Targeting dendritic cell mRNA delivery system as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses an mRNA delivery system for targeting dendritic cells as well as a preparation method and application of the mRNA delivery system. The mRNA delivery system is prepared by packaging tumor antigen mRNA into nano-lipid particles (LNP) and coupling the LNP with a specific antibody, and the mRNA delivery system remarkably improves the accuracy and effectiveness of immunotherapy through a'protection-targeting-activation 'triple mechanism, thereby providing technical support for realizing safer and more efficient anti-tumor immunotherapy clinically. On the basis, the invention further provides a vaccine for treating melanoma, and the vaccine has the effects of obviously promoting activation and maturation of DC cells, activating T cells in lymph nodes of a tested body in a tumor microenvironment, obviously improving the expression quantity of melanoma antigen mRNA in the spleen and the like. The goals of inhibiting the growth of melanoma and prolonging the survival time are achieved. The invention is applicable to the field of preparation of tumor vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically discloses an mRNA delivery system targeting dendritic cells, a preparation method and an application thereof. Background Art

[0002] Malignant tumors are currently the second leading cause of death worldwide. Clinically, cancer treatment options primarily include surgery, radiotherapy, chemotherapy, and novel therapies such as targeted biological therapies and immunotherapy. Immunotherapy aims to cure cancer by activating the host's anti-tumor immune system and altering the tumor-suppressing microenvironment. It primarily includes immune checkpoint inhibitors (such as PD-1 inhibitors) and cellular immunotherapy (such as chimeric antigen receptor T-cell therapy, or CAR-T). PD-1 monoclonal antibodies are currently widely used in the immunotherapy of various solid tumors. However, PD-1 monoclonal antibody therapy is effective in only a minority of patients, with the majority experiencing no response or recurrence after an initial response.

[0003] Dendritic cells (DCs), crucial members of the human immune defense system, can initiate antigen-specific immune responses in lymphoid tissues. They are the most powerful antigen-presenting cells (APCs) and are known as the "sentinels" of the immune system. They efficiently uptake, process, and present antigens. Immature DCs possess strong migratory abilities, while mature DCs effectively activate naive T cells, playing a central role in initiating, regulating, and maintaining immune responses. Therefore, using DCs as carriers for the preparation of DC cancer vaccines has become an effective approach for tumor immunotherapy.

[0004] DC-mediated tumor vaccines can be categorized as in vivo (in vivo) dendritic cell vaccines and in vitro (ex vivo) dendritic cell vaccines. Currently, all dendritic cell vaccines entering clinical trials are ex vivo vaccines. [GW1] offers the following advantages: high specificity, excellent flexibility, and the ability to optimize conditions in vitro, allowing for better control of dendritic cell maturation and antigen loading, resulting in improved therapeutic efficacy. However, the preparation of in vitro dendritic cell vaccines is cumbersome, requiring the extraction of mononuclear cells from the patient's blood or bone marrow, in vitro differentiation, dendritic cell culture, and cytokine stimulation to enhance their antigen presentation capacity. Tumor-associated antigens are then pulsed onto the dendritic cells, and the mature, antigen-loaded dendritic cells are then reinfused into the patient. The entire process involves isolation of the patient's own cells, in vitro preparation, and in vivo reinfusion, resulting in a low success rate. In vivo dendritic cell vaccines directly target and activate the patient's dendritic cells, eliminating the tedious steps of in vitro DC culture during preparation. This, in turn, reduces the production cost and price of DC vaccines and improves patient accessibility. However, dendritic cell vaccines in vivo generally have the defects of low antigen delivery efficiency, poor targeting and limited immune activation effect.

[0005] mRNA vaccines have become a key breakthrough in cancer treatment following immune checkpoint therapy due to their rapid customization, efficient immune activation, and low genotoxicity. However, most reported mRNA vaccines delivered via intravenous (IV) or intramuscular (IM) lipid nanoparticles (LNPs) exhibit very strong mRNA expression in the liver, hindering precise delivery. Summary of the Invention

[0006] In light of this, the present invention combines the advantages of mRNA-containing nanolipid particles and dendritic cancer vaccines, utilizing antibody-conjugated technology to deliver tumor antigen mRNA to dendritic cells and then to lymph nodes. This provides a dendritic cell-targeted mRNA delivery system, which can be used to prepare vaccines for treating solid tumors. The expression of tumor antigen mRNA induces a tumor-specific adaptive immune response, while the antigen is targeted and presented to dendritic cells, thereby enhancing the immune response and inhibiting tumor growth.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an mRNA delivery system targeting dendritic cells, comprising lipid nanoparticles loaded with tumor antigen mRNA, and antibodies coupled to the lipid nanoparticles; Wherein, the antibody is an antibody that can specifically bind to dendritic cell surface receptors.

[0008] This invention encapsulates tumor antigen mRNA in nanolipid particles and couples it to specific antibodies to create an mRNA delivery system. This system not only improves mRNA stability and promotes cellular uptake and cytoplasmic release, but also achieves efficient DC targeting and lymph node enrichment through receptor-specific binding, thereby optimizing the antigen presentation microenvironment and activating anti-tumor immune responses while also minimizing off-target effects. Through this triple mechanism of "protection-targeting-activation," the precision and effectiveness of immunotherapy are significantly improved, providing technical support for safer and more efficient anti-tumor immunotherapy in clinical practice.

