CN120678948A - MRNA delivery system of targeted dendritic cells and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of biomedicine and specifically discloses an mRNA delivery system targeting dendritic cells, its preparation method, and its application. The present invention encapsulates tumor antigen mRNA in nanolipid particles (LNPs) and couples them to specific antibodies to produce an mRNA delivery system. This system significantly improves the precision and effectiveness of immunotherapy through a triple mechanism of "protection-targeting-activation," providing technical support for achieving safer and more efficient anti-tumor immunotherapy in clinical practice. Based on this, the present invention also provides a vaccine for the treatment of melanoma, which has the effects of significantly promoting the activation and maturation of DC cells, activating T cells in the subject's lymph nodes within the tumor microenvironment, and significantly increasing the expression of melanoma antigen mRNA in the spleen, thereby achieving the goals of inhibiting melanoma growth and improving survival. The present invention is suitable for the preparation of tumor vaccines.

Description

MRNA delivery system of targeted dendritic cells and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, and particularly discloses an mRNA delivery system of a targeted dendritic cell, a preparation method and application thereof.

Background

Malignant tumors are the second leading cause of death in the world, and clinical tumor treatment means mainly comprise surgery, radiotherapy, chemotherapy and novel treatment methods including biological targeting treatment and immunotherapy. Immunotherapy is to activate host anti-tumor immunity, change the microenvironment for inhibiting tumor, and finally achieve the purpose of tumor treatment, and mainly comprises immune checkpoint inhibitor (such as PD1 inhibitor), cell immunotherapy (such as chimeric antigen receptor T cell therapy CAR-T), and the like. Treatment of solid tumors with PD1 mab is currently widely used in immunotherapy of a variety of solid tumors. However, PD1 mab treatment was effective in only a few patients, but not in most patients, or tumors began to recur after initial response.

Dendritic Cells (DCs), which are important members of the human immune defense system, are the most powerful Antigen Presenting Cells (APCs), known as the "sentinel" of the immune system, capable of initiating antigen-specific immune responses in lymphoid tissues. It can efficiently ingest, process and present antigens. Immature DCs have a strong migration capacity, whereas mature DCs can effectively activate naive T cells, in the central link of starting, regulating, and maintaining immune responses. Therefore, the selection of dendritic cells as a carrier for the preparation of DC cancer vaccines has become an effective means of tumor immunotherapy.

DC-mediated tumor vaccines can be divided into in vivo (in vivo) dendritic cell vaccines and in vitro (ex vivo) dendritic cell vaccines. The dendritic cell vaccines entering the clinical stage are all in vitro vaccines at present. The [ GW1] has the advantages of high specificity, good flexibility, capability of in vitro condition optimization, better control of dendritic cell maturation and antigen loading, and better treatment effect. However, in vitro dendritic cell vaccines are cumbersome to manufacture, requiring the extraction of monocytes from the patient's blood or bone marrow, in vitro differentiation, culturing of the dendritic cells, stimulation with cytokines to enhance their antigen presentation capacity, pulsing of tumor-associated antigens onto the dendritic cells, and finally reinfusion of the mature, antigen-loaded dendritic cells into the patient. The whole process involves the links of autologous cell separation, in-vitro preparation, in-vivo reinfusion and the like of patients, and has low success rate. The dendritic cell vaccine in the body can directly target and activate the dendritic cells in the patient, and the complicated step of in-vitro DC culture can be avoided during the preparation, so that the production cost and price of the DC vaccine can be reduced, and the accessibility of the patient can be improved. However, in vivo dendritic cell vaccines generally suffer from the disadvantages of low antigen delivery efficiency, poor targeting and limited immune activation.

MRNA vaccine is an important breakthrough direction following immune checkpoint therapy in cancer treatment with the advantages of rapid customization, efficient immune activation, low genotoxicity and the like. However, most reported Intravenous (IV) or Intramuscular (IM) injected nano-lipid particle (LNP) -based mRNA vaccines exhibit very strong mRNA expression in the liver and cannot be delivered accurately.

Disclosure of Invention

In view of this, the present invention combines the characteristics of nano-lipid particles containing mRNA and dendritic cancer vaccines, utilizes antibody coupling technology to deliver tumor antigen mRNA to dendritic cells and to direct to lymph nodes, provides an mRNA delivery system targeting dendritic cells, and applies it in preparing vaccines for treating solid tumors. The tumor specific adaptive immune response is induced by the expression of tumor antigen mRNA, and simultaneously, the antigen is presented to dendritic cells through targeting combination, so that the effect of enhancing the immune response is achieved, and the growth of tumors is further inhibited.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

In a first aspect, the invention provides a dendritic cell-targeted mRNA delivery system comprising a lipid nanoparticle loaded with tumor antigen mRNA, and an antibody coupled to the lipid nanoparticle;

wherein the antibody is an antibody that specifically binds to a dendritic cell surface receptor.

