MRNA-LNP vaccine targeting spleen cDC1 as well as preparation method and application of mRNA-LNP vaccine

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a spleen cDC1 targeting mRNA-LNP vaccine as well as a preparation method and application thereof. The targeting mRNA-LNP vaccine provided by the invention can be accurately targeted to a specific part through the target protein connected to the lipid nanoparticle (LNP), the effect that the nucleic acid drug mRNA exerts activity is improved, the whole LNP system is very stable, and the defect that the whole LNP system is possibly unstable due to the fact that the target protein is connected to the LNP in the prior art is overcome. Experimental results show that the LNP modified with the Syctatcher-XCL-1 fusion protein can be targeted to classical type I dendritic cells (cDC1) of the spleen, the growth of melanoma can be effectively inhibited, and the lifetime can be remarkably prolonged. The invention has important application value for development of therapeutic tumor vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to an mRNA-LNP vaccine targeting spleen cDC1 and a preparation method and application thereof. Background Art

[0002] Dendritic cells (DC) are a unique type of immune cells. As the strongest professional antigen-presenting cells in the body, they play a bridging role between innate immunity and adaptive responses. DC expresses special surface receptors and intracellular and extracellular pattern recognition receptors to sense different danger signals. By searching for antigens that can be used to activate effector cells at the site of infection or infiltrated pathological tissues, they initiate cellular and humoral immunity, which is crucial for the protection against infectious diseases and tumors. In vitro experiments have shown that loading tumor antigens on DC can trigger immune effects. In recent years, DC has been used as a carrier of tumor vaccines, and pulsed DCs loaded with various tumor antigens have been tested clinically. Although DC vaccines are considered to be promising therapies and have been proven to be effective in clinical trials, the preparation process of DC vaccines is cumbersome. It requires the isolation of DC or DC precursors from the patient's body, culture activation and antigen treatment, and then re-infusion into the body. It takes a long time and has high requirements. The re-infused DC cells have weak lymphatic homing ability. Therefore, DC tumor vaccines must introduce new methods to improve existing deficiencies and promote DC to play a stronger anti-tumor role.

[0003] DCs have different subsets, including conventional DCs, plasmacytoid DCs, monocyte-derived DCs, and Langerhans cells, which can activate different types of effector T cells and thus regulate immune responses. Conventional DCs are divided into classical type I DCs (cDC1) and classical type II DCs (cDC2). cDC1 can perform antigen cross-presentation to activate CD8 + T cells. Cytotoxic T lymphocytes (CTL cells) are commonly referred to as CD8 + T cells are a key component of the adaptive immune system and play an important role in the immune system's defense against pathogens (such as viruses, bacteria, and tumors). Together with NK cells (natural killer cells), they constitute an important line of defense for the body's anti-tumor immunity. cDC1 expresses XCR1 protein and exhibits chemotaxis under the action of chemokine XCL-1. Therefore, the preparation of tumor vaccines targeting cDC1 through the XCR1 / XCL-1 signaling axis is considered an effective method for anti-tumor.

[0004] LNP (lipid nanoparticle) is a nanoscale particle composed of lipids, which has unique advantages in delivering nucleic acid drugs such as mRNA. First, LNP is a spherical particle that can wrap mRNA inside to resist the action of nucleases; secondly, the LNP component is similar to the cell membrane, easy to fuse with the receptor cell, with high transfection efficiency, and is not restricted by the host, and can deliver mRNA fragments of different sizes. Modifying the target protein on LNP can enable the mRNA loaded by LNP to be accurately delivered to the target organ, thereby exerting better therapeutic effects. The existing technology connects the target protein to LNP (lipid nanoparticle), mainly by synthesizing the phospholipid DSPE-PEG2000 of LNP with NHS or MAL at the end, and with free groups containing -NH 2 or -SH target protein reaction to obtain target protein modified LNP. However, this technology requires the target protein to have a specific free group -NH 2 or -SH to react and bind with LNP, but some target proteins may not have -NH 2 or -SH, or multiple -NH 2 Or -SH, it cannot guarantee that all target proteins can be successfully linked to LNP, and it cannot guarantee the orientation of the target protein in LNP, which will cause the linked synthesized LNP to be unstable. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing DC tumor vaccine preparation method, such as the complexity of the LNP and target protein connection, and to provide a targeted mRNA-LNP vaccine.

[0006] The purpose of the present invention is to provide a method for preparing a targeted mRNA-LNP vaccine.