[0009] The present invention encapsulates tumor antigen mRNA in lipid nanoparticles, which not only improves the stability of mRNA by increasing resistance to nuclease degradation and improving pH buffering capacity, but also ensures efficient expression and presentation of tumor antigen proteins by enhancing the efficiency of dendritic cells' uptake of the corresponding antigen and reducing innate immune activation, thereby balancing immunogenicity and safety. In addition, the present invention couples lipid nanoparticles with specific antibodies to precisely target DC surface receptors, improving delivery specificity. Antibody-coupled LNPs can target lymph nodes through mechanisms such as lymphocyte homing receptor binding and DC migration guidance, enhancing T cell activation efficiency and optimizing the immune activation microenvironment.

[0010] Furthermore, the tumor antigen mRNA includes solid tumor antigen mRNA including melanoma antigen mRNA, lung cancer antigen mRNA, gastric cancer antigen mRNA, liver cancer antigen mRNA, intestinal cancer antigen mRNA, cervical cancer antigen mRNA, pancreatic cancer antigen mRNA or breast cancer antigen mRNA.

[0011] Furthermore, the antibody includes an anti-CLEC9A antibody.

[0012] Antibody coupling technology is used to couple LNP with antibodies against the specific antigen CLEC9A on dendritic cells, which can target cDC1. This allows the antigen to be highly specifically targeted and migrate to the dendritic cells in the lymph nodes, where it is internalized and processed by the cells and then presented to T cells, stimulating a strong immune response.

[0013] The results of animal experiments showed that, within the experimental range of the present invention, the expression level of related mRNA in the spleen of the mRNA group coupled with anti-CLEC9A antibodies was significantly enhanced compared with the mRNA group without antibody coupling, which was about 2.5 times that of the mRNA group without antibody coupling.

[0014] In a second aspect, the present invention further provides a method for preparing the above-mentioned mRNA delivery system targeting dendritic cells, the preparation method comprising the following steps: Step 1: dilute the tumor antigen mRNA with a buffer to obtain an aqueous phase; Step 2: Mix SM-102, DSPC, cholesterol, DMG-PEG2000, and maleimide-modified DSPE-PEG (DSPE-PEG-maleimide) to obtain a lipid phase solution; Step 3: mixing the aqueous phase solution and the lipid phase solution to obtain lipid nanoparticles loaded with tumor antigen mRNA; Step 4, thiolating the antibody to obtain a thiol-modified antibody; Step 5: Evenly mix the thiol-modified antibody and lipid nanoparticles loaded with tumor antigen mRNA, place them in an environment of 22°C to 28°C for antibody coupling reaction for 1.5h to 2.5h to obtain an mRNA delivery system targeting dendritic cells.

[0015] The present invention achieves precise and efficient delivery of tumor antigen mRNA to DCs through lipid component design, active targeting strategy of antibody coupling, and process parameter optimization, providing a technical path for the development of tumor vaccines that combines safety, controllability, and biological activity, and has important clinical translation potential.

[0016] Furthermore, the molar ratio of SM-102, DSPC, cholesterol, DMG-PEG2000 and maleimide-modified DSPE-PEG is (45-50): (10-15): (38-40): (1-2): (0.3-0.7).

[0017] SM-102 is a cationic lipid used to encapsulate mRNA and promote endocytosis; DSPC is a phospholipid that helps form the lipid bilayer and enhances stability; cholesterol enhances membrane rigidity and stability; DMG-PEG2000 is used to extend circulation time and reduce immune clearance; and maleimide-modified DSPE-PEG is used for antibody conjugation. By optimizing the types and amounts of these components, the resulting LNP parameters, such as particle size and charge, are controlled to ensure effective mRNA encapsulation and release.

[0018] Furthermore, the mass ratio of the tumor antigen mRNA in the aqueous solution to the lipid phase solution is 1:(20-25).

[0019] The cationic charge of the lipids must balance the negative charge of the mRNA to ensure efficient encapsulation. An imbalance in the charge ratio can lead to particle aggregation or charge anomalies, compromising delivery efficiency. Through optimization, the present invention achieves LNPs with an appropriate charge ratio, facilitating the formation of a product with uniform particle size and moderate surface potential, which facilitates uptake by dendritic cells via endocytosis.

[0020] Furthermore, the mass ratio of the thiol-modified antibody to the lipid nanoparticles loaded with tumor antigen mRNA is (0.9-1.1): (0.9-1.1).

[0021] The purpose of thiolation of the antibody is to avoid destroying the antigen binding domain (Fab segment) of the antibody and ensure that the antibody can still specifically recognize the DC surface receptor after coupling.

[0022] Furthermore, the buffer solution comprises a sodium citrate acidified buffer solution with a pH of 4 to 5.

[0023] Furthermore, the tumor antigen mRNA includes melanoma antigen mRNA.

[0024] Furthermore, the method for preparing melanoma antigen mRNA comprises the following steps: selecting melanoma specific antigen TRP2 180-188Three repeat fragments of the mRNA were connected with a linker (amino acid sequence GGGGS), followed by in vitro transcription, enzymatic capping, and methylation to produce TRP2 mRNA. Using TRP2 mRNA as the tumor antigen mRNA and an anti-CLEC9A antibody as the conjugated antibody, the resulting mRNA delivery system targeting dendritic cells was designated the CLEC9A / TRP2 mRNA delivery system.