According to the invention, tumor antigen mRNA is encapsulated into nano lipid particles and coupled with specific antibodies to prepare an mRNA delivery system, the system not only can improve the stability of mRNA and promote cell uptake and cytoplasmic release, but also can realize efficient targeting and lymph node enrichment of DC of the delivery system through receptor specific binding, thereby optimizing antigen presentation microenvironment, activating anti-tumor immune response, and reducing off-target effect. Through a triple mechanism of protection, targeting and activation, the accuracy and the effectiveness of immunotherapy are obviously improved, and technical support is provided for realizing safer and more efficient anti-tumor immunotherapy in clinic.

According to the invention, tumor antigen mRNA is encapsulated in the lipid nanoparticle, so that not only can the stability of the mRNA be improved by improving the resistance to nuclease degradation and the pH buffering capacity, but also the high-efficiency expression and presentation of tumor antigen proteins and the balance of immunogenicity and safety can be ensured by enhancing the uptake efficiency of dendritic cells on corresponding antigens, reducing the inherent immune activation and other effects. In addition, the specific antibody is coupled through the lipid nanoparticle, so that the DC surface receptor can be precisely targeted, the delivery specificity is improved, the LNP coupled with the antibody can target the lymph node through the mechanisms of lymph homing receptor binding, DC migration guiding and the like, the T cell activation efficiency is enhanced, and the immune activation microenvironment is optimized.

Further, the tumor antigen mRNA includes melanoma antigen mRNA, lung cancer antigen mRNA, stomach cancer antigen mRNA, liver cancer antigen mRNA, intestinal cancer antigen mRNA, cervical cancer antigen mRNA, pancreatic cancer antigen mRNA or breast cancer antigen mRNA.

Further, the antibodies include anti-CLEC 9A antibodies.

The antibody coupling technology is utilized to couple LNP and an antibody of a specific antigen CLEC9A on dendritic cells, so that the cDC1 can be targeted, the antigen can be highly specifically targeted and migrate to the dendritic cells in the lymph nodes, and the antigen is presented to T cells after endocytosis and treatment of the cells, so that a strong immune response is stimulated.

Animal experiment results show that in the experimental range of the invention, compared with the mRNA group which is not coupled with the antibody, the expression quantity of related mRNA in spleen of the mRNA group coupled with the anti-CLEC 9A antibody is obviously enhanced, which is about 2.5 times of that of the mRNA group not coupled with the antibody.

In a second aspect, the present invention also provides a method for preparing the above dendritic cell-targeted mRNA delivery system, the method comprising the steps of:

step one, diluting tumor antigen mRNA by using a buffer solution to obtain a water phase;

Mixing SM-102, DSPC, cholesterol, DMG-PEG2000 and maleimide modified DSPE-PEG (DSPE-PEG-maleimide) to obtain a lipid phase solution;

step three, mixing the aqueous phase solution and the lipid phase solution to obtain lipid nano particles loaded with tumor antigen mRNA;

step four, the antibody is thiolated to prepare a thiolated antibody;

And fifthly, uniformly mixing the sulfhydrylation antibody and the lipid nanoparticle loaded with tumor antigen mRNA, and placing the mixture in an environment of 22-28 ℃ for antibody coupling reaction for 1.5-2.5 h to obtain the mRNA delivery system of the targeted dendritic cells.

The invention realizes the accurate and efficient delivery of tumor antigen mRNA to DC through the design of lipid components, the active targeting strategy of antibody coupling and the optimization of technological parameters, provides a technical path with safety, controllability and biological activity for the development of tumor vaccines, and has important clinical transformation potential.

Further, the mass ratio of the SM-102 to the DSPC to the cholesterol to the DMG-PEG2000 to the maleimide modified DSPE-PEG is (45-50)/(10-15)/(38-40)/(1-2)/(0.3-0.7).

SM-102 is a cationic lipid for encapsulation of mRNA and promoting endocytosis, DSPC is a phospholipid to help form lipid bilayer, increase stability, cholesterol to enhance membrane rigidity and stability, DMG-PEG2000 for prolonged circulation time, reduce immune clearance, maleimide modified DSPE-PEG for coupling antibodies. Through optimizing the types and the amounts of the components, parameters such as particle size, charge and the like of the LNP are regulated and controlled, and effective encapsulation and release of mRNA are ensured.

Further, the mass ratio of the tumor antigen mRNA in the aqueous phase solution to the lipid phase solution is 1 (20-25).

The cationic charge of the lipid needs to reach charge balance with the negative charge of the mRNA, ensuring efficient encapsulation. Imbalance in charge ratio may lead to particle aggregation or charge anomalies, affecting delivery efficiency. According to the invention, LNP with proper charge ratio is obtained through optimization, which is favorable for forming products with uniform particle size and moderate surface potential and is favorable for dendritic cells to ingest through endocytosis.

Further, the mass ratio of the sulfhydrylation antibody to the lipid nanoparticle loaded with the tumor antigen mRNA is (0.9-1.1): 0.9-1.1.

Wherein the purpose of the antibody thiolation is to avoid disrupting the antigen binding domain (Fab fragment) of the antibody, ensuring that the antibody will still specifically recognize the DC surface receptor after conjugation.

