRNA delivery carriers, which have good safety and stability. By introducing anionic lipids DOPS, this invention can achieve targeted delivery to the spleen without excessive modification of the lipid nanoparticles, and can deliver the vaccine to antigen-presenting cells in the spleen, significantly improving the bioavailability of the mRNA vaccine. Moreover, DOPS LNPs can simultaneously induce Th1 and Th2 responses, which helps to promote a strong cytotoxic T lymphocyte response and humoral immune response.

[0070] (3) This invention utilizes DOPS LNPs with spleen-targeting and immune-activating capabilities to co-deliver manganese adjuvant and mRNA encoding tumor antigens. Efficient manganese ion loading is achieved through electrostatic adsorption with anionic lipid DOPSs. The optimized Mn@mRNA-LNP effectively encapsulates Mn... 2+ The adjuvant was delivered to antigen-presenting cells and promoted the expression of mRNA encoding anti-tumor antigens, enabling more efficient activation of the STING signaling pathway at lower doses. It upregulated the expression of IRF-7 and IFN-β in a short time, promoted the activation of DC cells and induced the production of cytotoxic T cells, and ultimately effectively inhibited the progression of established and postoperative B16-OVA tumors.

[0071] (4) This invention utilizes spleen-targeting LNP to simultaneously encapsulate tumor antigen mRNA and Mn 2+ The Mn@mRNA-LNP vaccine prepared using the (All-in-One) method showed better anti-tumor therapeutic effects in various mouse models, and the formulation could induce the production of long-term memory T cells, which helps prevent tumor recurrence.

[0072] (5) In this invention, a spleen-targeting mRNA tumor vaccine based on manganese ions is combined with anti-PD-1 for anti-tumor treatment. Although monotherapy with anti-PD-1 or Mn@Neo-mRNA-LNP can effectively alleviate tumor progression, the complete tumor regression rate is 0.0%. However, the combined treatment group of Mn@Neo-mRNA-LNP and anti-PD-1 antibody increased the percentage of complete tumor regression to 25.0%, significantly inhibited tumor progression, and prolonged the overall survival of mice. Attached Figure Description

[0073] Figure 1 This is a schematic diagram of the present invention, "A preparation technology and application of a spleen-targeting mRNA vaccine doped with metal ion adjuvants".

[0074] Figure 2 To characterize the physicochemical properties of the spleen-targeting LNP prepared in Example 2. Figure 2 A to Figure 2Figure C shows the results of evaluating the particle size, polydispersity index, and particle size changes over seven days in PBS for LNPs containing different proportions of 18PA / DOPG / DOPS.

[0075] Figure 3 Fluorescence images of major organs collected 6 hours after delivery of 0.1 mg / kg LucmRNA containing 10%, 15%, and 20% 18PA / DOPG / DOPS LNP in Example 3.

[0076] Figure 4 The results show the statistical results of fluorescence intensity of the spleen in Example 3.

[0077] Figure 5 This is a statistical graph showing the spleen targeting efficiency of 10%, 15%, and 20% 18PA / DOPG / DOPS LNPs, based on the fluorescence intensity of the liver, spleen, and lung organs, as described in Example 3.

[0078] Figure 6 This shows the transfection status of dendritic cells after co-incubation with 18PA / DOPG / DOPS LNP for 24 hours in Example 4.

[0079] Figure 7 This refers to the activation and antigen presentation of dendritic cells after the 18PA / DOPG / DOPS LNPs acted on them in Example 5. Figure 7 A is a flow cytometry atlas showing the expression of CD86 and SIINFEKL-H-2Kb in dendritic cells. Figure 7 B and 7C are the results of quantitative analysis by flow cytometry.

[0080] Figure 8 In Example 6, gene editing of spleen tissue was achieved in Ai9 mice by delivering Cre mRNA via 18PA / DOPG / DOPS LNP. Figure 8 A is a schematic diagram illustrating how the delivery of Cre mRNA formulation activated tdTom expression in Ai9 mice. Figure 8 B represents Ai9 mice that underwent tail vein injection of 18PA / DOPG / DOPS LNP (0.5 mg / kg Cre mRNA) for 48 hours, followed by fluorescence detection in the spleen. Figure 8 C represents the statistical results of fluorescence intensity in the spleen organs of each group.

[0081] Figure 9 In Example 6, the percentage of tdTom+ cells in specific cell subtypes (B, T, DC, and MΦ) in the spleen was determined by flow cytometry.

[0082] Figure 10In Example 7, serum was collected from mice immunized with 18PA / DOPG / DOPS LNP, and the OVA-specific IgG1 level in the induced serum was detected by ELISA. Figure 10 A) and IgG2c ( Figure 10 B) antibody titer.

[0083] Figure 11 The percentage of OVA-specific T cells in the spleen was determined by flow cytometry in Example 8. Figure 11 A is a representative flow cytometry atlas of H-2kb / SIINFEKL tetramer staining in CD8+ T cells from the spleen. Figure 11 B represents the quantitative analysis results of OVA-specific T cells.

[0084] Figure 12 In Example 9, the antigen-specific CTL response induced by 18PA / DOPG / DOPS LNP was evaluated by an in vivo cytotoxicity assay. Figure 12 A schematic diagram of the determination of OVA antigen-specific CTL response by CFSE method, and a representative flow cytometry atlas of OVA antigen-specific killing in the spleen of immunized mice. Figure 12 B) and quantitative percentage ( Figure 12 C).

[0085] Figure 13 To prepare and characterize different Mn@mRNA-LNP formulations in Example 10, the particle size of different Mn@mRNA-LNP formulations was detected by DLS. Figure 13 A) Polydispersion index ( Figure 13 B) and ζ potential ( Figure 13 C).

[0086] Figure 14 To investigate the determination of different amounts of adjuvant Mn in Example 10 using the Ribogreen method and ICP-MS. 2+ Mn@mRNA-LNP(DOPS / Mn) 2+ mRNA (w / w) Figure 14 A) and adjuvant Mn 2+ Packaging efficiency ( Figure 14 B).

[0087] Figure 15 To investigate the determination of DOPS and different metal ion adjuvants (Cu) by ICP-MS in Example 11 2+ Zn 2+ And Al 3 + Encapsulation efficiency of metal ion adjuvant at a mass ratio of 1:1.

[0088] Figure 16 The A549 cells in Example 12 were compared with those containing different amounts of adjuvant Mn. 2+ Cell viability after co-incubation with Mn@Luc-mRNA-LNP formulation for 24 hours ( Figure 16 A) and the status of transfection ( Figure 16 B).

[0089] Figure 17 In Example 13, different amounts of adjuvant Mn were used. 2+ Mn@mRNA-LNP(DOPS / Mn) 2+ Fluorescence images of major organs collected 6 hours after delivery of 0.1 mg / kg Luc mRNA (w / w). Figure 17 A) and the statistical results of fluorescence intensity of major organs ( Figure 17 B).

[0090] Figure 18 In Example 13, the fluorescence intensity of the liver, spleen, and lung organs was used to evaluate the levels of different masses of adjuvant Mn. 2+ A statistical graph showing the spleen targeting efficiency of Mn@Luc-mRNA-LNP.

[0091] Figure 19 Example 14 contains different amounts of adjuvant Mn 2+ The activation status of Mn@OVA-mRNA-LNP after acting on dendritic cells; among which... Figure 19 A is a flow cytometry atlas. Figure 19 B shows the expression of CD86 in dendritic cells and the results of quantitative analysis.

