An mRNA vaccine containing dual targets and its application

Abstract

The present invention provides an mRNA vaccine containing dual targets and its application, belonging to the technical field of medical preparations. The mRNA vaccine is transcribed from a target gene, and the target gene includes a CD20 single-chain antibody and a BCMA single-chain antibody. The nucleic acid artificial sequence of the CD20 single-chain antibody is as shown in SEQ ID NO.6 in the sequence listing, and the nucleic acid artificial sequence of the BCMA single-chain antibody is as shown in SEQ ID NO.9 in the sequence listing. The present invention uses the CD20 single-chain antibody and the BCMA single-chain antibody as dual targets, optimizes the nucleotide sequences of both, constructs a CAR structure and transcribes it into mRNA, which is delivered into the body by lipid nanoparticles (LNPs) to generate CAR-T cells for the treatment of systemic lupus erythematosus, overcoming side effects such as cytotoxicity brought by traditional autologous CAR-T therapy while improving the treatment effect.

Description

Technical Field

[0001] The present invention relates to an mRNA vaccine containing dual targets and its application, belonging to the technical field of medical preparations. Background Art

[0002] Systemic lupus erythematosus (SLE) is a diffuse and systemic autoimmune disease that can affect multiple organs and systems, and the skin and kidneys are the organs most frequently involved. As a highly heterogeneous autoimmune disease, its pathogenesis is complex and the cause is unknown. Current studies have pointed out that dysregulation of B cell activation plays a key role in the development of SLE. Abnormally activated B cells secrete autoantibodies, which cause damage to the functions of multiple organs through complement activation and antibody-dependent cell-mediated cytotoxicity (ADCC). At present, drugs targeting B cell targets are emerging continuously.

[0003] In recent years, CAR-T cell therapies targeting CD19, CD20, BCMA, etc. have shown good therapeutic effects on patients with systemic lupus erythematosus, bringing a new, safe and effective treatment option for patients with systemic lupus erythematosus. CD19 and CD20 are both proteins expressed on the surface of B cells, and they play important roles in the development and function of B cells. BCMA is a member of the tumor necrosis factor receptor superfamily, and both are highly expressed in SLE, so they have become important targets for the treatment of systemic lupus erythematosus.

[0004] Currently, the main treatment for systemic lupus erythematosus is the traditional autologous CAR-T therapy, and there is no report on the preparation of related therapeutic vaccines. Patent CN118290596A provides a chimeric antigen receptor targeting CD19 and CD22 and its CAR-T cells for the treatment of B cell malignancies and B cell-related autoimmune diseases, and it is determined that the cell therapy group with dual targets has much higher in vivo anti-tumor activity than the single-target treatment group. A paper published in The New England Journal of Medicine demonstrated the good and durable efficacy and safety of CAR-T treatment in systemic lupus erythematosus with immune thrombocytopenia, showing the application potential of CAR-T in the treatment of SLE and ITP (Li M, Zhang Y, Jiang N, et al. Anti-CD19 CAR T Cells in Refractory Immune Thrombocytopenia of SLE. [J]. The New England journal of medicine, 2024, 391(4): 376 - 378.).

[0005] Both of the above two methods apply CAR-T cells to the treatment of systemic lupus erythematosus. However, it usually requires collecting T cells from the patient's body, genetically engineering and amplifying them in vitro, and then reinfusing them into the patient's body, which takes several weeks to produce a sufficient number of cells. At the same time, before the redesigned cells can be introduced, the patient must also undergo chemotherapy as adjuvant therapy. The entire technological process is complex and time-consuming, so the cost is very high and the selling price is extremely high. Moreover, once the in vitro reprogrammed CAR-T cell therapy is infused into the human body, it can exist in the human body for several months or even years, and there may be various side effects on specific diseases.