Further, the buffer solution comprises a sodium citrate acidification buffer solution with the pH value of 4-5.

Further, the tumor antigen mRNA includes melanoma antigen mRNA.

Further, the preparation method of the melanoma antigen mRNA comprises the steps of selecting three repeated fragments of a specific antigen TRP2 ^180-188 of melanoma, connecting the three repeated fragments by a linker (amino acid sequence is GGGGS), and then performing in vitro transcription, enzymatic capping and methylation to obtain the TRP2 mRNA. The mRNA delivery system of the target dendritic cells prepared by taking TRP2 mRNA as tumor antigen mRNA and an anti-CLEC 9A antibody as a coupling antibody is named as CLEC9A/TRP2 mRNA delivery system.

Tyrosinase-related protein-2 (TRP 2) is a weakly immunogenic tumor-associated antigen, a natural antigen of the B16F10 tumor model. The present invention is described by way of example in terms of designing mRNA according to the antigen of a solid tumor of interest to produce a dendritic cell-targeted mRNA delivery system for the corresponding tumor. The invention takes the amino acid sequence of TRP2 ^180–188 as an antigen design target point of an mRNA tumor vaccine to design and synthesize a corresponding DNA sequence containing an HA tag.

Wherein the amino acid sequence of TRP2 ^180–188 is shown as SEQ ID No.1, and the DNA sequence of TRP2 ^180–188 containing the HA tag is shown as SEQ ID No. 2.

The average particle size of the CLEC9A/TRP2 mRNA delivery system is 120-150 nm.

In a third aspect, the invention provides the use of the dendritic cell-targeted mRNA delivery system described above for the preparation of a medicament for the treatment of solid tumors.

In a fourth aspect, the invention provides a vaccine for treating solid tumors, wherein the active ingredient of the vaccine comprises the mRNA delivery system for targeting dendritic cells, and the vaccine can be compounded with an adjuvant on the basis.

In a fifth aspect, the present invention provides a vaccine for treating melanoma, wherein the active ingredient of the vaccine comprises an mRNA delivery system of targeted dendritic cells prepared by taking melanoma antigen mRNA as a raw material.

Animal experiment results show that in the experimental range, the vaccine prepared by the CLEC9A/TRP2 mRNA delivery system has the effects of obviously promoting the activation and maturation of DC cells, activating T cells in lymph nodes of a test subject in a tumor microenvironment, obviously improving the expression level of melanoma antigen mRNA in spleen, obviously improving the number of tumor infiltration immune cells (such as T cells and DC cells) of mice and the like, and achieves the aims of inhibiting the growth of melanoma and improving the survival period.

In a sixth aspect, the present invention provides a pharmaceutical composition for treating melanoma, wherein the active ingredients of the pharmaceutical composition include the dendritic cell-targeted mRNA delivery system prepared from melanoma antigen mRNA as a raw material, and PD1 mab. Adjuvants or TLR agonists and the like may also be included in the active ingredients of the vaccine.

Compared with the independent use of PD1 monoclonal antibody or the use of melanoma antigen mRNA nano-lipid particles unconjugated with corresponding antibody, the CLEC9A/TRP2 mRNA delivery system provided by the invention has a synergistic effect with the PD1 monoclonal antibody in the process of treating melanoma, can obviously improve the ratio of activated T cells in organisms, particularly the ratio of IFNgamma ⁺/CD8⁺ T cells, and can effectively inhibit the growth of melanoma and improve the survival time of cancer-bearing animals.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of TRP2 epitope antigen in example 1 of the present invention;

FIG. 2 is a schematic diagram showing the structure of TRP2 mRNA vector in example 1 of the present invention;

FIG. 3 is a graph showing particle size distribution of TRP2 mRNA delivery system and CLEC9A/TRP2 mRNA delivery system in example 1 of the present invention, wherein FIG. 3A) represents particle size distribution of TRP2 mRNA-LNP and FIG. 3B) represents particle size distribution of CLEC9A/TRP2 mRNA-LNP;

FIG. 4 shows the expression of TRP2 mRNA in 293 cells of TRP2 mRNA-LNP according to example 1 of the present invention, wherein FIG. 4A) represents the expression of TRP2 ^180-188 X13 antigen and FIG. 4B) represents the expression of β -actin;

FIG. 5 shows the percentage of MHC II ⁺/CD11c⁺ DC cells in lymph nodes of different groups of mice in effect example 1 of the present invention;

FIG. 6 shows the percentage of CD80 ⁺/CD11c⁺ DC cells in lymph nodes of different groups of mice in effect example 1 of the present invention;

FIG. 7 shows the percentage of CD3 ⁺ T cells in the lymph nodes of different groups of mice in effect example 1 according to the present invention;

FIG. 8 shows the percentage of IFNgamma ⁺/CD8⁺ T cells in lymph nodes of different groups of mice according to effect example 1 of the present invention;