[0092] Figure 20 In Example 15, Mn@OVA-mRNA-LNP activated the STING signaling pathway by encapsulating manganese adjuvant. DC2.4 cells were co-incubated with Mn@mRNA-LNP for 8 h or 24 h, and IRF-7 was detected by RT-PCR. Figure 20 A) and IFN-β mRNA ( Figure 20 The expression of B).

[0093] Figure 21 To illustrate the activation and antigen cross-presentation of dendritic cells after Mn@OVA-mRNA-LNP treatment in Example 16, flow cytometry was used to visualize the CD86 (…) activating factor in dendritic cells. Figure 21 A) and CD40 Figure 21 B) Quantitative analysis results.

[0094] Figure 22To evaluate the T cell response induced by Mn@OVA-mRNA-LNP in the spleen in Example 17, the secretion of CD8+IFN-γ+ T cells in the spleen was measured by flow cytometry as the percentage of OVA-specific T cells. Figure 22 A is a representative flow cytometry atlas of H-2kb / SIINFEKL tetramer staining in CD8+IFN-γ+ T and CD8+ T cells in the spleen; Figure 22 B and 22C are the results of quantitative analysis of OVA-specific T cells.

[0095] Figure 23 This demonstrates the higher antigen-specific CTL response induced by Mn@OVA-mRNA-LNP in Example 17; Figure 23 A represents a flow cytometry map and quantitative percentage of CD8+ T cell-specific killing in the spleen of immunized mice. Figure 23 B).

[0096] Figure 24 This is an evaluation of the antitumor efficacy of Mn@OVA-mRNA-LNP in Example 18; Figure 24 A is a schematic diagram of Mn@OVA-mRNA-LNP administration; Figure 24 B is the average tumor growth curve of each group of mice.

[0097] Figure 25 The graph shows the survival time curves of mice in each group of the B16-OVA treatment model in Example 18.

[0098] Figure 26 The images shown are confocal images of CD4+T (red) and CD8+T (green) tumor tissues in mice on day 22 after different treatments, as evaluated by immunofluorescence experiments in Example 18.

[0099] Figure 27 In Example 18, flow cytometry was used to analyze the infiltration of effector T cells in the tumors of mice after receiving different treatments. Figure 27 A represents the quantitative analysis results of CD45+CD3+ in the tumor; Figure 27 B represents the quantitative analysis results of CD45+CD3+CD8+ T cells in the tumor.

[0100] Figure 28 This is an evaluation of the anti-tumor recurrence effect of Mn@OVA-mRNA-LNP after surgery in Example 18; Figure 28 A is a schematic diagram of the construction of a mouse tumor recurrence model after surgery; Figure 28 B is the average tumor growth curve of each group of mice.

[0101] Figure 29The survival curves of mice in each group in the B16-OVA postoperative recurrence model in Example 18 are shown.

[0102] Figure 30 This is a representative flow cytometry atlas of effector T cells and effector memory T cells in the peripheral blood of mice after different treatments, analyzed by flow cytometry in Example 18. Figure 30 A) and quantitative percentage ( Figure 30 B and Figure 30 C).

[0103] Figure 31 To assess the neoantigen-specific T cell response induced in vivo after immunization with the Neo-mRNA preparation in Example 19, representative flow cytometry maps of CD8+IFN-γ+ T cells secreted by spleen cells after different treatments were determined. Figure 31 A) and quantitative percentage ( Figure 31 (B and 31C).

[0104] Figure 32 In Example 19, Mn@Neo-mRNA-LNP can induce neoantigen-specific CTL responses; Figure 32 A represents the flow cytometry pattern and quantitative percentage of ADPGK / RESP1-specific killing in the spleen of immunized mice. Figure 32 (B and 32C).

[0105] Figure 33 This is an evaluation of the efficacy of Mn@Neo-mRNA-LNP against MC38 tumors in Example 20; Figure 33 A is a schematic diagram of Mn@Neo-mRNA-LNP administration; Figure 33 B is the average tumor growth curve of each group of mice.

[0106] Figure 34 The graph shows the survival time curves of mice in each group of the MC38 treatment model in Example 20. Detailed Implementation

[0107] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0108] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0109] The materials used in the following embodiments are as follows:

[0110] (1) ALC-0315, DSPC, and DMG-PEG (weight average molecular weight 2000) were purchased from Sinopec Xiamen Co., Ltd., cholesterol from Innochem Co., Ltd., 18PA from Macklin Co., Ltd., DOPG and MnCl2·4H2O from Sigma-Aldrich Co., Ltd., and DOPS from Aladdin Co., Ltd. Pur-A-Lyzer MIDIDialysis Kits (MWCO, 3.5kDa) and dialysis tubes from Sigma-Aldrich Co., Ltd., ovalbumin and OVA 257–263 (SIINFEKL) was purchased from Invivogen. LPS and cell viability assay kit CCK-8 was purchased from Beyotime; luciferase assay system and passive lysis buffer (5X) were purchased from Promega. mRNA in vitro transcription kit was purchased from Thermo Fisher. Tumor tissue digestion solution was purchased from Biogenous. Reps1 P45A With Adpgk R304M The polypeptide was synthesized by ChinaPeptides.

[0111] (2) CD11c-APC (Cat#: 17-0114-82, clone number: N418), CD-86-PE-Cy7 (Cat#: 25-0862-82, clone number: GL1), SIINFEKL / H-2Kb-PE (Cat#: 12-5743-81, clone number: 25-D1.16), CD45-FITC (Cat#: 11-0451-82, clone number: 30-F11), CD3-APC (Cat#: 17-0032-82, clone number: 17A2), B220-PE-Cy7 (Cat#: 25-0452-81, clone number: RA3-6B2), C The following CD11b-PE-Cy7 (Cat#: 25-0112-81, clone number: M1 / 70), CD40-PE (Cat#: 12-0401-82, clone number: 1C10), CD4-FITC (Cat#: 11-0041-82, clone number: GK1.5), CD8-PE (Cat#: 17-0032-82, clone number: 17A2), CD44-PE-Cy7 (Cat#: 25-0441-82, clone number: IM7), and CD62L-PerCP-Cy5.5 (Cat#: 45-0621-82, clone number: MEL-14) were all purchased from eBioscience. TMThe CD8-FITC (Cat#: MA5-16759, clone number: KT15) and T-Select H-2Kb OVA Tetramer-SIINFEKL-PE (Cat#: TS-5001-1) were purchased from MBL Company.

[0112] Example 1: mRNA Synthesis

[0113] The firefly luciferase (Luc), ovalbumin (OVA), cyclization recombinase (Cre), and neoantigen (Neo) mRNA involved in this invention are all synthesized through in vitro transcription.

[0114] First, the coding sequence fragments of the above-mentioned proteins were prepared by PCR (as shown in SEQ ID. NO. 1-4). These sequence fragments were then cloned into a template plasmid vector with an optimized 5'(3') untranslated region and a poly-A sequence, and the plasmid structure was determined by gene sequencing. After obtaining the plasmid containing the target gene fragment, it was digested into a linearized DNA template. Finally, the antigen-encoded mRNA was synthesized using in vitro transcription reagents. To reduce the immunogenicity of the mRNA and enhance its stability and transcription efficiency, the natural modified nucleoside N1-methylpseudouridine triphosphate (m1ψTP) was introduced to replace uridine triphosphate (UTP). Simultaneously, CleanCap was used for co-transcriptional capping during in vitro transcription, directly obtaining mRNA with the Cap-1 structure.