[0007] Another object of the present invention is to provide the use of the mRNA-LNP vaccine in the preparation of tumor prevention and treatment drugs.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] A targeted mRNA-LNP vaccine comprises lipid nanoparticles LNP and mRNA, wherein the lipid nanoparticles LNP comprise polyethylene glycol-lipid conjugates modified with SpyTag polypeptides, ionizable cationic lipids, amphiphilic phospholipids, steroidal lipids and polyethylene glycol-lipid conjugates; and the surface of the lipid nanoparticles LNP is modified with Spycatcher fusion protein.

[0010] The present invention modifies the SpyTag polypeptide on the polyethylene glycol-lipid conjugate to form a SpyTag polypeptide-modified polyethylene glycol-lipid conjugate. Further, under the action of Glu (glutamic acid) in the Spycatcher fusion protein, the Asp (aspartic acid) in the SpyTag polypeptide forms a covalent isopeptide covalent bond with the Lys (lysine) in the Spycatcher fusion protein, thereby anchoring the target protein fused in the Spycatcher fusion protein on the surface of the LNP containing the SpyTag polypeptide-modified polyethylene glycol-lipid conjugate, which is beneficial for subsequent mRNA delivery and targeting to a specific site.

[0011] Preferably, the structure of the ionizable cationic lipid (A1I4R22C18-2) is as follows:

[0012]

[0013] The LNP prepared by the ionizable cationic lipid (A1I4R22C18-2) of the present invention has better targeting to the spleen.

[0014] Preferably, the amphiphilic phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC).

[0015] Preferably, the polyethylene glycol-lipid conjugate is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).

[0016] Preferably, the steroidal lipid is cholesterol.

[0017] In the present invention, Spycatcher (surface protein of Streptococcus pyogenes) fusion protein is a protein that links Spycatcher to any protein other than itself.

[0018] Preferably, the Spycatcher fusion protein in the mRNA-LNP vaccine is Spycatcher-XCL-1 protein.

[0019] The spleen is the largest secondary lymphoid organ in the body, rich in DCs, B cells and T cells, and plays a very important role in the body's defense system, especially in exerting anti-tumor immune effects. The red pulp, marginal zone and central area of ​​the T cell zone of the spleen contain relatively abundant cDC1, which can absorb blood-derived antigens and present them to CD8 +T cells promote the formation of killer T cells, and XCL-1 is the main chemokine of cDC1, and its receptor protein XCR1 is expressed on cDC1. Therefore, in the present invention, a Spycatcher-XCL-1 protein is synthesized and anchored and modified on the surface of LNP, which can specifically target cDC1 in the spleen, effectively activate humoral and cellular immunity, and exert anti-tumor effects. The mRNA-LNP vaccine prepared by the present invention can target cDC1 in the spleen in vivo, and subsequently activate CD8 + T cells, which kill tumors.

[0020] Preferably, the molar ratio of the ionizable cationic lipid, the amphiphilic phospholipid, the steroidal lipid, the polyethylene glycol-lipid conjugate and the SpyTag polypeptide-modified polyethylene glycol-lipid conjugate is 48-50:8.5-10:36-38.5:0.1-2:0.1-2.

[0021] Preferably, the molar ratio of the ionizable cationic lipid, the amphiphilic phospholipid, the steroidal lipid, the polyethylene glycol-lipid conjugate and the SpyTag polypeptide-modified polyethylene glycol-lipid conjugate is 48-50:8.5-10:36-38.5:0.1-0.75:0.1-0.75.

[0022] Preferably, the molar ratio of the SpyTag polypeptide-modified polyethylene glycol-lipid conjugate to the Spycatcher fusion protein is 1:0.5-2.

[0023] Furthermore, the lipid nanoparticles are approximately spherical.

[0024] Furthermore, the particle size of the lipid nanoparticles is 50 to 250 nm; preferably, the particle size of the lipid nanoparticles is 80 to 190 nm; more preferably, the particle size of the lipid nanoparticles is 100 to 130 nm; specifically, the particle size of the lipid nanoparticles is 111 nm.

[0025] Furthermore, the Zeta potential of the lipid nanoparticles is -40mV to -3mV; preferably, the Zeta potential of the lipid nanoparticles is -28mV to -12mV; more preferably, the Zeta potential of the lipid nanoparticles is -20mV to -12mV; specifically, the Zeta potential of the lipid nanoparticles is -13.7mV.

[0026] Preferably, the mRNA encodes a tumor antigen.

[0027] More preferably, the mRNA is mRNA encoding chicken ovalbumin OVA (denoted as OVA mRNA).

[0028] In the present invention, the mRNA encoding ovalbumin OVA (OVA mRNA) is an mRNA encoding a known OVA protein sequence, wherein the nucleic acid sequence of OVA is derived from the OVA nucleic acid sequence in an existing gene database.

[0029] Preferably, the mRNA-LNP vaccine also includes a pharmaceutically acceptable excipient.