[0025] Tyrosinase-related protein-2 (TRP2) is a weakly immunogenic tumor-associated antigen and a natural antigen in the B16F10 tumor model. This invention uses this as an example to illustrate that mRNA can be designed based on the antigens of related solid tumors to prepare an mRNA delivery system targeting dendritic cells for the corresponding tumors. 180–188 The amino acid sequence of was used as the antigen design target for mRNA tumor vaccine, and the corresponding DNA sequence containing HA tag was designed and synthesized.

[0026] Among them, TRP2 180–188 The amino acid sequence of TRP2 containing HA tag is shown in SEQ ID No.1. 180–188 The DNA sequence is shown as SEQ ID No.2.

[0027] The average particle size of the CLEC9A / TRP2 mRNA delivery system is 120nm~150nm.

[0028] In a third aspect, the present invention provides use of the above-mentioned mRNA delivery system targeting dendritic cells in the preparation of drugs for treating solid tumors.

[0029] In a fourth aspect, the present invention provides a vaccine for treating solid tumors, wherein the active ingredients of the vaccine include the above-mentioned mRNA delivery system targeting dendritic cells, and the vaccine can also be compounded with an adjuvant on this basis.

[0030] In a fifth aspect, the present invention provides a vaccine for treating melanoma, wherein the active ingredient of the vaccine includes an mRNA delivery system targeting dendritic cells prepared using melanoma antigen mRNA as raw material.

[0031] The results of animal experiments show that, within the experimental scope of the present invention, the vaccine prepared with the CLEC9A / TRP2 mRNA delivery system has the effects of significantly promoting the activation and maturation of DC cells, activating T cells in the lymph nodes of the subjects in the tumor microenvironment, significantly increasing the expression of melanoma antigen mRNA in the spleen, and significantly increasing the number of tumor-infiltrating immune cells (such as T cells and DC cells) in mice, thereby achieving the goal of inhibiting the growth of melanoma and improving survival.

[0032] In a sixth aspect, the present invention provides a pharmaceutical composition for treating melanoma, wherein the active ingredients of the pharmaceutical composition include the aforementioned dendritic cell-targeted mRNA delivery system prepared using melanoma antigen mRNA as raw material and a PD-1 monoclonal antibody. The active ingredients of the vaccine may also include an adjuvant or a TLR agonist.

[0033] Compared with the use of PD1 monoclonal antibody alone or the use of melanoma antigen mRNA nanolipid particles not coupled with the corresponding antibody, the CLEC9A / TRP2 mRNA delivery system provided by the present invention combined with PD1 monoclonal antibody has a synergistic effect in the treatment of melanoma, which can significantly increase the proportion of activated T cells in the body, especially IFNγ + / CD8 + The proportion of T cells can effectively inhibit the growth of melanoma and increase the survival of cancer-bearing animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 Schematic diagram of the TRP2 epitope antigen map in Example 1 of the present invention; Figure 2 Schematic diagram of the TRP2 mRNA vector structure in Example 1 of the present invention; Figure 3 This is a particle size distribution diagram of the TRP2 mRNA delivery system and the CLEC9A / TRP2 mRNA delivery system in Example 1 of the present invention, wherein: Figure 3 A) represents the particle size distribution of TRP2 mRNA-LNP. Figure 3 B) Representative particle size distribution of CLEC9A / TRP2 mRNA-LNPs; Figure 4 The expression of TRP2 mRNA in TRP2 mRNA-LNP in 293 cells in Example 1 of the present invention; wherein, Figure 4 A) represents TRP2 180-188 ×3 Antigen expression, Figure 4 B) represents the expression of β-actin; Figure 5 The MHCII in the lymph nodes of different groups of mice in Example 1 of the present invention + / CD11c + The percentage of DC cells in the total DC cells; Figure 6 CD80 in the lymph nodes of different groups of mice in Example 1 of the present invention + / CD11c + The percentage of DC cells in the total DC cells; Figure 7 CD3 in the lymph nodes of different groups of mice in Example 1 of the present invention + The percentage of T cells in total T cells; Figure 8 The IFNγ in the lymph nodes of different groups of mice in Example 1 of the present invention + / CD8 + The percentage of T cells in total T cells; Figure 9 CD3 infiltrated into mouse melanoma tissue in G3 and G4 groups in Example 1 of the present invention + T cells and CD11c + Fluorescence images of dendritic cells; Figure 10 These are representative pictures of mice in different groups on day 17 in Example 2 of the present invention; Figure 11 This is a graph showing the changes in tumor volume of mice in different groups from day 7 to day 17 in Example 2 of the present invention; Figure 12 This is a survival curve diagram of mice in different groups in Example 3 of the present invention; Figure 13 The expression of Fluc mRNA in different organs in different groups of mice in Example 4 of the present invention; Figure 14 This is a graph showing the relative expression ratios of Fluc mRNA in the lymph nodes and livers of different groups of mice in Effective Example 4 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] In order to better illustrate the embodiments of the present invention, further examples are given below.

[0038] The method for constructing a tumor xenograft mouse melanoma model in the present invention is as follows: Tumor cell implantation: 2×10 5 B16 cells were injected subcutaneously into the right flank of mice (C57BL / 6) along with 1:1 Matrigel (total volume 200 μL). Tumor size was measured with a caliper every two to three days and the formula (a2 × b) / 2 (a, width; b, length), and the tumor volume was calculated.