[0115] The amino acid sequence encoding the Luc-mRNA is SEQ ID NO.1:

[0116] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV

[0117] Amino acid sequence of the OVA - mRNA coding sequence SEQ ID.NO.2:

[0118] MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP

[0119] Amino acid sequence of the Cre-mRNA coding sequence SEQ ID.NO.3:

[0120] MPKKKRKVANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD

[0121] Amino acid sequence of the Neo-mRNA coding sequence SEQ ID.NO.4:

[0122] MDFTGSNGDPSSPYSLHYLSPTGVNEYKARDETAALLNSAVLGAAPLFVPPADSKLLSFMAPIDHTTMSDDARTELFRSGRVLELFRAAQLANDVVLQIMELCGATRDIDPSSSVLFEYMEKPDFSLFSPEAGQSLVISASIIVFNLLELEGDYRGIPVHLELASMTNMELMSSIVHQQVFPT

[0123] Example 2: Preparation and characterization of spleen-targeting LNPs

[0124] ALC-0315, DSPC, cholesterol, DMG-PEG, and anionic lipids (18PA, DOPG, or DOPS) were dissolved in ethanol at specific molar percentages. DOPG was directly soluble in ethanol along with other lipids, while 18PA and DOPS required complete dissolution in anhydrous tetrahydrofuran before the addition of ethanol, resulting in a tetrahydrofuran to ethanol volume ratio of 2:1 in the final 18PA and DOPS anionic lipid solution. The mRNA encoding the antigen (specifically, Luc-mRNA, OVA-mRNA, Cre-mRNA, or Neo-mRNA prepared in Example 1) was dissolved in 10 mM citrate buffer (pH 4.0). The mRNA was then rapidly mixed into the lipid solution at a volume ratio of 3:1 (mRNA:lipid, vol / vol), resulting in a final weight ratio of 40 / 1 (total lipid / mRNA, wt / wt). The above solution was then incubated at room temperature for 15 min. The resulting solution was placed in a Pur-A-Lyzer Maxi 3500 dialysis tube and dialyzed with a large volume of 0.01M PBS for at least 2 hours to remove ethanol, yielding spleen-targeted LNPs, which were then stored at 4°C for later use. Spleen-targeted LNPs (18PA / DOPG / DOPS LNPs, abbreviated as 18PA, DOPG, and DOPS, respectively) obtained by dynamic light scattering (DLS) characterization are shown in [link to documentation]. Figure 2 A to Figure 2 C indicates that the spleen-targeted LNP obtained in this embodiment has a particle size range of 140-200 nm and a polydispersity index of 0.1-0.3, indicating that the size of the above nanoparticles is relatively uniform. At the same time, the particle size change of the above LNP was evaluated after being placed at 4°C for seven days. The experimental results showed that the particle size of the above LNP did not change significantly within seven days, indicating that it has good stability.

[0125] Table 1: Molar percentage of each component in spleen-targeted LNP

[0126]

[0127] Example 3: Screening for spleen-targeted LNPs

[0128] The 18PA / DOPG / DOPS LNPs prepared in Example 2, containing different ratios, were injected into C57BL / 6 mice (18-20g) via tail vein at a dose of 0.1 mg / kg Luc mRNA. Six hours later, 100 μL of D-fluorescein potassium salt (30 mg / mL) was injected intraperitoneally. The mice were then imaged using the IVIS in vivo imaging system (Perkin Elmer). Once the fluorescence intensity stabilized, the mice were euthanized, and their major organs (heart, liver, spleen, lungs, and kidneys) were removed and imaged. The fluorescence intensity was analyzed using in vivo imaging software (Perkin Elmer).

[0129] Figure 3 These are fluorescence images of various organs. Figure 4 Statistical analysis and comparison of fluorescence intensity in various organs. Figure 5 The spleen-targeting efficiency of LNPs containing different proportions of 18PA / DOPG / DOPS was analyzed. The results showed that LNPs containing different percentages of 18PA / DOPG / DOPS could effectively and specifically target and deliver to the spleen. The fluorescence intensity in the spleen was significantly higher than that of the traditional ALC-0315LNP (liver-targeting LNP). Furthermore, it was found that the spleen-targeting effect was optimal when the molar percentage of anionic lipids was 15%. Therefore, subsequent experiments used LNPs containing 15% anionic lipids.

[0130] Example 4: Transfection experiment of dendritic cells

[0131] Bone marrow-derived dendritic cells (BMDCs) were extracted from mice and processed at a density of 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in white 96-well plates. 50 μL of LNP containing 15% 18PA / DOPG / DOPS (obtained in Example 2) was added to each well, and 0.2 μg of Luc-mRNA was immobilized in each well. After 24 h of incubation, the supernatant was discarded, and 20 μL of 1X cell lysis buffer (Promega) was added to each well. After lysis for 20 min, 15 μL of the lysis buffer was transferred to each well in a white 96-well plate, and 50 μL of ONEGlo™ luciferase substrate was added to each well to detect luciferase expression. The results are as follows: Figure 6 As shown, Luc-mRNA alone cannot be effectively taken up by BMDCs, but using 15% 18PA / DOPG / DOPS LNP as an mRNA delivery vector can effectively enhance the delivery of Luc-mRNA to BMDCs, thereby improving the transfection efficiency of Luc-mRNA.

[0132] Example 5: Dendritic cell activation and antigen presentation experiment

[0133] In this example, OVA was used as the template antigen. The short peptide (SIINFEKL) at positions 257-264 of the OVA protein contains an antigenic epitope that can be recognized by MHC I, and is a commonly used antigen in research. OVA-mRNA (amino acid sequence as shown in SEQ ID NO.2) was synthesized as described in Example 1. Therefore, immature BMDCs were used at a density of 1 × 10⁻⁶ mRNA per well. 6 Cells were seeded at a density of 100 μg / well in 24-well plates, and then LNP (containing 2.0 μg OVA-mRNA) and LPS (2.0 μg / well) containing 15% 18PA / DOPG / DOPS obtained in Example 2 were added and the plates were treated at 37°C for 48 hours. Cells were collected by centrifugation at 800g for 5 minutes. The collected BMDCs were then labeled with CD11c-APC, CD-86-PE-Cy7, and SIINFEKL / H-2Kb-PE fluorescent antibodies in the dark for 20 minutes. After washing the cells three times with 0.01M PBS, the expression levels of CD11c+CD86+ and CD11c+SIINFEKL-H-2Kb+ on the BMDCs were analyzed by flow cytometry. For detailed flow cytometry data, please refer to [link to flow cytometry diagram]. Figure 7 In combination with the above Figure 7 Results A through 7C show that the expression levels of the NC group (the group without any added substances) were similar to those of the OVA-mRNA group, indicating that it could not upregulate CD86 expression. However, the LNP groups of 18PA / DOPG / DOPS could all upregulate the percentage of the related protein CD86. At the same time, compared with the control group, the 18PA / DOPG / DOPS LNP groups could effectively increase the percentage of the OVA peptide (SIINFEKL)-MHC-I complex, indicating that 18PA / DOPG / DOPS LNP can promote the maturation of BMDCs and the presentation of related antigens.