[0030] Preferably, the mRNA-LNP vaccine is injected by tail vein injection or intramuscular injection.

[0031] At the same time, the present invention provides a method for preparing the mRNA-LNP vaccine, comprising the following steps:

[0032] S1. Take an ionizable cationic lipid, an amphiphilic phospholipid, a steroidal lipid, a polyethylene glycol-lipid conjugate and a SpyTag polypeptide-modified polyethylene glycol-lipid conjugate and add anhydrous ethanol and mix well to obtain an ethanol phase;

[0033] S2. Dissolve the mRNA in a buffer solution with a pH of 3.5 to 4.5 to obtain an aqueous phase, mix the ethanol phase of S1 with the aqueous phase, let stand, dialyze, take the dialyzed solution, add the Spycatcher fusion protein, incubate, and obtain the mRNA-LNP vaccine.

[0034] Furthermore, the buffer solution is one of sodium citrate solution, sodium acetate, PBS or Tris solution.

[0035] Furthermore, the volume mass ratio of the ethanol phase, the aqueous phase and the mRNA is 1 μL: (2.5-3.5) μL: (0.2-0.3) μg.

[0036] Furthermore, the standing temperature is 25-30° C. (room temperature).

[0037] Furthermore, the standing time is 10 to 20 minutes.

[0038] Furthermore, the incubation is carried out at 3 to 5° C. for 8 to 24 hours.

[0039] Additionally, the present invention protects the use of the mRNA-LNP vaccine in the preparation of tumor prevention and treatment drugs.

[0040] The present invention has the following beneficial effects:

[0041] (1) The present invention provides a targeted mRNA-LNP vaccine, in which a polyethylene glycol-lipid conjugate modified with a SpyTag polypeptide is added to prepare lipid nanoparticles (LNP), and then a covalent isopeptide covalent bond is formed by using the Lys of the Spycatcher protein and the Asp of the SpyTag under the action of the Glu of the Spycatcher protein, and finally the target protein fused in the Spycatcher fusion protein is anchored on the surface of the LNP, which is beneficial for the subsequent mRNA delivery to target a specific part. The mRNA-LNP vaccine of the present invention can accurately deliver the loaded mRNA to a specific part through the target protein connected to the LNP, and the entire LNP system is very stable, which solves the defect of the prior art that the target protein connected to the LNP may cause the LNP system to be unstable.

[0042] (2) The targeted mRNA-LNP vaccine prepared by the present invention can be modified with XCL-1 protein on the surface of LNP, which can enable the vaccine to be accurately delivered to cDC1 cells in the spleen. At the same time, cDC1 cells perform antigen cross-presentation to activate CD8 + The killing effect of T cells can play a better anti-tumor effect.

[0043] (3) The mRNA-LNP vaccine of the present invention is easy to prepare, has stable quality, and is easy to industrialize and produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The particle size distribution diagram of the mRNA-LNP prepared in Example 1 of the present invention and Comparative Example 1.

[0045] Figure 2 This is a morphological characterization diagram of the mRNA-LNP prepared in Example 1 of the present invention.

[0046] Figure 3 This is an in vivo expression imaging diagram of the Luciferase mRNA-encapsulated LNP prepared in Example 2 of the present invention after tail vein injection into mice.

[0047] Figure 4 This is a graph showing the level of LNP phagocytosis by spleen cDC1 after tail vein injection of DIR-labeled mRNA-LNP prepared in Example 3 of the present invention into mice.

[0048] Figure 5 This is a statistical graph of IgG antibody titer data in mice after injection of XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0049] Figure 6The effect memory CD8 in the spleen of mice after injection of XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1 + Statistical graph of T cell level data.

[0050] Figure 7 This is a statistical graph of the activation level of cDC1 in the spleen of mice after injection of XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0051] Figure 8 This is a graph showing the tumor growth curves of B16F10-OVA tumor-bearing mice after injection of XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0052] Fig. 9 This is a survival curve of B16F10-OVA tumor-bearing mice after injection of XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0053] Fig.10 The cytotoxic CD8 T cells in the tumor site of B16F10-OVA mice after injection of XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1 + Statistical graph of T cell level data.

[0054] Note: Figures 4 to 8 and Fig.10 *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 indicate significant differences compared with the Control group. DETAILED DESCRIPTION

[0055] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0056] In the specific embodiments and drawings of the present invention, CD8 + 、CD11c + 、CD86 + 、CD107a + Indicates that the corresponding CD8, CD11c, CD86, and CD107a protein molecules are positively expressed.