[0006] mRNA vaccines have the following advantages: high safety, no potential risk of infection or insertional mutation; high effectiveness, the mRNA is constructed onto the vector molecule, can be rapidly taken up and expressed in the cytoplasm, and achieve effective in vivo delivery; high stability, various gene modifications make the mRNA more stable and highly translatable; can continuously optimize the encoded antigen by changing the nucleic acid sequence to achieve antigen-specific optimization; short time-consuming. The above advantages can exactly make up for the limitations of traditional CAR-T cell therapy.

[0007] There is no reported therapeutic mRNA vaccine for systemic lupus erythematosus in the prior art. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the present invention provides a dual-target mRNA vaccine and its application, achieving the following invention purposes: the dual-target mRNA vaccine has a high transfection efficiency for T cells and has an obvious therapeutic effect on systemic lupus erythematosus.

[0009] To solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A dual-target mRNA vaccine, the mRNA vaccine is transcribed from a target gene; the target gene includes a CD20 single-chain antibody and a BCMA single-chain antibody; the nucleic acid artificial sequence of the CD2 single-chain antibody is as shown in SEQ ID NO.6 in the sequence listing, and the nucleic acid artificial sequence of the BCMA single-chain antibody is as shown in SEQ ID NO.9 in the sequence listing.

[0011] The target gene is sequentially concatenated by the following modules: 5′–UTR, leader, CD20 single-chain antibody, linker, BCMA single-chain antibody, CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, 3′–UTR.

[0012] The nucleic acid artificial sequence of the target gene is as shown in SEQ ID NO.2 in the sequence listing.

[0013] The mRNA vaccine is an mRNA vaccine encapsulated by LNP.

[0014] The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, and DSPE-PEG-maleimide, and the mass ratio is (2.9-3.1):(1.35-1.39):(0.34-0.36):(0.72-0.76):(0.15-0.18).

[0015] The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, and DSPE-PEG-maleimide, and the mass ratio is 3:1.37:0.35:0.74:0.16.

[0016] The application of the mRNA vaccine in the preparation of a drug for treating systemic lupus erythematosus.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention uses CD20 single-chain antibody and BCMA single-chain antibody as double targets, optimizes the nucleotide sequences of both, constructs a CAR structure and transcribes it into mRNA, which is delivered into the body by lipid nanoparticles (LNP) to generate CAR-T cells for treating systemic lupus erythematosus, overcoming side effects such as cytotoxicity brought by traditional autologous CAR-T therapy while improving the treatment effect.

[0019] The mRNA vaccine of the present invention has a high transfection efficiency for T cells, can extend the survival period of systemic lupus erythematosus, improve its survival rate, and reduce the values of systemic lupus erythematosus-related indexes in its serum. Description of the Drawings

[0020] Figure 1 It is a bar graph of the toxicity evaluation of LNP / mRNA-1 vaccine and LNP / mRNA-2 vaccine on T cells;

[0021] Figure 2 It is a flow cytometry diagram of the expression rates of CD20 and BCMA in T cells transfected with LNP / mRNA-1 vaccine;

[0022] Among them, A is a flow cytometry diagram of the expression rate of CD20 in T cells transfected with LNP / mRNA-1 vaccine; B is a flow cytometry diagram of the expression rate of BCMA in T cells transfected with LNP / mRNA-1 vaccine;

[0023] Figure 3 It is a flow cytometry diagram of the expression rates of CD20 and BCMA in T cells transfected with LNP / mRNA-2 vaccine;

[0024] Among them, A is a flow cytometry plot of the expression rate of CD20 in T cells transfected with the LNP / mRNA-2 vaccine; B is a flow cytometry plot of the expression rate of BCMA in T cells transfected with the LNP / mRNA-2 vaccine;

[0025] Figure 4 is a line graph of the survival rate and survival period of the mice in Example 5;

[0026] Figure 5 is a bar graph of the systemic lupus erythematosus-related indicators of the mice in Example 6;