[0134] Example 6: Screening of LNP-transfected cell subtypes in the spleen

[0135] In this example, genetically engineered B6.Cg-Gt(ROSA)26Sor was selected. tm9(CAG-tdTomato)Hze / J mice (Ai9 mice) are used as model mice. Their LoxP-side stop cassettes prevent tdTom protein expression, while the absence of these stop cassettes allows for efficient tdTom protein expression. Therefore, they are commonly used to detect gene-edited cells. Delivery of Cre recombinase mRNA is one of the main ways to activate tdTom fluorescence in edited cells. Figure 8A). In this example, to test the ability of 18PA / DOPG / DOPS LNP-edited mice in the spleen, Cre mRNA preparations were prepared as described in Example 2 above, and administered via tail vein injection (0.5 mg / kg Cre mRNA). Two days later, Ai9 mice were euthanized (n=3 per group), and the spleen was imaged using the IVIS Lumina system (Perkin Elmer). The results are as follows: Figure 8 As shown, 18PA / DOPG / DOPS LNP can effectively edit the spleen organ.

[0136] Subsequently, the LNP-transfected cell subtypes in the spleen were further detected, specifically tdTom+ cells. The spleens were collected, and freshly collected spleens were added to a six-well plate containing 1 mL of RPMI 1640 complete medium. The medium was mixed with 1 mL of sterile syringe until it became turbid, and the spleens were thoroughly ground. The medium containing spleen cells was passed through a 40 μm sieve to remove large pieces of tissue. The spleen cells were then obtained by centrifugation at 800 g and 4 °C for 5 min. The spleen cells were washed three times with cold 0.01 M PBS. Finally, the spleen cells were incubated with corresponding antibodies (CD45-FITC, CD3-APC, B220-PE-Cy7, CD3-APC, CD11c-APC, and CD11b-PE-Cy7) in the dark for 20 min. After incubation, wash the cells three times and resuspend them in 100 μL of 0.01 M PBS. Add 1.0 μL of DAPI (1 mg / mL) to the solution and incubate for 10 min. The cells are then ready for analysis. (Based on the above...) Figure 9 The results showed that DCs and MΦ cells in the spleen of Ai9 mice treated with 18PA / DOPG / DOPS LNP were tdTom+ cells, indicating that 18PA / DOPG / DOPS LNP was mainly transfected by antigen-presenting cells in the spleen.

[0137] Example 7: ELISA assay for detecting OVA-specific antibodies in the serum of immunized mice

[0138] Six- to eight-week-old C57BL / 6 mice (18-20g) were injected via tail vein on days 0 and 5 with a 18PA / DOPG / DOPS LNP formulation loaded with OVA mRNA (1.0 mg / kg OVA mRNA, 3 mice per group). On day 14, 100 μL of blood was collected via tail vein. The collected whole blood was incubated at 4°C for 30 min, followed by centrifugation at 3000 rpm for 10 min in a pre-chilled centrifuge to collect serum. The collected serum was stored at -20°C for later use. The levels of antigen-specific IgG1 and IgG2c in the serum were measured using ELISA. For ELISA, OVA protein was dissolved in 50 mM carbonate buffer to a final concentration of 20 μg / mL, and then pre-coated with the OVA protein solution in 96-well plates (Nunc) and incubated overnight at 4°C. After incubation, the mice were washed three times with PBST (containing 0.05% Tween-20, pH 7.4) and blocked with PBST containing 10% FBS for 1 hour. After blocking, the mice were washed once with PBST. The collected serum from the immunized mice was serially diluted (from 2... 7 Dilute to 2 21 The diluted serum was then added to the wells and incubated at 37°C for 2 hours. After incubation, the wells were washed three times, and 100 μL of HRP-conjugated IgG1 and IgG2c antibodies (1:10000 dilution) were added. Incubation continued for 1 hour under the same conditions. After incubation, the wells were washed three times, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. Incubation was carried out at 37°C for 5-10 minutes, followed by the addition of 100 μL of stop solution. The optical density was measured at 450 nm using an ELISA reader. IgG1 antibody was mainly induced by CD4+ helper T cells type 2 (Th2), while IgG2c antibody was produced and bound by CD4+ helper T cells type 1 (Th1). Figure 10 Results A and 10B show that mice inoculated with the OVA mRNA formulation all produced high OVA-specific IgG1 titers. It is also noteworthy that 18PA LNP did not show a significant IgG2c response, while DOPG / DOPS LNP produced a high IgG2c response, indicating that DOPG / DOPS LNP can simultaneously induce Th1 and Th2 responses.

[0139] Example 8: In vivo OVA-specific CD8+ T detection

[0140] On days 0 and 5, 6-8 week old C57BL / 6 mice were intravenously injected with 18PA / DOPG / DOPS LNP formulation loaded with OVA mRNA (1.5 mg / kg OVA mRNA, 4 mice per group). On day 10, the immunized mice were euthanized, and splenocytes were collected according to the method described in Example 6. The collected splenocytes were then labeled by incubating with T-Select H-2KbOVA Tetramer-SIINFEKL-PE fluorescent antibody in the dark for 20 min. After washing the cells three times with 0.01M PBS, CD8-FITC was added again and incubated at 4°C for 20 min. After washing the cells three times with 0.01M PBS, the expression level of OVA-specific CD8+T was analyzed by flow cytometry. For specific flow cytometry atlases, please refer to [link to flow cytometry analysis]. Figure 11 A, in combination with the above Figure 11 Results B showed that 18PA / DOPG / DOPS LNPs significantly promoted the proliferation of OVA-specific CD8+ T cells, with DOPS LNP inducing a greater number of OVA-specific CD8+ T cells compared to 18PA / DOPG LNPs.

[0141] Example 9: Identification of in vivo cytotoxic killing effect

[0142] On days 0 and 5, C57BL / 6 mice (6-8 weeks old) were intravenously injected via tail vein with 18PA / DOPG / DOPS LNP formulation loaded with OVA mRNA (0.75 mg / kg OVA mRNA, 4 mice per group). Nine days later, untreated (unimmunized) mice were euthanized, and splenocytes were collected according to the method described in Example 6. The collected splenocytes were counted, and then incubated with 10 μM OVA257-263 short peptide in complete culture medium at 37°C for 2 h. After incubation, cells without peptide pulse and those with peptide pulse were incubated with 0.05 μM Carboxyfluorescein succinimidyl ester (CFSE) in complete culture medium for 15 min, respectively. After incubation, equal numbers (1.5 × 10⁻⁶) of cells were incubated with 10 μM OVA257-263 short peptide in complete culture medium. 7 0.05 μM CFSE-labeled un-OVA peptide-pulsed cells (each spleen cell) were mixed with 0.5 μM CFSE-labeled OVA peptide-pulsed cells and injected via the tail vein into immunized mice. Eighteen hours later, spleen cells from these immunized mice were collected for flow cytometry analysis. Figure 12 A). The percentage of target cell-specific killing by OVA peptide pulses was calculated using the following equation: Specific lysis percentage = (1 - Experimental positive rate / Non-transfer control positive rate) × 100%. See the detailed flow cytometry atlas for more information. Figure 12 B, in combination with the above Figure 12 Results from B and 12C show that both DOPG / DOPS LNP significantly increased the percentage of OVA-specific killing, while 18PA LNP only slightly increased the OVA-specific killing efficiency compared to the PBS group. It is also worth noting that DOPS LNP has a higher OVA-specific killing efficiency compared to DOPG LNP.