FIG. 9 is a fluorescent chart of CD3 ⁺ T cells and CD11c ⁺ dendritic cells infiltrated into a melanoma tissue of a mouse in the G3 group and the G4 group in effect example 1 of the present invention;

FIG. 10 is a representative picture of mice of different groups on day 17 in effect example 2 of the present invention;

FIG. 11 is a graph showing tumor volume change between day 7 and day 17 of mice of different groups in effect example 2 of the present invention;

FIG. 12 is a graph showing survival of mice of different groups in effect example 3 of the present invention;

FIG. 13 shows the expression of Fluc mRNA in different organs of mice of different groups in effect example 4 of the present invention;

FIG. 14 is a graph showing the ratio of the relative expression amounts of Fluc mRNA in lymph node to liver of mice of different groups in effect example 4 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

In order to better illustrate the embodiments of the present invention, the following is further illustrated by examples.

The construction method of the tumor xenograft mouse melanoma model in the invention comprises the following steps:

Tumor cell implantation 2X 10 ⁵ B16 cells were subcutaneously injected into the right posterior abdomen of mice (C57 BL/6) together with 1:1 matrigel (total 200. Mu.L). Tumor size was measured every two to three days with calipers and tumor volume was calculated using the formula (a ² ×b)/2 (a, width; b, length).

When the tumor volume reached about 50mm ³ -7 days after tumor inoculation, mice were randomly grouped according to tumor size (n=8 mice per group), and according to experimental design, mice were inoculated with PBS, TRP2 mRNA-LNP, PD1, CLEC9A/TRP2 mRNA-LNP or CLEC9A/TRP2 mRNA-lnp+pd1, respectively, at a frequency of 3-5 days/time intervals for two injections. Wherein, PBS treatment group served as control group.

Example 1

1. The present example provides a dendritic cell-targeted mRNA delivery system comprising lipid nanoparticles loaded with tumor antigen mRNA, and an antibody coupled to the lipid nanoparticles;

wherein the tumor antigen mRNA is TRP2 mRNA and the antibody is an anti-CLEC 9A antibody.

2. The embodiment also provides a preparation method of the dendritic cell-targeted mRNA delivery system, which comprises the following steps:

step one, three repeated fragments of specific antigen TRP2 of melanoma are selected, connected by a connector, then in vitro transcription is carried out, TRP2 mRNA is obtained, and then the mRNA is diluted by a buffer solution to obtain an aqueous phase, wherein the specific contents are as follows:

S11, the amino acid sequence of TRP2 ^180–188 is used as an antigen design target point of the mRNA tumor vaccine, and a corresponding DNA sequence containing an HA tag is designed and synthesized. Three repeated fragments of TRP2 ^180–188 (without linker in the middle) were then ligated in the middle of TRP2 repeated fragment and HA tag (amino acid sequence of linker GGSLGGGGSGGGGGS) using the Gibson assembly method, and the DNA sequences of the N-terminal signal peptide and MHC-I transport signal (MHC-I TRAFFICKING SIGNAL, MITD) were inserted into pmRVac vector, DNA template transcribed in vitro. Wherein, the schematic diagram of TRP2 epitope antigen is shown in FIG. 1. pmRVac the vector contains a T7 promoter sequence, a 5'UTR, a 3' UTR, a 110nt segmented polyA and a sapI restriction enzyme site for transcription termination.

S12. Cap1 mRNA was generated using the vaccinia capping system. After denaturing the uncapped transcripts by heating, vaccinia capping enzyme, 2' -O methyltransferase, GTP, S-adenosylmethionine (SAM) and capping buffer are added to establish a capping reaction. The reaction mixture was incubated at 37 ℃ for 1 hour. Subsequently, the template DNA was removed by DNase I treatment. Schematic representation of TRP2 mRNA vector is shown in figure 2. Cap1 mRNA is produced 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 magnesium-containing buffer to establish an in vitro transcription reaction. The reaction mixture was incubated at 37 ℃ for 2 hours. For m1Psi modified transcript synthesis, m1Psi was used instead of UTP in vitro transcription.

S13, further purifying the capped transcripts by using magnetic beads. The isolated mRNA was eluted with sodium citrate buffer acidic buffer at pH 4 and stored at-80℃to give TRP2 mRNA mother liquor. mRNA concentration and purity were measured on an ultraviolet-visible spectrophotometer. mRNA integrity was measured by denaturing agarose gel analysis. mRNA sequencing is used to identify mRNA molecules.

S14, determining that the tumor antigen mRNA sequence is correct by adopting Sanger sequencing, the length of the mRNA is correct, and the integration of the mRNA is kept good. The absorbance ratio (A ₂₆₀/A₂₈₀) of mRNA at A ₂₆₀ and A _280nm was 2.12, indicating that the synthesized mRNA was high in purity and little contamination of DNA and protein.

And re-suspending the TRP2 mRNA mother solution in a 50mM NaAc solution with the pH of 4 to obtain a TRP2 mRNA solution, namely an aqueous phase solution.