[0057] The OVA mRNA used in the examples of the present invention was purchased from Shenzhen Hongxin Biotechnology Co., Ltd., with the product number HT22-PL05R. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0058] Example 1 Preparation of XCL-1-Tag-LNPs Encapsulating OVA mRNA

[0059] The mRNA-LNP vaccine described in Example 1 was prepared according to the following method:

[0060] S1. An ionizable cationic lipid (A1I4R22C18-2), distearoylphosphatidylcholine (DSPC), cholesterol, dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) and SpyTag polypeptide-modified DMG-PEG2000-SpyTag were added to ethanol at a molar ratio of 50:10:38.5:0.75:0.75 to obtain an ethanol phase;

[0061] S2. Take 5 μg of OVA mRNA and add it to sodium citrate solution (pH=4) to obtain an aqueous phase, a total of 60 μL, then add 20 μL of ethanol phase, mix at room temperature, let stand for 15 minutes, load the solution into a 1000Da dialysis membrane, and dialyze in PBS with pH=7.4 at 4°C for 2 hours. After the dialysis, collect the solution, add Spycatcher-XCL-1 protein (the molar ratio of DMG-PEG2000-SpyTag: Spycatcher-XCL-1 protein is 1:0.5), and incubate at 4°C for 16 hours to obtain the mRNA-LNP vaccine (XCL-1-Tag-LNP) targeting spleen cDC1.

[0062] Example 2 Preparation of XCL-1-Tag-LNPs Encapsulating Luciferase mRNA

[0063] According to the preparation method of Example 1, XCL-1-Tag-LNP (containing 5 μg of Luciferase mRNA) encapsulating Luciferase mRNA was prepared for observing the expression of XCL-1-Tag-LNP in vivo.

[0064] Example 3

[0065] DIR-dyed XCL-1-Tag-LNP was prepared. The preparation method was different from that in Example 1 in that after obtaining the ethanol phase in step S1, DIR dye was added to the ethanol phase (0.1 μL DIR dye was added to every 20 μL ethanol phase), and the remaining preparation steps were consistent with those in Example 1.

[0066] Comparative Example 1 Preparation of Tag-LNPs Encapsulating OVA mRNA

[0067] S1. An ionizable cationic lipid (A1I4R22C18-2), distearoylphosphatidylcholine (DSPC), cholesterol, dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) and SpyTag polypeptide-modified DMG-PEG2000-SpyTag were added to ethanol at a molar ratio of 50:10:38.5:0.75:0.75 to obtain an ethanol phase;

[0068] S2. Take 5 μg of OVA mRNA and add it to 60 μL of sodium citrate solution (pH = 4) to obtain the aqueous phase, then add 20 μL of ethanol phase, mix at room temperature, let stand for 15 minutes, load the solution into a 1000Da dialysis membrane, and dialyze it in PBS with pH = 7.4 at 4°C for 2 hours. After the dialysis, collect the solution, add Spycatcher protein (the molar ratio of DMG-PEG2000-SpyTag: Spycatcher protein is 1:0.5), and incubate at 4°C for 16 hours to obtain the mRNA-LNP vaccine (Tag-LNP).

[0069] The difference from Example 1 is that the XCL protein is not modified.

[0070] Comparative Example 2 Preparation of Tag-LNPs Encapsulating Luciferase mRNA

[0071] The preparation method is different from Example 2 in that the XCL protein is not modified, that is, in step S2, Spycatcher-XCL-1 is replaced by Spycatcher protein, and the rest of the experimental methods and steps are the same.

[0072] Comparative Example 3

[0073] The preparation method is different from that of Example 3 in that the XCL protein is not modified, that is, in step S2, Spycatcher-XCL-1 is replaced by Spycatcher protein, and the rest of the experimental methods and steps are the same.

[0074] Characterization analysis:

[0075] Experimental materials: XCL-1-Tag-LNP and Tag-LNP prepared in Example 1 and Comparative Example 1.

[0076] The average particle size and Zeta potential of XCL-1-Tag-LNP particles were tested using a Malvern particle size analyzer. Figure 1 and as shown in Table 1. The prepared particles have uniform particle size, good dispersibility and negative charge.

[0077] Table 1 Average particle size and Zeta potential of XCL-1-Tag-LNP and Tag-LNP

[0078] sample Diameter(nm) PDI Zeta(mV) Tag-LNP 104±2 0.13±0.02 -29.2±0.8 XCL-1-Tag-LNP 111±1 0.14±0.01 -13.7±0.2

[0079] 10 μL of XCL-1-Tag-LNP solution was dripped onto the electron microscope copper grid and allowed to stand at room temperature for 1 min. The liquid was then removed with filter paper. 10 μL of 3% phosphotungstic acid was dripped onto the grid and stained for 1 min. The liquid was removed with filter paper and dried in a 37°C oven for one day. The electron microscope was then used to take photos. The experimental results are shown in Figure 1. Figure 2 As shown in the figure, it can be seen that XCL-1-Tag-LNP is spherical.