[0143] Example 10: Preparation and characterization of different Mn@mRNA-LNP formulations

[0144] Based on the results of Examples 7 to 9, it is shown that DOPS LNP can induce both Th1 and Th2 responses simultaneously and can induce antigen-specific T cell responses more efficiently. Therefore, DOPS LNP was selected as the optimal formulation. Subsequent experiments were all based on the preparation of Mn@mRNA-LNP by encapsulating manganese adjuvant in the DOPS LNP formulation.

[0145] ALC-0315, DSPC, cholesterol, DMG-PEG, and DOPS were mixed in a specific molar percentage (39.35:7.99:36.99:1.36:15.00). ALC-0315, DSPC, cholesterol, and DMG-PEG were directly soluble in ethanol, while the anionic lipid DOPS required complete dissolution in anhydrous tetrahydrofuran before the addition of ethanol, resulting in a tetrahydrofuran to ethanol volume ratio of 2:1 in the final DOPS anionic lipid solution. MnCl2 solutions with different mass ratios than DOPS and mRNA were dissolved in 10 mM citrate buffer (pH 4.0), and Mn@mRNA-LNPs were prepared as described in Example 2. The size, polydispersity index, and zeta potential of the Mn solutions containing different masses of adjuvants were examined by DLS. 2+ Mn@mRNA-LNP(DOPS / Mn) 2+ (,w / w), see Figure 13 A to Figure 13 B, all Mn@mRNA-LNP formulations showed no significant changes in particle size and polydispersity index. Figure 13 C shows the zeta potential of all Mn@mRNA-LNP formulations, demonstrating the zeta potential as Mn... 2+ The concentration of Mn increased with increasing concentration, but all Mn@mRNA-LNP formulations still carried a negative charge. These results indicate that Mn... 2+The incorporation of [a specific ingredient] did not significantly affect the physicochemical properties of DOPS LNPs. Subsequently, the mRNA and adjuvant Mn content of all Mn@mRNA-LNP formulations were determined using the Ribogreen method and inductively coupled plasma-mass spectrometry (ICP-MS). 2+ The encapsulation efficiency (EE) is shown in Figure 14A. As Mn... 2+ As the concentration of adjuvant increases, the encapsulation efficiency of mRNA decreases, especially when DOPS and Mn... 2+ At mass ratios of 1:1 and 1:2, there was no significant effect on mRNA encapsulation efficiency, and ICP-MS results showed that ( Figure 14 B) All Mn@mRNA-LNP formulations can effectively encapsulate Mn. 2+ Adjuvant, wherein DOPS is used with adjuvant Mn 2+ When the mass ratio is 1:1, Mn 2+ The optimal packaging efficiency can reach 65.97%.

[0146] Example 11: Delivery of different metal ion adjuvants using the DOPS LNP delivery system

[0147] DOPS LNPs were prepared as described in Example 10, and different metal ion adjuvants (CuCl2, ZnCl2, or AlCl3) and mRNA at a mass ratio of 1:1 with DOPS were dissolved in 10 mM citrate buffer (pH 4.0). LNPs were then prepared as described in Example 2. The encapsulation efficiency of different metal ions was subsequently detected by inductively coupled plasma-mass spectrometry (ICP-MS). Figure 15 As shown, the DOPSLNP delivery system can effectively encapsulate Cu 2+ Zn 2+ And Al 3+ The payload efficiencies were 28.55%, 51.94%, and 51.32%, respectively.

[0148] Example 12: Evaluation of transfection efficiency of different Mn@mRNA-LNP formulations

[0149] A549 cells (human non-small cell lung cancer cell line) were cultured at 0.8 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1000 cells per well in 96-well plates, and then 50 μL of the adjuvant Mn obtained in Example 10 with different masses was added. 2+The Mn@mRNA-LNP formulation was used to immobilize 0.2 μg of Luc-mRNA in each well. Cell viability was then assessed using the CCK-8 assay, and luciferase expression was detected as described in Example 4. The results are as follows: Figure 16 As shown in A, all Mn@mRNA-LNP formulations and those without Mn are compared. 2+ Compared to the Mn@Luc-mRNA-LNP formulation, the cell viability remained around 80%, indicating that Mn... 2+ The incorporation of Mn@Luc-mRNA-LNP formulations did not increase cytotoxicity, and all Mn@Luc-mRNA-LNP formulations exhibited good biocompatibility. Furthermore, it was found that when Mn... 2+ As the concentration of Mn@Luc-mRNA-LNP gradually increased, the transfection efficiency first increased and then decreased. Specifically, when the concentrations of DOPS and Mn... 2+ When the mass ratio is 1:1, the transfection efficiency of this formulation reaches its peak. Figure 16 B).

[0150] Example 13: Evaluation of spleen targeting efficiency of different Mn@mRNA-LNP formulations

[0151] The adjuvant Mn prepared in Example 10 contained different masses of adjuvant Mn. 2+ Mn@mRNA-LNP(DOPS / Mn) 2+ Following the mRNA encapsulation efficiency measured in Example 10, a uniform mRNA dosage (0.1 mg / kg Luc mRNA) was applied, and the fluorescence intensity of the major organs of mice treated with the above formulation was analyzed as described in Example 3. The results are as follows: Figure 17 As shown in A and 17B, all Mn@Luc-mRNA-LNP formulations and those without adjuvant Mn 2+ Compared to the Mn@mRNA-LNP formulation, the fluorescence intensity in the spleen was comparable, and the spleen targeting efficiency showed no significant change. Figure 18 This indicates that the adjuvant Mn 2+ The incorporation of [the substance] does not affect the expression efficiency of Luc-mRNA in the spleen or the spleen targeting efficiency.

[0152] Example 14: Evaluation of the ability of different Mn@mRNA-LNP formulations to promote dendritic cell maturation

[0153] The adjuvant Mn prepared in Example 10 contained different masses of adjuvant Mn. 2+ Mn@OVA-mRNA-LNP(DOPS / Mn) 2+After incubating immature BMDCs at 37°C for 48 hours as described in Example 5 (w / w), the cells were collected and labeled with CD11c-APC and CD-86-PE-Cy7 by incubation in the dark for 20 min. The cells were washed three times with 0.01M PBS, and the expression levels of CD11c+CD86+ on the BMDCs were analyzed by flow cytometry. See the detailed flow cytometry atlas for more information. Figure 19 A. Combining the above Figure 19 Results A and 19B show that, compared to the unencapsulated adjuvant Mn... 2+ Compared to the Mn@OVA-mRNA-LNP formulation, all formulations encapsulating adjuvant Mn 2+ The Mn@OVA-mRNA-LNP formulation significantly increased CD86 expression. It is also noteworthy that only a small amount of adjuvant Mn was added. 2+ (DOPS / Mn 2+ The use of ,w / w,1:1) can further promote the maturation of BMDCs.