[0039] When the tumor volume reaches about 50 mm 5-7 days after tumor inoculation 3 At the time of the study, mice were randomly divided into groups (n=8 mice per group) according to tumor size. According to the experimental design, mice were vaccinated with PBS, TRP2 mRNA-LNP, PD1, CLEC9A / TRP2 mRNA-LNP, or CLEC9A / TRP2 mRNA-LNP+PD1, with an injection frequency of 3-5 days per injection for a total of two injections. The PBS-treated group served as the control group.

[0040] Example 1 1. This embodiment provides an mRNA delivery system targeting dendritic cells, which includes lipid nanoparticles loaded with tumor antigen mRNA and antibodies coupled to the lipid nanoparticles; Among them, the tumor antigen mRNA is TRP2 mRNA, and the antibody is anti-CLEC9A antibody.

[0041] Second, this embodiment also provides a method for preparing the above-mentioned mRNA delivery system targeting dendritic cells, the preparation method comprising the following steps: Step 1: The present invention selects three repeat fragments of the melanoma-specific antigen TRP2, connects them with a linker, and then performs in vitro transcription to obtain TRP2 mRNA, which is then diluted with a buffer to obtain an aqueous phase. The specific contents are as follows: S11. The present invention uses TRP2 180–188 The amino acid sequence of TRP2 was used as the antigen design target of mRNA tumor vaccine, and the corresponding DNA sequence containing HA tag was designed and synthesized. 180–188 The repeat fragment (without a linker in the middle) was then added between the TRP2 repeat fragment and the HA tag (the amino acid sequence of the linker is GGSLGGGGSGGGGGS), and the DNA sequence of the N-terminal signal peptide and MHC-I trafficking signal (MITD) was inserted into the pmRVac vector, and the DNA template for in vitro transcription was used. The schematic diagram of the TRP2 epitope antigen map is shown in the figure. Figure 1 The pmRVac vector contains a T7 promoter sequence, a 5'UTR, a 3'UTR, a 110nt polyA segment, and a SapI restriction enzyme site for transcription termination.

[0042] S12. Cap1 mRNA was generated using the vaccinia capping system. After denaturing the uncapped transcript by heat, vaccinia capping enzyme, 2'-O methyltransferase, GTP, S-adenosylmethionine (SAM), and capping buffer were added to establish the capping reaction. The reaction mixture was incubated at 37°C for 1 hour. Subsequently, the template DNA was removed by DNase I treatment. Schematic diagram of the TRP2 mRNA vector is shown in Figure 1. Figure 2 As shown in the figure. Cap1 mRNA was generated by T7 in vitro transcription, followed by enzymatic capping and methylation. To generate uncapped in vitro RNA transcripts, a linear DNA template was mixed with T7 RNA polymerase, nucleotide triphosphates (NTPs), and a magnesium-containing buffer to set up an in vitro transcription reaction. The reaction mixture was incubated at 37°C for 2 hours. For the synthesis of m1Psi-modified transcripts, m1Psi was used instead of UTP for in vitro transcription.

[0043] S13. Further purify the capped transcript using magnetic beads. Elute the isolated mRNA with pH 4 sodium citrate buffer and store at -80°C to obtain a TRP2 mRNA stock solution. Measure mRNA concentration and purity using a UV-visible spectrophotometer. Assess mRNA integrity by denaturing agarose gel analysis. Identify mRNA molecules using mRNA sequencing.

[0044] S14. Sanger sequencing was used to confirm that the tumor antigen mRNA sequence was correct, the mRNA length was correct, and the mRNA integration was well maintained. 260 and A 280nm The absorbance ratio at 260 / A 280 ) was 2.12, indicating that the synthesized mRNA was of high purity with minimal contamination by DNA and protein.

[0045] The TRP2 mRNA mother solution was resuspended in a 50 mM NaAc solution at pH 4 to obtain a TRP2 mRNA solution, which was an aqueous phase solution.

[0046] Among them, TRP2 180–188 The amino acid sequence is shown in SEQ ID No. 1 and is as follows: SVYDFFVWL; HA-tagged TRP2 180–188 The DNA sequence is shown in SEQ ID No. 2, which is as follows: ATGAGCGTGTATGATTTTTTTGTGTGGCTGTAA.

[0047] Step 2: Mix SM102, DSPC, cholesterol, DMG-PEG200, and maleimide-modified DSPE-PEG (DSPE-PEG-maleimide) at a molar ratio of 50:10:38:1.5:0.5 and dissolve thoroughly in 1400 μL of anhydrous ethanol to prepare a lipid phase solution. SM102 is present at 42 μmol.

[0048] Step 3: Using microfluidics technology, the lipid phase solution and the aqueous phase solution were mixed to generate 2286 μg of lipid nanoparticles loaded with TRP2 mRNA, which were recorded as TRP2 mRNA-LNP.

[0049] The molar mass of TRP2 mRNA was calculated based on the N:P ratio of the cationic lipid (SM102) in TRP2 mRNA (N:P = 6:1). The total mass of mRNA required was calculated based on the molecular mass of TRP2 mRNA. The total mass of the lipid phase solution was calculated based on the molar ratios of the five lipid components. The actual amounts and ratios of TRP2 mRNA and lipid phase solution were calculated based on a ratio of 1:23.273 for the total mass of mRNA:total mass of the lipid phase solution. When preparing TRP2 mRNA-LNPs, the ethanol must be removed promptly and the final solution must be dissolved in 15 mM Tris-HCl + 15 mM sodium acetate for later use. The concentration of TRP2 mRNA-LNPs was 544 ng / μL.