[0080] Experimental Example 1

[0081] Experimental materials: XCL-1-Tag-LNP and Tag-LNP prepared in Example 2 and Comparative Example 2.

[0082] The XCL-1-Tag-LNP and Tag-LNP prepared in Example 2 and Comparative Example 2 were respectively injected into C57BL / 6 mice via the tail vein (5 μg mRNA / mouse, 3 mice in each group), and Luciferase substrate (D-Luciferin Sodium Salt, D-luciferin sodium salt) was injected intraperitoneally 6 hours later. The mice were killed 10 minutes after the injection, and the heart, liver, spleen, lung, and kidney organs were removed for organ imaging.

[0083] The experimental results are as follows Figure 3 As shown, there was no fluorescence expression in the heart, liver, lung and kidney of the mouse. The results showed that Luciferase mRNA was mainly expressed in the spleen, proving that the mRNA-LNP vaccine prepared by the present invention has good targeting to the spleen.

[0084] Experimental Example 2: In vivo cellular distribution of DIR-stained XCL-1-Tag-LNP

[0085] Experimental materials: XCL-1-Tag-LNP and Tag-LNP prepared in Example 3 and Comparative Example 3.

[0086] XCL-1-Tag-LNP and Tag-LNP containing DIR dye prepared in Example 3 and Comparative Example 3 were respectively injected into C57BL / 6 mice via the tail vein (5 μg mRNA / mouse, 5 mice in each group), and 100 μL PBS was injected into the tail vein of the Control group. The mice were killed 4 hours after the above tail vein injection, the spleen was taken, ground into a single cell suspension, filtered with a 70 μm mesh, centrifuged, the supernatant was removed, 5 mL of red blood cell lysis solution was added, and the mixture was allowed to stand at room temperature for 8 minutes, centrifuged for 5 minutes, and 5 mL of PBS was added to resuspend, centrifuged, and the supernatant was removed. 100 μL of PBS containing live dead dye (biolegend, item number 423102) was added, and placed at 4°C for 20 minutes. Centrifuged for 5 minutes, the supernatant was removed, 1 mL of flow buffer was added to resuspend, centrifuged for 5 minutes, and the supernatant was removed. Add 100 μL of flow buffer containing 1 μg CD16 / 32 blocking antibody. Place at 4°C for 20 minutes. Centrifuge for 5 minutes, remove the supernatant, add 100 μL of flow buffer containing 1 μg CD8 antibody, 0.25 μg MHCII and 0.25 μg CD11c antibody, incubate at 4°C for 30 minutes, centrifuge, remove the supernatant, add 1 mL of flow buffer to resuspend, centrifuge, remove the supernatant, add 400 μL of flow buffer to resuspend, and detect the DIR fluorescence in cDC1 using a flow cytometer. The results are shown in Figure 4 It was shown that mRNA-LNP modified with XCL-1 could promote its uptake by cDC1.

[0087] Experimental Example 3 Detection of OVA-specific IgG antibody titer in mouse serum

[0088] Experimental materials: XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0089] Experimental method: After adaptive feeding, C57BL / 6 mice were randomly divided into 4 groups, 5 mice in each group:

[0090] Control group: mice were injected with 100 μL PBS via tail vein once every two weeks for a total of three injections, and were kept normally during the injection period;

[0091] Tag-LNP (iv) group: mice were injected with Tag-LNP prepared in Comparative Example 1 via tail vein, with a dose of 5 μg mRNA / mouse, once every two weeks, for a total of three injections, and were kept normally during the injection period;

[0092] XCL-1-Tag-LNP (im) group: mice were injected intramuscularly with XCL-1-Tag-LNP prepared in Example 1, with a dose of 5 μg mRNA / mouse, once every two weeks, for a total of three injections, and were fed normally during the injection period;

[0093] XCL-1-Tag-LNP (iv) group: mice were injected with XCL-1-Tag-LNP prepared in Example 1 via tail vein, with a dose of 5 μg mRNA / mouse, once every two weeks, for a total of three injections, and were kept normally during the injection period.

[0094] On the seventh day after the second injection, blood was collected from the eye sockets, serum was separated, and the IgG antibody titer in the serum was detected using an Elisa kit.