[0154] Example 15: In vitro assessment of IRF-7 and IFN-β gene expression by RT-PCR

[0155] The combined results of Example 10 and Examples 12 to 14 indicate that Mn@mRNA-LNP(DOPS / Mn) 2+ (w / w, 1:1) can further efficiently encapsulate the adjuvant Mn without affecting mRNA encapsulation. 2+ Furthermore, this formulation exhibited good transfection efficiency, spleen targeting efficiency, and the ability to promote BMDC maturation; therefore, it was selected as the optimal formulation for subsequent experiments. First, in this example, the ability of Mn@OVA-mRNA-LNP to activate the STING signaling pathway was evaluated. The expression of IRF-7 and IFN-β genes in the STING signaling pathway was detected by RT-PCR. Therefore, 2 × 10⁶ cells / well were seeded in 24-well plates. 5DC2.4 cells were incubated at 37°C for 24 hours. Then, the cells were co-incubated with PBS, free MnCl2 (Mn), DOPS LNP (DOPS), DOPS LNP+MnCl2 (DOPS+Mn, indicating that MnCl2 is not loaded into DOPS LNPs but rather the DOPS LNPs are mixed thoroughly with MnCl2), and Mn@OVA-mRNA-LNP for 8 or 24 hours. After incubation, cells were collected, and total mRNA was isolated using the RNA isolater Total RNA Extraction Reagent (Vazyme) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using a high-capacity cDNA reverse transcription kit (Vazyme). RT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) and... Detection was performed using a 96-fold real-time PCR detection system. 96SW 1.1 software was used to analyze relative gene expression. Results are as follows: Figure 20 As shown in A and 20B, the Mn@OVA-mRNA-LNP group upregulated the expression of IRF-7 and IFN-β mRNA at an early time point of 8h, and after 24h, Mn@OVA-mRNA-LNP induced a higher response compared to other groups.

[0156] Example 16: Experiment on Mn@mRNA-LNP promoting dendritic cell activation

[0157] As described in Example 5, immature BMDCs were incubated with the groupings described in Example 15 at 37°C for 48 hours (LPS as a positive control group). The cells were then collected and labeled with CD11c-APC, CD-86-PE-Cy7, and MHC II-FITC by incubation in the dark for 20 minutes. After washing the cells three times with 0.01M PBS, the expression levels of CD11c+CD86+ and CD11c+CD40+ on the BMDCs were analyzed by flow cytometry. Combined with the above... Figure 21 Results A and 21B show that after 48 hours of co-incubation of immature BMDCs with Mn@OVA-mRNA-LNP, the expression of BMDC activation markers (CD86 and CD40) was significantly increased in the Mn@OVA-mRNA-LNP group compared with the Mn group, DOPS group, and DOPS+Mn group.

[0158] Example 17: Evaluation of T cell responses evoked by Mn@mRNA-LNP in the spleen

[0159] Six- to eight-week-old C57BL / 6 mice were selected and divided into five groups as described in Example 15. Different OVA mRNA preparations were injected into these mice via tail vein on days 0 and 5, respectively. On day 10, the immunized mice were euthanized, and spleen cells were collected according to the method described in Example 6. Finally, flow cytometry was used to analyze the secretion of CD8+IFN-γ+ T cells and the expression of OVA-specific T cells in the spleen after vaccination. For the detection of CD8+IFN-γ+ T cells, the collected spleen cells were labeled with CD8-FITC and incubated in the dark for 20 min. After washing the cells three times with 0.01M PBS, the cells were resuspended in 200 μL of pre-chilled Fixation / permeabilization solution and incubated at 4°C for 30 min. After incubation, the cells were collected by centrifugation at 1000g for 5 min and then pre-diluted to 1×BD perm / Wash. TM The cells were washed twice, and IFN-γ-PE-Cy7 flow cytometry antibody was added to the cells. They were then incubated again for 20 minutes under the same conditions. The detection method for OVA-specific T cells was as described in Example 8. For detailed flow cytometry atlases, please refer to... Figure 22 A, in combination with the above Figure 22 Results B and 22C show that the Mn@OVA-mRNA-LNP group further promoted the secretion of the pro-inflammatory cytokine IFN-γ and the proliferation of OVA-specific T cells compared with other groups, indicating that the Mn@OVA-mRNA-LNP group can more effectively activate the T cell response in the spleen and induce the proliferation of specific T cells.

[0160] Subsequently, the in vivo cytotoxic killing ability of Mn@OVA-mRNA-LNP was further evaluated. Six- to eight-week-old C57BL / 6 mice were divided into five groups as described in Example 15, and the detection method was as described in Example 9. For specific flow cytometry atlases, please refer to [link to example]. Figure 23 A, the result is as follows Figure 23 As shown in Figure B, neither the Mn group nor the PBS group induced OVA-specific killing. The DOPS group and the DOPS+Mn group showed similar high OVA-specific killing efficiency. However, the OVA-specific killing efficiency of Mn@OVA-mRNA-LNP was 62.28±7.64%, which was significantly higher than that of NC (10.50±4.95%), Mn group (16.84±12.05%), DOPS group (47.37±4.74%), and DOPS+Mn group (44.53±7.28%). This indicates that the Mn@OVA-mRNA-LNP group can induce a higher cytotoxic T lymphocyte (CTL) response.

[0161] Example 18: Evaluation of the antitumor and immunomodulatory effects of Mn@mRNA-LNP

[0162] Immunocomplished C57BL / 6 mice (6-8 weeks old) weighing 22-25g were selected and 1.5×10⁻⁶ mice were injected. 5 B16-OVA cells cultured under standard conditions were subcutaneously inoculated into the right groin of mice. When the tumor grew to 50-100 mm... 3 Following the grouping method described in Example 15, mice were divided into PBS group, Mn group, DOPS group, DOPS+Mn group, and Mn@OVA-mRNA-LNP group. Every four days, mice were injected via tail vein with the different OVA mRNA formulations (2.0 mg / kg OVA mRNA, 8 mice per group), for a total of two injections, 200 μL per mouse. The PBS was 0.01 M PBS, and the adjuvant Mn was added to the Mn and DOPS+Mn groups. 2+ The dosage can be determined based on the adjuvant Mn at a dose of 2.0 mg / kg OVA mRNA as measured in Example 10. 2+ Encapsulation efficiency was calculated ( Figure 24 A). After drug administration, the growth of the mice was continuously observed. Tumor size was measured every other day, and the weight of the tumor-bearing mice was weighed. Tumor volume and survival curves were then calculated. Tumor volume (V) = (Longest diameter of tumor (mm) × Shortest diameter of tumor (mm)) 2 ) / 2, survival time is defined as the period from the start of tumor inoculation in mice until death or when the tumor volume exceeds the animal ethics guidelines of the institution (>1500mm). 3 The time interval is as follows. Figure 24 As shown in Figure B, tumors in both the PBS and Mn groups grew rapidly and their progression could not be inhibited. The DOPS and DOPS+Mn groups effectively slowed tumor growth, but ultimately failed to inhibit it. However, the Mn@OVA-mRNA-LNP group significantly inhibited tumor growth and effectively prolonged the survival of mice compared to other groups. Figure 25 ).