Wherein the amino acid sequence of TRP2 ^180–188 is shown as SEQ ID No.1, and is specifically shown as SVYDFFVWL;

the DNA sequence of TRP2 ^180–188 containing the HA tag is shown as SEQ ID No.2, and is specifically shown as ATGAGCGTGTATGATTTTTTTGTGTGGCTGTAA.

Step two, mixing the SM102, DSPC, cholesterol, DMG-PEG200 and maleimide modified DSPE-PEG (DSPE-PEG-maleimide) according to the mass ratio of 50:10:38:1.5:0.5, and fully dissolving in 1400 mu L of absolute ethyl alcohol to prepare a lipid phase solution. Wherein SM102 is 42. Mu. Mol.

Step three, mixing the lipid phase solution with the aqueous phase solution by a microfluidic technology to generate 2286 mug of lipid nanoparticles loaded with TRP2 mRNA, which is marked as TRP2 mRNA-LNP.

The molar mass of TRP2 mRNA was calculated from the ratio of the number of N-containing to the number of P-containing cationic lipids (SM 102) in TRP2 mRNA to N: p=6:1. The total mass required for mRNA was calculated from the molecular mass of the bound TRP2 mRNA, the total mass required for the lipid phase solution was calculated from the mass ratio of the 5 lipid component substances, and the actual amounts and proportions of TRP2 mRNA and lipid phase solution were calculated in the ratio of the total mass required for mRNA to the total mass of lipid phase solution=1:23.273. The preparation of TRP2 mRNA-LNP requires the timely removal of ethanol, which is finally dissolved in 15 mM Tris-HCl+15 mM sodium acetate solution for later use. The concentration of TRP2 mRNA-LNP was 544 ng/. Mu.L.

Step four, preparing a sulfhydrylation antibody in the step, which comprises the following specific contents:

2286 μg of anti-CLEC 9A antibody (10 mM HEPES,0.15M NaCl, pH 7.5) is taken, and SATA is weighed according to the mass ratio of SATA to anti-CLEC 9A antibody=9:1 and dissolved in DMSO to obtain 18 mmol/L SATA solution;

Mixing the SATA solution and the antibody solution, reacting for 0.5h at room temperature, ultrafiltering with a 10K ultrafiltration tube, changing the solution with PBS buffer solution with pH of 7.4 for 3-4 times, centrifuging at 9400 rpm for 7min at 8 ℃, concentrating, and fixing the volume to 200 μL with PBS buffer solution to obtain CLEC9A antibody-SATA;

Dissolving 0.5M hydroxylamine hydrochloride in PBS buffer solution containing 25mM EDTA and having pH of 7.3 to form 200 mu L of 0.5M hydroxylamine hydrochloride solution, reacting with the CLEC9A antibody-SATA at room temperature for 2h, performing ultrafiltration on the CLEC9A antibody-SATA by a 10K ultrafiltration tube after deprotection, changing the solution of the PBS buffer solution containing 10mM EDTA for 3-4 times, centrifuging for 7min at 8 ℃, and obtaining the sulfhydrylation antibody (CLEC 9A antibody-SH) after volume-fixing to 300 mu L.

And fifthly, uniformly mixing the sulfhydrylation antibody and TRP2 mRNA-LNP with equal mass, placing the mixture in a 25 ℃ environment for 2 hours to perform antibody coupling reaction, and then, allowing the reaction to be more complete overnight at 4 ℃ to obtain an mRNA delivery system of the targeted dendritic cells, wherein the mRNA delivery system is named CLEC9A/TRP2 mRNA-LNP.

The particle size of CLEC9A/TRP2 mRNA-LNP prepared as described above, and the expression level of TRP2 mRNA in 293 cells were also examined in this example, specifically as follows:

(1) This example examined the effect of conjugated antibodies on the particle size of the resulting mRNA delivery system and examined the particle size distribution of TRP2 mRNA-LNP and CLEC9A/TRP2 mRNA-LNP, the comparison of which is shown in FIG. 3, wherein FIG. 3A) represents the particle size distribution of TRP2 mRNA-LNP and FIG. 3B) represents the particle size distribution of CLEC9A/TRP2 mRNA-LNP. The average particle size of TRP2 mRNA-LNP unconjugated to the anti-CLEC 9A antibody was 72nm, and the average particle size of CLEC9A/TRP2 mRNA-LNP conjugated to the anti-CLEC 9A antibody was 139nm. In addition, it can be seen from fig. 3 that the particle size of the produced nano-lipid particles is significantly increased and the overall particle size distribution is wider after the antibody is conjugated.