[0095] On the seventh day after the third injection, the mice were killed, the spleens of the mice were taken, ground into single cell suspension, filtered with a 70μm mesh, centrifuged, the supernatant was removed, 5mL of red blood cell lysis buffer was added, and the suspension was allowed to stand at room temperature for 8min, centrifuged for 5min, 5mL of PBS was added to resuspend, centrifuged, the supernatant was removed, 100μL of PBS containing live dead dye (biolegend, catalog number 423102) was added, and the suspension was placed at 4℃ for 20min. Centrifuged for 5min, the supernatant was removed, 1ml of flow buffer was added to resuspend, centrifuged for 5min, and the supernatant was removed. 100μL of flow buffer containing 1μg of CD16 / 32 blocking antibody was added. The suspension was placed at 4℃ for 20min. Centrifuge for 5 minutes, remove the supernatant, add 100 μL of flow buffer containing 1 μg CD3 antibody, 0.25 μg CD8 antibody, 0.25 μg CD44 antibody and 0.25 μg CD62L antibody, incubate at 4°C for 30 minutes, centrifuge, remove the supernatant, add 1 mL of flow buffer to resuspend, centrifuge, remove the supernatant, add 400 μL of flow buffer to resuspend, and detect by flow cytometer.

[0096] Experimental results:

[0097] Figure 5 The titer of IgG antibody in the serum of each group of mice was 7 days after the second injection. Figure 5 It can be seen that each group of mRNA-LNP can induce antibody titers. The mRNA delivered by the LNP of the present invention is expressed as protein in the body and induces humoral immunity.

[0098] Effector memory CD8 + T cells (TEM) are one of the important cells in anti-tumor immunity and anti-infection immunity, effector memory CD8 + The increase in the number of T cells is more helpful in resisting diseases. The present invention uses live and dead dyes to circle live cells, and then circle CD3 +positive cells, namely T cells, and then through CD8 + Circle the CD8 in the T cell + T cells, and then through CD44 + Circle CD8 and CD62L expression + Effector memory CD8 in T cells + T cells (Tem). Figure 6 Shows the effector memory CD8 in the spleen of mice seven days after the third injection + As can be seen from the figure, the XCL-1-Tag-LNP (iv) group, i.e., tail vein injection of XCL-1 modified LNP, can significantly promote the effector memory CD8 + The level of T cells in the control group was significantly different (p<0.001), and the tail vein injection of XCL-1-Tag-LNP was more effective than the intramuscular injection of XCL-1-Tag-LNP. However, the two groups had no significant effect on increasing CD8 + The effects at the T cell level were better than those in the Tag-LNP (iv) group.

[0099] Experimental Example 4

[0100] Experimental materials: XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0101] Experimental method: After adaptive feeding, C57BL / 6 mice were randomly divided into 4 groups, 5 mice in each group:

[0102] Control group: mice were injected with 100 μL PBS via tail vein and kept normally during the injection period;

[0103] Tag-LNP (iv) group: mice were injected with Tag-LNP prepared in Comparative Example 1 via tail vein, with a dose of 5 μg mRNA / mouse, and were kept normally during the injection period;

[0104] XCL-1-Tag-LNP (im) group: mice were intramuscularly injected with XCL-1-Tag-LNP prepared in Example 1, with a dose of 5 μg mRNA / mouse, and were kept normally during the injection period;

[0105] XCL-1-Tag-LNP (iv) group: mice were injected with XCL-1-Tag-LNP prepared in Example 1 via tail vein, with a dose of 5 μg mRNA / mouse. Mice were kept normally during the injection period.

[0106] After 24 hours of the above treatment, the mice were killed, the spleen was removed, ground into a single cell suspension, filtered with a 70μm mesh, centrifuged, the supernatant was removed, 5mL of red blood cell lysis buffer was added, and the suspension was allowed to stand at room temperature for 8min, centrifuged for 5min, 5mL of PBS was added to resuspend, centrifuged, the supernatant was removed, 100μL of PBS containing live dead dye (biolegend, catalog number 423102) was added, and the suspension was placed at 4℃ for 20min. Centrifuged for 5min, the supernatant was removed, 1mL of flow buffer was added to resuspend, centrifuged for 5min, and the supernatant was removed. 100μL of flow buffer containing 1μg CD16 / 32 blocking antibody was added. The suspension was placed at 4℃ for 20min. Centrifuge for 5 minutes, remove the supernatant, add 100 μL of flow buffer containing 0.25 μg CD8 antibody, 0.25 μg CD11c antibody, 0.25 μg MHCII antibody and 0.25 μg CD86 antibody, incubate at 4°C for 30 minutes, centrifuge, remove the supernatant, add 1 mL of flow buffer to resuspend, centrifuge, remove the supernatant, add 400 μL of flow buffer to resuspend, and detect by flow cytometer.