[0163] The antitumor immune response to Mn@mRNA-LNP was evaluated, and the infiltration of CD4+ / CD8+ T cells in tumor tissue sections of each group was analyzed using tissue multichannel / multicolor immunofluorescence. Results are as follows: Figure 26As shown, the PBS group and Mn group showed less fluorescence signal of CD4+ T (red fluorescence) and CD8+ T (green fluorescence) cells, indicating less T cell infiltration in the tumor. The DOPS group and DOPS+Mn group showed increased fluorescence signal of CD4+ T and CD8+ T cells, while the Mn@OVA-mRNA-LNP group showed significantly increased fluorescence signal of CD4+ T and CD8+ T cells, indicating that the Mn@OVA-mRNA-LNP group effectively induced T cell responses and promoted effector T cell infiltration at the tumor site compared to other groups. Subsequently, tumor tissues from mice in each group (n=4 per group) were collected after 22 days of treatment. The tumor tissues were placed in complete culture medium, and under aseptic conditions, the tumor tissues were thoroughly minced. Enzymatic digestion solution containing 0.2 mg / mL DNase I, hyaluronidase, and 1 mg / mL type IV collagenase was added. Digestion was carried out at 37°C in a shaker for 1-2 hours. After the culture medium became turbid, RPMI-1640 medium containing 10% FBS was added to terminate the digestion. The single-cell suspension of the above-mentioned tumor tissue was passed through a 40 μm sieve to remove undigested large tissue fragments. The cells were centrifuged at 800g for 5 min at 4°C, and washed three times with 0.01M PBS buffer to obtain the single-cell suspension of tumor tissue. These cells were then labeled and incubated with CD45-PE-Cy7, CD3-APC, and CD8-FITC in the dark for 20 min. After washing the cells three times with 0.01M PBS, the proportion of CD8+ T cells infiltrating the tumor was analyzed using flow cytometry. Figure 27 As shown, the percentages of CD45+CD3+ and CD45+CD3+CD8+ T cells in the tumor were increased by 3.84 and 4.54 times, respectively, compared with PBS in the Mn@OVA-mRNA-LNP group, indicating that Mn@OVA-mRNA-LNP effectively increased the infiltration of cytotoxic T lymphocytes (CTLs), further stimulating the anti-tumor immune response, thereby inhibiting or even killing the tumor.

[0164] Currently, surgery remains the preferred treatment for cancer in clinical practice. However, despite continuous advancements in diagnostic and treatment methods, postoperative tumor recurrence and metastasis remain major causes of death for cancer patients. Therefore, we constructed a mouse model of postoperative tumor recurrence to evaluate the inhibitory effect of Mn@OVA-mRNA-LNP on postoperative recurrent tumors. Six- to eight-week-old C57BL / 6 mice were randomly divided into two groups (PBS group and Mn@OVA-mRNA-LNP group). On day 0, 1.5 × 10⁻⁶ Mn@OVA-mRNA-LNP was subcutaneously injected into the right groin of the mice. 5 B16-OVA cells cultured under standard conditions. When the tumor grew to approximately 500 mm... 399% of the tumor was surgically removed, leaving 1% as a residual tumor. The wound was then sutured with a wound clip to simulate postoperative tumor recurrence (day 19). Mn@OVA-mRNA-LNP formulation was administered via tail vein to mice on days 22, 25, and 28 postoperatively. After administration, the growth of the mice was continuously observed. Tumor size and body weight were measured every other day, and tumor volume and survival curves were statistically analyzed. Figure 28 A). The result is as follows Figure 28 As shown in B and 29, compared with the PBS group, the Mn@OVA-mRNA-LNP group effectively delayed tumor recurrence and significantly prolonged the postoperative survival of mice. Subsequently, mice in each group were anesthetized, and whole blood was collected from each group using anticoagulant tubes containing heparin sodium. The collected whole blood was centrifuged at 8000g for 5 min in a centrifuge pre-cooled to 4℃. Then, five volumes of erythrocyte lysis buffer were added and thoroughly mixed. After standing for 5-10 min, the mixture was centrifuged at 800g for 5 min at 4℃. The cells were washed three times with 0.01M PBS buffer to obtain peripheral blood T lymphocytes. The corresponding flow cytometry antibodies (CD3-APC, CD4-FITC, CD8-PE, CD44-PE-Cy7, and CD62L-PerCP-Cy5.5) were added to these cells and incubated for 20 min in the dark. After incubation, the cells were washed twice with PBS buffer and resuspended in 200 μL of PBS buffer. Flow cytometry was then used to analyze the secretion of memory T cells in the peripheral blood of mice post-surgery. Figure 30 As shown in A to 30C, the Mn@OVA-mRNA-LNP group significantly increased the proportion of effector memory T cells in peripheral blood compared with the PBS group, indicating that mice treated with Mn@OVA-mRNA-LNP have better anti-tumor immune memory.

[0165] Example 19: Detection of neoantigen-specific T cells

[0166] In this embodiment, seven neoantigen sequences of MC38 (mouse colon cancer cell line) were screened (the MC38 neoantigen is composed of CPNE1, IRGQ, AATF, RESP1, MED12, DPAGT1, and ADPGK genes, as shown in Table 2), and these sequences were synthesized into Neo-mRNA as described in Example 1. Subsequently, the neoantigen-specific T-cell response induced by Neo-mRNA in vivo was further detected. First, on days 0 and 5, 6-8 week old C57BL / 6 mice were injected via tail vein with Neo-mRNA preparation (Mn@mRNA-LNP, 2.0 mg / kg Neo-mRNA). On day 10, the immunized mice were euthanized, and mouse spleen cells were extracted. The obtained spleen cells were divided into two groups, A and B. Group A contained the above-mentioned spleen cells (2.0 × 10⁶).6 BMDCs (2.0 × 10⁶ cells) that had been pre-incubated with ADPGK or RESP1 peptides for 48 h were added to a splenocyte sample. 5 Group B cells were co-incubated again for 48 hours with the above-mentioned spleen cells (2.0 × 10⁶ cells). 6 ADPGK or RESP1 peptide (2.0 μg / mL) was added to each spleen cell and incubated for 48 h, with dimethyl sulfoxide (DMSO) as a negative control. Cells from groups A and B were then collected, and the secretion of CD8+IFN-γ+ T cells was analyzed as shown in Example 17. For detailed flow cytometry data, please refer to [link to flow cytometry data]. Figure 31 A, in combination with the above Figure 31 Results B and 31C show that T cells extracted after inoculation with Neo-mRNA preparations, when stimulated again by ADPGK or RESP1 neoantigen peptides or by BMDCs incubated with ADPGK or RESP1 neoantigen peptides, showed a significant increase in CD8+IFN-γ+ T cell secretion. This demonstrates that Neo-mRNA preparations can induce the production of neoantigen-specific T cells and activate specific T cell immune responses.

[0167] Subsequently, the in vivo cytotoxic killing ability of the Neo-mRNA formulation was further evaluated. Six- to eight-week-old C57BL / 6 mice were randomly divided into three groups: a PBS group, a Mn@Luc-mRNA-LNP group, and a Mn@Neo-mRNA-LNP group. The detection method was the same as described in Example 9, except that 20 μM ADPGK or RESP1 peptide was used to pulse splenocytes. For specific flow cytometry atlases, please refer to [link to relevant documentation]. Figure 32 A, the result is as follows Figure 32 As shown in B and 32C, neither the Mn@Luc-mRNA-LNP group nor the PBS group induced ADPGK / RESP1-specific killing. However, the ADPGK-specific killing efficiency of Mn@Neo-mRNA-LNP was 45.41±15.06% and the RESP1-specific killing efficiency was 32.42±5.02%, which were significantly higher than those of the PBS group and the Mn@Luc-mRNA-LNP group. This indicates that the Mn@Neo-mRNA-LNP group can activate neoantigen-specific T cell responses and induce higher CTL responses.