[0050] Step 4: This step is to prepare the thiol-modified antibody. The specific contents are as follows: Take 2286 μg of anti-CLEC9A antibody (10 mM HEPES, 0.15 M NaCl, pH 7.5), weigh SATA at a molar ratio of 9:1, and dissolve it in DMSO to obtain an 18 mmol / L SATA solution. The SATA solution and antibody solution were mixed and reacted at room temperature for 0.5 h. The mixture was then ultrafiltered using a 10K ultrafiltration tube. The PBS buffer solution, pH 7.4, was exchanged 3-4 times. The solution was centrifuged at 9400 rpm for 7 min at 8°C. After concentration, the volume was fixed to 200 μL using PBS buffer to obtain CLEC9A antibody-SATA. Dissolve 0.5 M hydroxylamine hydrochloride in PBS buffer containing 25 mM EDTA, pH 7.3 to form 200 μL of 0.5 M hydroxylamine hydrochloride solution, and react with the above-mentioned CLEC9A antibody-SATA at room temperature for 2 hours. After deprotection of CLEC9A antibody-SATA, ultrafiltration was performed using a 10K ultrafiltration tube. The solution was exchanged 3-4 times with PBS buffer containing 10 mM EDTA, and centrifuged at 8°C for 7 minutes. After changing the solution and making the volume to 300 μL, the thiol-ylated antibody (CLEC9A antibody-SH) was obtained for use.

[0051] Step 5. The above-mentioned thiol-modified antibody was evenly mixed with an equal mass of TRP2 mRNA-LNP, placed in a 25°C environment for 2 hours for antibody coupling reaction, and then incubated at 4°C overnight to allow the reaction to be more complete, thereby obtaining an mRNA delivery system targeting dendritic cells, which was recorded as CLEC9A / TRP2 mRNA-LNP.

[0052] This example also examined the particle size of the CLEC9A / TRP2 mRNA-LNP prepared above and the expression level of TRP2 mRNA in 293 cells, as follows: (1) This example investigates the effect of antibody coupling on the particle size of the prepared mRNA delivery system, and investigates the particle size distribution of TRP2 mRNA-LNP and CLEC9A / TRP2 mRNA-LNP. The particle size distribution comparison of the two is shown in the figure below. Figure 3 As shown, where: Figure 3 A) represents the particle size distribution of TRP2 mRNA-LNP. Figure 3 B) represents the particle size distribution of CLEC9A / TRP2 mRNA-LNP. The average particle size of TRP2 mRNA-LNP without anti-CLEC9A antibody conjugation is 72nm, while the average particle size of CLEC9A / TRP2 mRNA-LNP conjugated with anti-CLEC9A antibody is 139nm. Figure 3 It can also be seen that after coupling with antibodies, the particle size of the prepared nanolipid particles increased significantly, and the overall particle size distribution became wider.

[0053] (2) In this embodiment, 2 μg (in terms of mRNA) of the TRP2 mRNA-LNP prepared above was used to transfect 293 cells. After 24 hours and 48 hours of transfection, the cell contents were extracted for protein, and the transient expression of TRP2 antigen in the cells was detected by Western blotting. At the same time, β-actin was used as a control to normalize the expression level of mRNA or protein. The expression of TRP2 mRNA in 293 cells in TRP2 mRNA-LNP was as follows: Figure 4 As shown, Figure 4 A) represents TRP2180-188 ×3 Antigen expression, lane Blank represents nanolipid particles prepared without TRP2 mRNA vector loading, No.1 to No.4 are different TRP2 mRNA-LNP clones; Figure 4 B) represents the expression of β-actin. Lane Blank represents the nanolipid particles prepared without loading the TRP2 mRNA vector. No.1~No.4 are different TRP2 mRNA-LNP clones.

[0054] Depend on Figure 4 It can be seen that TRP2 mRNA-LNP can specifically express the antigen corresponding to the melanoma tumor epitope antigen TRP2-related mRNA in cells.

[0055] Example 2 This embodiment provides an mRNA delivery system targeting dendritic cells, which includes lipid nanoparticles loaded with tumor antigen mRNA and antibodies coupled to the lipid nanoparticles; Among them, the tumor antigen mRNA is TRP2 mRNA, and the antibody is anti-CLEC9A antibody.

[0056] This example also provides a method for preparing the mRNA delivery system targeting dendritic cells. The method is substantially the same as that in Example 1, with the only difference being slight differences in the following three steps. Other preparation parameters are the same as those in Example 1: (1) In step 2, SM102, DSPC, cholesterol, DMG-PEG200, and maleimide-modified DSPE-PEG were mixed at a molar ratio of 48:12:40:1:0.3, with SM102 at 40.3 μmol. (2) In step 3, the actual amount and ratio of TRP2 mRNA and lipid phase solution were calculated based on the ratio of total mRNA mass to total lipid phase solution mass = 1:20; (3) Step 5: Mix the thiol-modified antibody and TRP2 mRNA-LNP at a mass ratio of 0.9:1.1, place the mixture at 22°C for 2.5 hours to carry out the antibody coupling reaction, and then place it at 4°C overnight to allow the reaction to be more complete. The mRNA delivery system targeting dendritic cells was obtained, which was recorded as CLEC9A / TRP2 mRNA-LNP.