[0107] The results are as follows Figure 7 The results showed that the XCL-1-Tag-LNP (iv) group, i.e., tail vein injection of XCL-1 modified LNP, can effectively induce CD86 protein expression in spleen cells (mainly cDC1 cells). The above experimental results indicate that XCL-1 modified LNP promotes the activation of cDC1 cells more than LNP without protein modification. At the same time, different administration methods of XCL-1 modified LNP affect its activation effect on cDC1, and the tail vein method has a stronger activation effect than the intramuscular injection method.

[0108] Experimental Example 5 Treatment Effects on Tumor-bearing Mice

[0109] Experimental materials: XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0110] Experimental method: After C57BL / 6 mice were adaptively raised, each C57BL / 6 mouse was subcutaneously injected with 5*10 6 B16F10-OVA cells (B16F10-OVA cells are B16F10 mouse melanoma tumor cell line with overexpression of OVA gene) were randomly divided into 4 groups after tumor formation six days later, with 7 mice in each group:

[0111] Control group: On day 0, mice were first injected with 100 μL PBS via tail vein, once every 7 days, for a total of three injections, and were kept normally during the injection period;

[0112] Tag-LNP (iv) group: On day 0, mice were first injected with Tag-LNP prepared in Comparative Example 1 through the tail vein at a dose of 5 μg mRNA / mouse, once every 7 days, for a total of three injections, and were fed normally during the injection period;

[0113] XCL-1-Tag-LNP (im) group: On day 0, mice were injected intramuscularly for the first time with XCL-1-Tag-LNP prepared in Example 1, with a dose of 5 μg mRNA / mouse, once every 7 days, for a total of three injections, and were fed normally during the injection period;

[0114] XCL-1-Tag-LNP (iv) group: On day 0, mice were first injected with XCL-1-Tag-LNP prepared in Example 1 via tail vein at a dose of 5 μg mRNA / mouse. The injection frequency was once every 7 days for a total of three injections. The mice were fed normally during the injection period.

[0115] During the above treatment process, the volume changes of the tumor were recorded every two days. After the drug injection, the mice were fed normally every day and their survival rate was recorded.

[0116] Experimental results:

[0117] from Figure 8 It can be seen that after the mice form tumors, the injection of XCL-1-Tag-LNP and Tag-LNP can effectively reduce the size of the tumor. Among them, the XCL-1-Tag-LNP (iv) group has the best effect, which means that under the same injection method, the injection of XCL-1 modified LNP can better inhibit the growth of tumors.

[0118] from Fig. 9 The figure shows the survival of mice after tumor formation. It can be seen from the figure that the control group mice that were raised normally without any operation after tumor formation had a survival rate of only 50% at 14 days. The survival rate of mice injected with Tag-LNP and XCL-1-Tag-LNP after tumor formation was significantly improved at the same number of days. Among them, the XCL-1-Tag-LNP (iv) group had the best effect in prolonging the survival rate of mice, with a survival rate of 50% at 36 days, which was significantly better than the XCL-1-Tag-LNP (im) group and the Tag-LNP (iv) group.

[0119] Experimental Example 6 Cytotoxic CD8 in tumor sites of tumor-bearing mice + T cell level

[0120] Experimental materials: XCL-1-Tag-LNP prepared in Example 1 and Tag-LNP prepared in Comparative Example 1.

[0121] Experimental method: After C57BL / 6 mice were adaptively raised, each C57BL / 6 mouse was subcutaneously injected with 5*10 6 After six days of tumor formation, the B16F10-OVA cells were randomly divided into four groups, with seven cells in each group:

[0122] Control group: mice were raised normally;

[0123] Tag-LNP (iv) group: mice were injected with the Tag-LNP prepared in Comparative Example 1 via tail vein, with a dose of 5 μg mRNA / mouse;

[0124] XCL-1-Tag-LNP (im) group: mice were intramuscularly injected with XCL-1-Tag-LNP prepared in Example 1, with a dose of 5 μg mRNA / mouse;

[0125] XCL-1-Tag-LNP (iv) group: The mice were injected with XCL-1-Tag-LNP prepared in Example 1 via tail vein, with a dose of 5 μg mRNA / mouse.

[0126] After the above grouping treatment, mice were killed on the 7th day, tumor sites were taken, ground into single cell suspension, filtered with a 70μm mesh, centrifuged, supernatant removed, 5mL flow PBS was added for resuspending, centrifuged, supernatant removed, 100μL PBS containing live dead dye (biolegend, catalog number 423102) was added, and placed at 4℃ for 20min. Centrifuged for 5min, supernatant removed, 1mL flow buffer was added for resuspending, centrifuged for 5min, and supernatant removed. 100μL flow buffer containing 1μg CD16 / 32 blocking antibody was added. Placed at 4℃ for 20min. Centrifuge for 5 minutes, remove the supernatant, add 100 μL of flow buffer containing 0.25 μg CD45 antibody, 1 μg CD3 antibody, 0.25 μg CD8 antibody and 0.25 μg CD107a antibody, incubate at 4°C for 30 minutes, centrifuge, remove the supernatant, add 1 mL of flow buffer to resuspend, centrifuge, remove the supernatant, add 400 μL of flow buffer to resuspend, and detect by flow cytometer.