[0168] Table 2: The seven amino acid sequences of the MC38 neoantigen encoded by Neo-mRNA

[0169]

[0170] Example 20: Evaluation of the antitumor efficacy of Mn@Neo-mRNA-LNP

[0171] Select 22-25g immunocompetent C57BL / 6 mice and administer 2.0×10⁻⁶ mice. 5 One MC38 cell cultured under standard conditions was subcutaneously inoculated into the right inguinal region of mice. When the tumor grew to 50-100 mm... 3 Mice were divided into four groups: PBS group, anti-PD-1 group, Mn@Neo-mRNA-LNP group, and Mn@Neo-mRNA-LNP+anti-PD-1 group (n=8 per group). Mice were administered Mn@Neo-mRNA-LNP (2.0 mg / kg Neo mRNA) via tail vein injection and anti-PD-1 antibody (2.5 mg / kg) via intraperitoneal injection on days 10, 13, 17, and 20, for a total of four times. Figure 33 A). After the drug administration was completed, the growth of the mice was continuously observed. The size of the tumors was measured every other day, the weight of the tumor-bearing mice was weighed, and the tumor volume and survival curves were statistically analyzed. The results are as follows: Figure 33 As shown in Figure B, tumor progression was faster in the PBS group, with a complete tumor inhibition rate of 0.0%. While the anti-PD-1 group and the Mn@Neo-mRNA-LNP group effectively slowed tumor growth, their complete tumor inhibition rates were only 0.0%. In contrast, the Mn@Neo-mRNA-LNP combined with the anti-PD-1 antibody significantly inhibited tumor growth, achieving a complete tumor inhibition rate of 25.0%. Furthermore, compared to the PBS group, the anti-PD-1 group, and the Mn@Neo-mRNA-LNP group, the Mn@Neo-mRNA-LNP combined with the anti-PD-1 antibody group effectively prolonged the survival time of mice. Figure 34 This indicates that the combination of Mn@Neo-mRNA-LNP and therapeutic PD-1 antibody further enhances the anti-tumor efficacy of Mn@Neo-mRNA-LNP.

[0172] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A spleen-targeting lipid nanoparticle loaded with mRNA and adjuvant metal ions, comprising spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions; The spleen-targeting lipid nanoparticles carrying mRNA include spleen-targeting lipid nanoparticles and mRNA; the raw materials of the spleen-targeting lipid nanoparticles include ionizable lipids, cofactor phospholipids, steroidal lipids, polyethylene glycol-lipid conjugates, and targeted functional lipids. in, The targeted functional lipid is anionic lipid, and the anionic lipid is sodium salt of 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine. The anionic lipids account for 10-20% of the total lipids in the raw material. The adjuvant metal ion is a manganese ion; The mass ratio of the anionic lipid to manganese ions is 1:(1-10). The ionizable lipid is ALC-0315 (((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)). The auxiliary phospholipid was selected as DSPC (1,2-distearate-sn-glycero-3-phosphatidylcholine). The steroidal lipid is cholesterol; The polyethylene glycol-lipid conjugate is DMG-PEG2000 (1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol-2000). The ionizable lipids account for 20-60% of the total lipids in the raw material. The auxiliary phospholipids account for 1-20% of the total lipids in the raw materials; The steroidal lipids account for 20-60% of the total lipids in the raw material. The polyethylene glycol-lipid conjugate accounts for 0.1-10% of the total lipids in the raw material.

2. The spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions according to claim 1, characterized in that: The mRNA is any mRNA molecule that can encode one or more tumor antigens.

3. The spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions according to claim 1 or 2, characterized in that: The ratio of the total mass of lipid molecules to the mass of mRNA in the spleen-targeting lipid nanoparticles containing mRNA and adjuvant metal ions is (10-50):

1.

4. The spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions according to claim 3, characterized in that: The ionizable lipids account for 30-50% of the total lipids in the raw material. And / or, the auxiliary phospholipids account for 2-10% of the total lipids in the raw material; And / or, the steroidal lipids account for 30-50% of the total lipids in the raw material; And / or, the polyethylene glycol-lipid conjugate accounts for 0.5-5% of the total lipids in the raw material.

5. The spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions according to claim 4, characterized in that: The anionic lipids account for 15% of the total lipids in the raw material. And / or, the ionizable lipids account for 39.35% of the total lipids in the feedstock; And / or, the auxiliary phospholipids account for 7.99% of the total lipids in the raw material; And / or, the steroidal lipids account for 36.29% of the total lipids in the raw material; And / or, the polyethylene glycol-lipid conjugate accounts for 1.36% of the total lipids in the raw material.

6. The spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions according to claim 1, characterized in that: The manganese ions are provided by divalent manganese salts, including any one of manganese chloride, manganese nitrate, and manganese sulfate.

7. The spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions according to claim 6, characterized in that: The mass ratio of the anionic lipid to manganese ions is 1:(1-5). And / or, the manganese ions are provided by manganese chloride.

8. The spleen-targeting lipid nanoparticles encapsulating mRNA and adjuvant metal ions according to claim 7, characterized in that: The mass ratio of the anionic lipid to manganese ions is 1:

1.

9. A method for preparing spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions as described in any one of claims 1-8, comprising the following steps: Step (B1): Ionizable lipids, cofactor phospholipids, steroidal lipids, polyethylene glycol-lipid conjugates, and targeted functional lipids are dissolved in an organic solvent to obtain a lipid organic phase; Step (B2): Dissolve the adjuvant metal ions and mRNA in an appropriate buffer solution to obtain an aqueous solution; Step (B3): The lipid organic phase from step (B1) and the aqueous phase solution from step (B2) are mixed and incubated to obtain a solution of spleen-targeting lipid nanoparticles loaded with mRNA and adjuvant metal ions.

10. The preparation method according to claim 9, characterized in that: The organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, tetrahydrofuran, and diethyl ether; And / or, the pH of the buffer solution is 2.0-4.0; And / or, the mass ratio of anionic lipids to adjuvant metal ions in the lipid organic phase is 1:(1-10). And / or, the total mass ratio of lipid molecules in the lipid organic phase to the mass ratio of mRNA in the mRNA solution is (10-50):1; And / or, the volume ratio of the lipid organic phase to the mRNA solution is 1:(1-5). And / or, the incubation conditions are static incubation at room temperature for 10-15 minutes.

11. The preparation method according to claim 10, characterized in that: The pH value of the buffer solution is 4.0; And / or, the mass ratio of anionic lipids to adjuvant metal ions in the lipid organic phase is 1:(1-5). And / or, the total mass ratio of lipid molecules in the lipid organic phase to the mass ratio of mRNA in the mRNA solution is 40:1; And / or, the volume ratio of the lipid organic phase to the mRNA solution is 1:

3.

12. The preparation method according to claim 11, characterized in that: The mass ratio of anionic lipids to adjuvant metal ions in the lipid organic phase is 1:

1.

13. The use of spleen-targeting lipid nanoparticles carrying mRNA and adjuvant metal ions as described in any one of claims 1-8 in the preparation of vaccines for tumor immunotherapy.

14. The application according to claim 13, characterized in that: The tumors include, but are not limited to, one or more of melanoma, colon cancer, liver cancer, breast cancer, ovarian cancer, stomach cancer, prostate cancer, and lung cancer.

15. A pharmaceutical composition for treating tumors, comprising spleen-targeting lipid nanoparticles carrying mRNA and adjuvant metal ions as described in any one of claims 1-8, and an immune checkpoint inhibitor.

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