[0057] Example 3 This embodiment provides an mRNA delivery system targeting dendritic cells, which includes lipid nanoparticles loaded with tumor antigen mRNA and antibodies coupled to the lipid nanoparticles; Among them, the tumor antigen mRNA is TRP2 mRNA, and the antibody is anti-CLEC9A antibody.

[0058] This example also provides a method for preparing the mRNA delivery system targeting dendritic cells. The method is substantially the same as that in Example 1, with the only difference being slight differences in the following three steps. Other preparation parameters are the same as those in Example 1: (1) In step 2, SM102, DSPC, cholesterol, DMG-PEG200, and maleimide-modified DSPE-PEG were mixed at a molar ratio of 45:15:39:2:0.7, with SM102 at 37.8 μmol. (2) In step 3, the actual amount and ratio of TRP2 mRNA and lipid phase solution were calculated based on the ratio of total mRNA mass to total lipid phase solution mass = 1:25; (3) Step 5: Mix the thiol-modified antibody and TRP2 mRNA-LNP at a mass ratio of 1.1:0.9, place the mixture at 28°C for 1.5 hours to carry out the antibody coupling reaction, and then place it at 4°C overnight to allow the reaction to be more complete. The mRNA delivery system targeting dendritic cells was obtained, which was recorded as CLEC9A / TRP2 mRNA-LNP.

[0059] Effect Example 1 The effect of the CLEC9A / TRP2 mRNA-LNP prepared in Example 1 on activating DC immune cell activation and T cell immune response was investigated in the present invention. The specific contents are as follows: In this study, a mouse melanoma model was established and the tumor volume reached approximately 50 mm 5 days after tumor inoculation. 3 Mice were randomly divided into five groups (n = 8 mice per group) based on tumor size and vaccinated with PBS, TRP2 mRNA-LNP, CLEC9A / TRP2 mRNA-LNP, or CLEC9A / TRP2 mRNA-LNP + PD1. The PBS-treated group served as the control group. The five groups of mice are designated G1 to G5, respectively. The injected drugs and doses for each group are shown in Table 1.

[0060] Table 1

[0061] Tumor progression and weight were assessed every 3 days by measuring tumor size.On the third day after the second vaccine injection, mice were sacrificed, lymph nodes and spleens were removed, and DCs and T cells were isolated and analyzed by flow cytometry.

[0062] (1) MHCII in lymph nodes of mice in different groups + / CD11c + The percentage of DC cells in the total DC cells is as follows Figure 5 As shown, CD80 in lymph nodes of mice in different groups+ / CD11c + The percentage of DC cells in the total DC cells is as follows Figure 6 shown.

[0063] Depend on Figure 5-6 It can be seen that compared with the G1 group and the G3 group, the CLEC9A / TRP2 mRNA-LNP provided by the present invention can better promote the activation and maturation of DC cells. + / CD11c + The percentage of CLEC9A / TRP2 mRNA-LNP in total DC cells increased from 33% in the PBS group to 91%. After the combination of CLEC9A / TRP2 mRNA-LNP and PD1 monoclonal antibody, activated DC cells (MHCII + / CD11c + ) was slightly lower than that when CLEC9A / TRP2 mRNA-LNP was used alone.

[0064] Activated DC cells (CD80 + / CD11c + The percentage of CLEC9A / TRP2 mRNA-LNP in total DC cells increased from 0.3% in the PBS group to 6%. When CLEC9A / TRP2 mRNA-LNP was combined with PD1 monoclonal antibody, PD1 monoclonal antibody could further activate DC cells (CD80 + / CD11c + ) increased to 14%.

[0065] From the above, it can be seen that compared with the injection of PBS or TRP2 mRNA-LNP not coupled to anti-CLEC9A antibody, the CLEC9A / TRP2 mRNA-LNP provided by the present invention can more effectively activate DC immune cells and promote the maturation of DC cells.

[0066] (2) CD3 in lymph nodes of mice in different groups + The percentage of T cells in the total T cells Figure 7 As shown, IFNγ in the lymph nodes of mice in different groups + / CD8 + The percentage of T cells in the total T cells Figure 8 shown.

[0067] Depend on Figure 7-8 As shown, TRP2 mRNA-LNP (9.1%) and CLEC9A / TRP2 mRNA-LNP (7.3%) increased the percentage of CD3+ T cells in the spleen compared with PBS treatment (5.8%). The results also showed that CLEC9A / TRP2 mRNA-LNP significantly increased IFNγ + / CD8 +The percentage of T cells increased from 20.8% (PBS) to 37.5% (CLEC9A / TRP2 mRNA-LNP). The combined treatment of CLEC9A / TRP2 mRNA-LNP and PD1 monoclonal antibody increased IFNγ + / CD8 + The T cell expression level was increased by 20.8% compared with PBS treatment.

[0068] (3) The expression of CD3 in G3 and G4 groups was investigated by tumor tissue sectioning and fluorescence staining. + T cells and CD11c + Can dendritic cells infiltrate into mouse melanoma tissue? + T cells and CD11c + Fluorescence images of dendritic cells Figure 8 shown.