(2) In this embodiment, 293 cells were transfected with the TRP2 mRNA-LNP 2. Mu.g (in terms of mRNA) prepared as described above, and the contents of the cells were subjected to protein extraction 24 hours and 48 hours after transfection, respectively, to detect transient expression of TRP2 antigen in the cells by Western hybridization. Meanwhile, beta-actin (Beta-actin) is used as a control to normalize the expression level of mRNA or protein. Wherein the expression of TRP2 mRNA in the TRP2 mRNA-LNP in 293 cells is shown in FIG. 4, wherein FIG. 4A) represents the expression of TRP2 ^180-188 X3 antigen, lane Blank represents nano-lipid particles prepared without loading TRP2 mRNA vector, no. 1-No. 4 are different TRP2 mRNA-LNP clones, FIG. 4B) represents the expression of beta-actin, lane Blank represents nano-lipid particles prepared without loading TRP2 mRNA vector, and No. 1-No. 4 are different TRP2 mRNA-LNP clones.

As can be seen from fig. 4, TRP2 mRNA-LNP can specifically express an antigen corresponding to the melanin tumor epitope antigen TRP 2-related mRNA in cells.

Example 2

The present example provides a dendritic cell-targeted mRNA delivery system comprising lipid nanoparticles loaded with tumor antigen mRNA, and an antibody coupled to the lipid nanoparticles;

wherein the tumor antigen mRNA is TRP2 mRNA and the antibody is an anti-CLEC 9A antibody.

The present example also provides a method for preparing the dendritic cell-targeted mRNA delivery system described above, which is substantially the same as example 1, except that the following three steps are slightly different, and other preparation parameters are the same as example 1:

(1) In the second step, the SM102, DSPC, cholesterol, DMG-PEG200 and maleimide modified DSPE-PEG are mixed according to the mass ratio of substances of 48:12:40:1:0.3, and the SM102 is 40.3 mu mol;

(2) Calculating the actual dosage and proportion of TRP2 mRNA and the lipid phase solution according to the ratio of the total mass of the mRNA to the total mass of the lipid phase solution=1:20;

(3) Step five, uniformly mixing the thiolated antibody and TRP2 mRNA-LNP according to the mass ratio of 0.9:1.1, placing the mixture in a 22 ℃ environment for 2.5h for antibody coupling reaction, and then, allowing the reaction to be more complete at 4 ℃ overnight to obtain an mRNA delivery system of the targeted dendritic cells, wherein the mRNA delivery system is named CLEC9A/TRP2 mRNA-LNP.

Example 3

The present example provides a dendritic cell-targeted mRNA delivery system comprising lipid nanoparticles loaded with tumor antigen mRNA, and an antibody coupled to the lipid nanoparticles;

wherein the tumor antigen mRNA is TRP2 mRNA and the antibody is an anti-CLEC 9A antibody.

The present example also provides a method for preparing the dendritic cell-targeted mRNA delivery system described above, which is substantially the same as example 1, except that the following three steps are slightly different, and other preparation parameters are the same as example 1:

(1) In the second step, the SM102, DSPC, cholesterol, DMG-PEG200 and maleimide modified DSPE-PEG are mixed according to the mass ratio of 45:15:39:2:0.7, and the SM102 is 37.8 mu mol;

(2) Calculating the actual dosage and proportion of TRP2 mRNA and the lipid phase solution according to the ratio of the total mass of the mRNA to the total mass of the lipid phase solution=1:25;

(3) Step five, uniformly mixing the thiolated antibody and TRP2 mRNA-LNP according to the mass ratio of 1.1:0.9, placing the mixture in a 28 ℃ environment for 1.5h to perform antibody coupling reaction, and then enabling the reaction to be more complete at 4 ℃ overnight to obtain an mRNA delivery system of the targeted dendritic cells, wherein the mRNA delivery system is named CLEC9A/TRP2 mRNA-LNP.

Effect example 1

The effect example of the present invention examined the effect of CLEC9A/TRP2 mRNA-LNP prepared in example 1 in activating DC immune cell activation and T cell immune response, and the specific contents are as follows:

After constructing the mouse melanoma model in this effect example, when the tumor volume reached about 50mm ³ days after tumor inoculation, the mice were randomly divided into five groups (n=8 mice per group) according to tumor size, PBS, TRP2 mRNA-LNP, CLEC9A/TRP2 mRNA-LNP or CLEC9A/TRP2 mRNA-lnp+pd1 were used as vaccines, and five groups of mice were inoculated respectively, wherein the PBS treatment group was used as a control group. Five groups of mice were sequentially designated as G1 group through G5 group, and the respective groups of injections and dosages are shown in Table 1.

TABLE 1

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, DC cells and T cells were isolated, and flow cytometry analysis was performed.

(1) The percentage of MHCII ⁺/CD11c⁺ DC cells in the lymph nodes of the different groups of mice was shown in FIG. 5, and the percentage of CD80 ⁺/CD11c⁺ DC cells in the lymph nodes of the different groups of mice was shown in FIG. 6.

From FIGS. 5-6, it can be seen that CLEC9A/TRP2 mRNA-LNP provided by the present invention promotes activation and maturation of DC cells better than those of the G1 group and the G3 group. Wherein the percentage of activated DC cells (mhc ii ⁺/CD11c⁺) in total DC cells can be increased from 33% to 91% in the PBS group. However, the ratio of activated DC cells (MHCII ⁺/CD11c⁺) was slightly lower than CLEC9A/TRP2 mRNA-LNP alone after combined use of CLEC9A/TRP2 mRNA-LNP with PD1 mab.