[0127] Experimental results:

[0128] Tumor infiltrating CD8 + T cells belong to "tumor infiltrating lymphocytes", which are a heterogeneous group of lymphocytes with anti-tumor effects present in tumors, among which the cytotoxic CD8 + T cells (also called cytotoxic T lymphocytes, CTL) are a major killer cell that secretes various cytokines to participate in immune responses and has a killing effect on certain viruses and tumor cells. However, the killer CD8 +T cell function can be inhibited, such as by overexpression of PD-L1, which puts them in a state of exhaustion and makes them unable to kill tumor cells. + The protein molecule of T cells is expressed on the surface of cells after being activated, and its content level is similar to that of CD8 + The toxicity of T cells in killing tumors is proportional.

[0129] The present invention uses live-dead dye to circle live cells, and then circles CD45 + and CD3 + Double positive cells are T cells, and then through CD8 + Circle the CD8 in the T cell + T cells, and then through CD107a + Circle CD8 + CD8 in T cells has cytotoxic function + T cells are CTLs. CD107a is a cell-killing marker. The more CD107a there is, the stronger its ability to kill tumors is. Fig.10 Cytotoxic CD8 + T cell level. As can be seen from the figure, the CD8 + CD107a in T cells + The percentage is low, which means that after the mice grow tumors, without any treatment, CD8 + The tumor infiltration of T cells was low. However, after treatment with Tag-LNP and XCL-1-Tag-LNP, the CD8 + CD107a in T cells + The percentage increased significantly, among which the XCL-1-Tag-LNP (iv) group had the best effect. The above results show that the injection of Tag-LNP and XCL-1-Tag-LNP can significantly promote the killing of CD8 + XCL-1 modified LNPs can significantly promote the infiltration of cytotoxic CD8 T cells in tumor sites. + The infiltration of T cells can effectively kill tumor cells and thus inhibit tumor growth.

[0130] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A targeted mRNA-LNP vaccine, characterized in that: The invention comprises lipid nanoparticles LNP and mRNA. The lipid nanoparticles LNP comprise polyethylene glycol-lipid conjugates modified with SpyTag polypeptide, ionizable cationic lipids, amphiphilic phospholipids, steroidal lipids and polyethylene glycol-lipid conjugates; and the surface of the lipid nanoparticles LNP is modified with Spycatcher fusion protein.

2. The mRNA-LNP vaccine according to claim 1, characterized in that The structure of the ionizable cationic lipid is shown below:

3. The mRNA-LNP vaccine according to claim 1, characterized in that The Spycatcher fusion protein is Spycatcher-XCL-1 protein.

4. The mRNA-LNP vaccine according to claim 1, characterized in that The molar ratio of the ionizable cationic lipid, the amphiphilic phospholipid, the steroidal lipid, the polyethylene glycol-lipid conjugate and the SpyTag polypeptide-modified polyethylene glycol-lipid conjugate is 48-50:8.5-10:36-38.5:0.1-2:0.1-2.

5. The mRNA-LNP vaccine according to claim 1, characterized in that The molar ratio of the SpyTag polypeptide-modified polyethylene glycol-lipid conjugate to the Spycatcher fusion protein is 1:0.5-2.

6. The mRNA-LNP vaccine according to claim 1, characterized in that The amphiphilic phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine or dipalmitoylphosphatidylcholine.

7. The mRNA-LNP vaccine according to claim 1, characterized in that The polyethylene glycol-lipid conjugate is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000.

8. The mRNA-LNP vaccine according to claim 1, characterized in that The mRNA encodes a tumor antigen.

9. The method for preparing the mRNA-LNP vaccine according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. Take an ionizable cationic lipid, an amphiphilic phospholipid, a steroidal lipid, a polyethylene glycol-lipid conjugate and a SpyTag polypeptide-modified polyethylene glycol-lipid conjugate and add anhydrous ethanol and mix well to obtain an ethanol phase; S2. Dissolve the mRNA in a buffer solution with a pH of 3.5 to 4.5 to obtain an aqueous phase, mix the ethanol phase of S1 with the aqueous phase, let stand, dialyze, take the dialyzed solution, add the Spycatcher fusion protein, incubate, and obtain the mRNA-LNP vaccine.

10. Use of the mRNA-LNP vaccine according to any one of claims 1 to 8 in the preparation of tumor prevention and treatment drugs.

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