[0069] Depend on Figure 8 It can be seen that CD3+T cells and CD11c + Dendritic cells can infiltrate melanoma tumors in mice, and the number of infiltrating lymphocytes significantly increased in tumors treated with the G4 group (CLEC9A / TRP2 mRNA-LNP). Because tumor-infiltrating lymphocytes are negatively correlated with tumor growth, it is speculated that CLEC9A / TRP2 mRNA-LNP can inhibit melanoma growth.

[0070] In summary, within the experimental scope of the present invention, the CLEC9A / TRP2 mRNA-LNP provided by the present invention can effectively activate DC immune cell activation, enhance T cell activation efficiency, and optimize the immune activation microenvironment; in addition, CLEC9A / TRP2 mRNA-LNP can also promote CD3 + T cells and CD11C + Dendritic cells infiltrate tumor cells and have the potential to better inhibit tumor growth.

[0071] Effect Example 2 This study investigated the inhibitory effect of CLEC9A / TRP2 mRNA-LNP prepared in Example 1 on melanoma. It also investigated whether different injection methods (such as intravenous injection or subcutaneous injection) had an effect on the tumor inhibition. The details are as follows: In this study, a mouse melanoma model was established and the tumor volume reached 50 mm 5 days after tumor inoculation. 3Around 3 dpi, the mice were randomly divided into six groups (n=8 mice per group) according to the tumor size, and PBS, TRP2 mRNA-LNP, CLEC9A / TRP2 mRNA-LNP, or CLEC9A / TRP2 mRNA-LNP+PD1 were used as vaccines to inoculate the six groups of mice, of which the PBS group served as the control group.

[0072] The six groups of mice were respectively designated as G1 to G6. The injected drugs and dosages of different groups are shown in Table 2.

[0073] Table 2

[0074] Tumor progression and weight were assessed by measuring tumor size every 3 days, and tumor size was recorded from day 7 to day 17. Representative images of mice in different groups on day 17 are shown in the figure. Figure 10 As shown in the figure, the changes in tumor volume of mice in different groups from day 7 to day 17 are shown in the figure. Figure 11 shown.

[0075] Depend on Figure 10-11 It was found that monotherapy with PD-1 monoclonal antibody (group G2) had no significant tumor inhibitory effect compared with PBS (group G1). TRP2 mRNA-LNPs unconjugated to anti-CLEC9A antibodies (group G3) inhibited tumor growth compared with PBS (P < 0.05). Intravenous injection of CLEC9A / TRP2 mRNA-LNPs alone, and intravenous or subcutaneous injection of CLEC9A / TRP2 mRNA-LNPs in combination with PD-1 monoclonal antibody, significantly inhibited tumor growth compared with the PBS group. Furthermore, the combination of CLEC9A / TRP2 mRNA-LNPs and PD-1 monoclonal antibody was found to have a slightly better inhibitory effect on melanoma than CLEC9A / TRP2 mRNA-LNPs alone.

[0076] Effect Example 3 This effect example investigates the effect of CLEC9A / TRP2 mRNA-LNP prepared in Example 1 on the survival of melanoma mice. The specific contents are as follows: Establish a mouse melanoma model until the tumor volume reaches approximately 50 mm 3 The model mice were randomly divided into four groups (8 mice in each group) and injected with PBS, TRP2 mRNA-LNP, CLEC9A / TRP2 mRNA-LNP, or CLEC9A / TRP2 mRNA-LNP+PD1, respectively. The four groups of mice were respectively recorded as G1 to G4. The injection drugs and doses of different groups are shown in Table 3.

[0077] Table 3

[0078] The tumor size was measured and recorded every 2-3 days. 2 The mice were humanely killed and the survival rate of the mice was finally calculated. The survival curves of mice in different groups are shown in the figure below. Figure 10 shown.

[0079] Depend on Figure 12 It can be seen that CLEC9A / TRP2 mRNA-LNP increased the survival time of B16 tumor mice from 18 days in the G1 group to 25 days, with a survival improvement rate of up to 38.8%.

[0080] Effect Example 4 This study used FireflyLuciferase (FLuc) protein as a fluorescent marker to investigate the distribution of LNPs in different organs after conjugation with anti-CLEC9A antibodies. The details are as follows: Eighteen 8-week-old male ICR mice were randomly divided into three groups (n=6). PBS, FluCell mRNA-LNP, or CLEC9A / Fluc mRNA-LNP were injected via the tail vein at a dose of 5 μg / mouse (based on mRNA). PBS served as the control group.

[0081] The preparation method of CLEC9A / Fluc mRNA-LNP is basically the same as the preparation method of CLEC9A / TRP2 mRNA-LNP described in Example 1, except that an equal amount of Fluc mRNA is used instead of TRP2 mRNA. The preparation method of Fluc mRNA includes synthesizing luciferase-encoding mRNA by in vitro transcription, modifying the 5' end with an ARCA cap, and optimizing the 3' end with a poly A tail to obtain Fluc mRNA. The preparation method of Fluc mRNA-LNP is basically the same as the preparation method of CLEC9A / Fluc mRNA-LNP, with the only difference being that steps 4 and 5 are not performed, that is, thiol-linked antibodies are not prepared, and the prepared Luciferase mRNA-containing LNP is not coupled with antibodies, ultimately producing Fluc mRNA-LNP.