The percentage of activated DC cells (CD 80 ⁺/CD11c⁺) in total DC cells can be increased from 0.3% to 6% in the PBS group. However, when CLEC9A/TRP2 mRNA-LNP was used in combination with PD1 mab, PD1 mab could further increase the duty cycle of activated DC cells (CD 80 ⁺/CD11c⁺) to 14%.

From the above, compared with the injection of PBS or the uncoupling of TRP2 mRNA-LNP of anti-CLEC 9A antibody, the CLEC9A/TRP2 mRNA-LNP provided by the invention can activate DC immune cells more effectively and promote the maturation of DC cells.

(2) The percentage of CD3 ⁺ T cells in the lymph nodes of the different groups of mice versus total T cells is shown in fig. 7, and the percentage of ifnγ ⁺/CD8⁺ T cells in the lymph nodes of the different groups of mice versus total T cells is shown in fig. 8.

As shown in fig. 7-8, TRP2 mRNA-LNP (9.1%) and CLEC9A/TRP2 mRNA-LNP (7.3%) increased the percentage of cd3+ T cells in the spleen compared to PBS treatment (5.8%). The results also show that CLEC9A/TRP2 mRNA-LNP significantly increased the percentage of ifnγ ⁺/CD8⁺ T cells from 20.8% (PBS) to 37.5% (CLEC 9A/TRP2 mRNA-LNP). CLEC9A/TRP2 mRNA-LNP and PD1 mab combination treatment increased ifnγ ⁺/CD8⁺ T cell expression levels by 20.8% over PBS treatment.

(3) By tumor tissue section and fluorescent staining, it was examined whether CD3 ⁺ T cells and CD11c ⁺ dendritic cells in G3 and G4 could infiltrate into mouse melanoma tissue. The fluorescence patterns of CD3 ⁺ T cells and CD11c ⁺ dendritic cells infiltrated into mouse melanoma tissue in the G3 and G4 groups are shown in fig. 8.

As can be seen from fig. 8, cd3+ T cells and CD11c ⁺ dendritic cells were able to infiltrate into mouse melanoma, and the number of infiltrated lymphocytes was significantly increased in tumors treated with group G4 (CLEC 9A/TRP2 mRNA-LNP). Because tumor infiltrating lymphocytes are inversely related to tumor growth, CLEC9A/TRP2 mRNA-LNP can be presumed to inhibit melanoma growth.

In addition, the CLEC9A/TRP2 mRNA-LNP can promote the infiltration of CD3 ⁺ T cells and CD11C ⁺ dendritic cells into tumor cells, and has the potential of better inhibiting the growth of the tumor.

Effect example 2

The effect example examines the inhibition effect of CLEC9A/TRP2 mRNA-LNP prepared in example 1 on melanoma, and examines whether different injection modes (such as intravenous injection or subcutaneous injection) have influence on the inhibition effect of tumors, and the specific contents are as follows:

After the mouse melanoma model was constructed in this effect example, when the tumor volume reached about 50mm ³ days after tumor inoculation, the mice were randomly divided into six groups (n=8 mice per group) according to tumor size, PBS, TRP2 mRNA-LNP, CLEC9A/TRP2 mRNA-LNP or CLEC9A/TRP2 mRNA-lnp+pd1 were used as vaccines, and six groups of mice were inoculated respectively, wherein PBS group was used as a control group.

Six groups of mice were sequentially identified as group G1 through group G6, and the injected drugs and dosages of the different groups are shown in Table 2.

TABLE 2

Tumor progression and weight were assessed by measuring tumor size every 3 days, and tumor size was recorded from day 7 to day 17. Wherein representative pictures of mice of different groups at day 17 are shown in fig. 10, and tumor volume change patterns of mice of different groups at day 7-day 17 are shown in fig. 11.

As can be seen from fig. 10-11, PD1 mab treatment (group G2) did not have significant tumor suppression compared to PBS (group G1). The TRP2 mRNA-LNP (G3 group) unconjugated with anti-CLEC 9A antibody can inhibit the growth of tumor (P < 0.05) compared with PBS, and the CLEC9A/TRP2 mRNA-LNP alone by intravenous injection or subcutaneous injection can inhibit the growth of tumor remarkably compared with PBS group by combining the CLEC9A/TRP2 mRNA-LNP with PD1 monoclonal antibody. Furthermore, it was found that the combined use of CLEC9A/TRP2 mRNA-LNP and PD1 mab has slightly better melanoma inhibitory effect than CLEC9A/TRP2 mRNA-LNP alone.

Effect example 3

The effect example examined the effect of CLEC9A/TRP2 mRNA-LNP prepared in example 1 on survival of melanoma mice, and the specific contents are as follows:

The melanoma model of the mice was constructed until the tumor volume reached about 50mm ³, the model mice were randomly divided into four groups (8 mice per group), and the four groups of mice respectively injected with PBS, TRP2 mRNA-LNP, CLEC9A/TRP2 mRNA-LNP or CLEC9A/TRP2 mRNA-LNP+PD1 were respectively and sequentially designated as G1 group to G4 group, and the injection drugs and dosages of the different groups are shown in Table 3.