[0082] Six hours after injection, mice were intraperitoneally injected with the luciferin substrate Luciferin (150 mg / kg). After 5-10 minutes of substrate distribution, the mice were sacrificed and the blood was removed by cardiac perfusion with PBS. The heart, liver, spleen, lung, kidney, and lymph node tissues were collected for ex vivo tissue BLI imaging. The relative expression of Fluc mRNA was determined by the fluorescence intensity in different organs. The expression of Fluc mRNA in different organs of mice in different groups is shown in the figure below. Figure 13 The relative expression ratios of Fluc mRNA in lymph nodes and liver of mice in different groups are shown in Figure 14 shown.

[0083] Depend on Figure 13-14 It can be seen that the fluorescent protein in the Fluc mRNA-LNP group not coupled with anti-CLEC9A antibody is expressed more in the liver. However, liver expression may induce nonspecific immune activation or liver toxicity, and the liver is not an ideal site for initiating anti-tumor immunity.

[0084] CLEC9A / Fluc mRNA-LNPs were able to target the spleen, with the relative expression of Fluc mRNA in lymph nodes and liver being approximately 2.5 times that of the Fluc mRNA-LNP group. This indicates that LNPs conjugated with anti-CLEC9A antibodies can target the spleen, not only more efficiently stimulating anti-tumor T cell responses but also significantly reducing mRNA exposure in the liver and minimizing toxicity. Furthermore, targeted delivery allows the target mRNA to achieve effective immunogenicity at a lower dose, reducing drug dosage and production costs.

[0085] In summary, the present invention provides a dendritic cell-targeted mRNA delivery system, using anti-CLEC9A antibodies and TRP2 mRNA as examples. This system, used alone or in combination with PD-1 monoclonal antibodies as the active ingredient in a melanoma vaccine, effectively activates the patient's own immune system, promotes DC maturation, enhances T cell activation efficiency, significantly inhibits melanoma growth, and improves the survival rate of tumor-bearing mice. It possesses significant scientific research value and potential for clinical translation.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A mRNA delivery system targeting dendritic cells, characterized in that: The invention comprises lipid nanoparticles loaded with tumor antigen mRNA and antibodies coupled to the lipid nanoparticles; Wherein, the antibody is an antibody that can specifically bind to dendritic cell surface receptors.

2. The mRNA delivery system targeting dendritic cells according to claim 1, wherein The tumor antigen mRNA includes solid tumor antigen mRNA including melanoma antigen mRNA, lung cancer antigen mRNA, gastric cancer antigen mRNA, liver cancer antigen mRNA, intestinal cancer antigen mRNA, cervical cancer antigen mRNA, pancreatic cancer antigen mRNA or breast cancer antigen mRNA; and / or The antibodies include anti-CLEC9A antibodies.

3. The method for preparing the mRNA delivery system targeting dendritic cells according to claim 1 or 2, wherein: The preparation method comprises the following steps: Step 1: dilute the tumor antigen mRNA with a buffer to obtain an aqueous phase; Step 2: Mix SM-102, DSPC, cholesterol, DMG-PEG2000 and maleimide-modified DSPE-PEG to obtain a lipid phase solution; Step 3: mixing the aqueous phase solution and the lipid phase solution by microfluidic technology to obtain lipid nanoparticles loaded with tumor antigen mRNA; Step 4, thiolating the antibody to obtain a thiolated antibody; Step 5: Evenly mix the thiol-modified antibody and lipid nanoparticles loaded with tumor antigen mRNA, and place them in an environment of 22° C. to 28° C. for 1.5 h to 2.5 h to obtain an mRNA delivery system targeting dendritic cells.

4. The method for preparing the mRNA delivery system targeting dendritic cells according to claim 3, wherein: The molar ratio of SM-102, DSPC, cholesterol, DMG-PEG2000 and maleimide-modified DSPE-PEG is (45-50): (10-15): (38-40): (1-2): (0.3-0.7); and / or The mass ratio of the tumor antigen mRNA in the aqueous solution to the lipid phase solution is 1:(20-25); and / or The mass ratio of the thiol-modified antibody to the lipid nanoparticles loaded with tumor antigen mRNA is (0.9-1.1): (0.9-1.1).

5. The method for preparing the mRNA delivery system targeting dendritic cells according to claim 3, wherein: The buffer solution comprises a sodium citrate acidified buffer solution having a pH of 4 to 5; and / or The tumor antigen mRNA includes melanoma antigen mRNA.

6. The method for preparing the mRNA delivery system targeting dendritic cells according to claim 5, wherein: The method for preparing melanoma antigen mRNA comprises the following steps: selecting a melanoma specific antigen TRP2 180-188 The three repeat fragments were connected with a linker, and then transcribed in vitro, enzymatically capped and methylated to obtain TRP2 mRNA.

7. Use of the mRNA delivery system targeting dendritic cells as claimed in claim 1 or 2 in the preparation of drugs for treating solid tumors.

8. A vaccine for treating solid tumors, characterized in that: The active ingredients of the vaccine include the mRNA delivery system targeting dendritic cells as claimed in claim 1 or 2.

9. A vaccine for treating melanoma, characterized in that: The active ingredients of the vaccine include the mRNA delivery system targeting dendritic cells prepared by the preparation method according to claim 5 or 6.

10. A pharmaceutical composition for treating melanoma, characterized in that: The active ingredients of the pharmaceutical composition include the mRNA delivery system targeting dendritic cells prepared by the preparation method according to claim 5 or 6 and PD1 monoclonal antibody.

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