TABLE 3 Table 3

Tumor size was measured every 2-3 days and recorded, and when tumor size reached 2000mm ², mice were humane sacrificed and survival rate of mice was finally calculated. The survival plots for the different groups of mice are shown in figure 10.

From FIG. 12, it is clear that CLEC9A/TRP2 mRNA-LNP increased survival of B16 tumor mice from day 18 of the G1 group to day 25, with a survival improvement of 38.8%.

Effect example 4

The effect example adopts FireflyLuciferase (FLuc) protein as a fluorescent marker, and the distribution condition of LNP (low-density polyethylene) prepared after coupling an anti-CLEC 9A antibody in different organs is examined, wherein the specific content is as follows:

18 male ICR mice of 8 weeks old were taken and randomly divided into three groups (n=6). PBS, fluc mRNA-LNP or CLEC9A/Fluc mRNA-LNP was injected by tail vein at a dose of 5. Mu.g/dose of mRNA, PBS was used as a control group.

The preparation method of CLEC9A/Fluc mRNA-LNP is basically the same as that of CLEC9A/TRP2 mRNA-LNP described in example 1, except that equal amount of Fluc mRNA is used instead of TRP2 mRNA, wherein the preparation method of Fluc mRNA comprises synthesizing coding mRNA of Luciferase (Luciferase) by in vitro transcription, and preparing Fluc mRNA by 5 '-end ARCA cap modification and 3' -end polyA tail optimization;

The preparation method of the Fluc mRNA-LNP is basically the same as the preparation method of the CLEC9A/Fluc mRNA-LNP, and the difference is that the preparation method is not carried out in the fourth step and the fifth step, namely, the sulfhydrylation antibody is not prepared, the prepared LNP containing the Luciferase mRNA is not coupled with the antibody, and finally the Fluc mRNA-LNP is prepared.

Mice were given an intraperitoneal injection of fluorescein substrate Luciferin (150 mg/kg) 6 hours after injection, after 5-10 minutes of substrate distribution, then the mice were sacrificed and after blood was cleared by cardiac perfusion with PBS, heart, liver, spleen, lung, kidney and lymph node tissues were collected for in vitro tissue BLI imaging. The relative expression levels of Fluc mRNA were judged by fluorescence intensities in different organs. The expression of Fluc mRNA in different organs in different groups of mice is shown in figure 13. The ratio of the relative expression levels of Fluc mRNA in lymph nodes to liver in mice of different groups is shown in fig. 14.

From fig. 13-14, it can be seen that fluorescent proteins in the Fluc mRNA-LNP group, which are not conjugated to anti-CLEC 9A antibodies, are more expressed in the liver, however liver expression may trigger nonspecific immune activation or hepatotoxicity, and the liver is not an ideal site for initiating anti-tumor immunity.

Whereas CLEC9A/Fluc mRNA-LNP was able to target the spleen, the relative expression level ratio of Fluc mRNA in lymph node to liver was about 2.5 times that of Fluc mRNA-LNP group. It is known that LNP after coupling anti-CLEC 9A antibody can target spleen, not only can more efficiently start anti-tumor T cell response, but also can remarkably reduce mRNA exposure of liver and toxicity, and in addition, targeting delivery can enable target mRNA to achieve effective immunogenicity at lower dosage, and reduce drug consumption and production cost.

Taken as a summary, the present invention provides an mRNA delivery system targeting dendritic cells, taking anti-CLEC 9A antibodies, TRP2 mRNA as an example. The system can be used as an active ingredient of a melanoma vaccine, can be used alone or in combination with PD1 monoclonal antibody, can effectively activate the autoimmune system of a patient, promote the maturation of DC cells and enhance the activation efficiency of T cells, remarkably inhibit the growth of melanoma, improve the survival rate of tumor-bearing mice, and has important scientific research value and clinical transformation potential.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

Claims (10)

1. A 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. 2. The mRNA delivery system targeting dendritic cells according to claim 1, wherein the tumor antigen mRNA comprises a 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, characterized in that 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 thiol-modified 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 a dendritic cell-targeted mRNA delivery system 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 comprises a sodium citrate acidified buffer having a pH of 4 to 5; and/or The tumor antigen mRNA includes melanoma antigen mRNA. 6. The method for preparing a dendritic cell-targeted mRNA delivery system according to claim 5, wherein the method for preparing the melanoma antigen mRNA comprises the following steps: selecting three repeat fragments of the melanoma-specific antigen TRP2 ^180-188 , connecting them with a linker, and then performing in vitro transcription, enzymatic capping, and methylation 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 ingredient of the vaccine comprises the mRNA delivery system targeting dendritic cells according to claim 1 or 2. 9. A vaccine for treating melanoma, characterized in that the active ingredient of the vaccine comprises 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.