[0027] Among them, A is a bar graph of the IgG content in the mouse serum; B is a bar graph of the IgM content in the mouse serum; C is a bar graph of the anti-dsDNA content in the mouse serum; D is a bar graph of the IL-6 content in the mouse serum. Detailed implementation manners

[0028] Example 1 mRNA construction containing the CAR (CD20-BCMA) structure

[0029] The respective modules of the target gene and its nucleic acid artificial sequences are as follows:

[0030] (1) 5′–UTR, whose nucleic acid artificial sequence is as shown in SEQ ID NO.3 in the sequence listing;

[0031] (2) Leader, whose nucleic acid artificial sequence is as shown in SEQ ID NO.4 in the sequence listing;

[0032] (3) ScFv(CD20), whose nucleic acid artificial sequence before optimization is as shown in SEQ ID NO.5 in the sequence listing; the optimized nucleic acid artificial sequence is as shown in SEQ ID NO.6 in the sequence listing;

[0033] (4) Linker, whose nucleic acid artificial sequence is as shown in SEQ ID NO.7 in the sequence listing;

[0034] (5) ScFv(BCMA), whose nucleic acid artificial sequence before optimization is as shown in SEQ ID NO.8 in the sequence listing; the optimized nucleic acid artificial sequence is as shown in SEQ ID NO.9 in the sequence listing;

[0035] (6) CD8 Hinge region, whose nucleic acid artificial sequence is as shown in SEQ ID NO.10 in the sequence listing;

[0036] (7) CD28 transmembrane region, whose nucleic acid artificial sequence is as shown in SEQ ID NO.11 in the sequence listing;

[0037] (8) CD28-4-1BB costimulatory region, the artificial nucleic acid sequence of which is shown as SEQ ID NO.12 in the sequence listing;

[0038] (9) CD3ζ intracellular region, the artificial nucleic acid sequence of which is shown as SEQ ID NO.13 in the sequence listing;

[0039] (10) 3′–UTR, the artificial nucleic acid sequence of which is shown as SEQ ID NO.14 in the sequence listing.

[0040] A. Construction of recombinant plasmid PVAX1-CAR-1

[0041] The artificial nucleic acid sequences of 5′–UTR, leader, ScFv(CD20), linker, ScFv(BCMA), CD8Hinge region, CD28 transmembrane region, CD28-4-1BB costimulatory region, CD3ζ intracellular region, and 3′–UTR were ligated in sequence to construct a recombinant plasmid, in which the artificial nucleic acid sequences of ScFv(CD20) and ScFv(BCMA) before optimization were used.

[0042] Specifically as follows:

[0043] The artificial nucleic acid sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14 were ligated in sequence. The ligated sequence is shown as SEQ ID NO.1 in the sequence listing. Entrust Nanjing Genscript Biotech Co., Ltd. to synthesize its entire expression cassette and insert it into the standard vector pUC57 vector, named pUC-CAR-1. The pUC-CAR-1 vector and the PVAX1 vector were double digested with FastDigest BamH I (purchased from Thermo Fisher) and Fast Digest Not I (purchased from Thermo Fisher). The linearized DNA fragments were recovered by gel cutting and ligated overnight at 16°C to form the PVAX1-CAR-1 expression vector. The above PVAX1-CAR-1 expression vector was transformed into E.coli (DH5α), positive clones were picked, identified by PCR, and then the plasmids were extracted and sent to Nanjing Genscript Biotech Co., Ltd. for sequencing. The sequencing identification was correct, that is, PVAX1-CAR-1 was successfully constructed. The PVAX1-CAR-1 recombinant plasmid was extracted from the positive clones, diluted to 2 μg / μL, and stored at -80°C for later use.

[0044] B. Construction of recombinant plasmid PVAX1-CAR-2

[0045] The nucleic acid artificial sequences of 5′–UTR, guide sequence, ScFv(CD20), linker, ScFv(BCMA), CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, and 3′–UTR were successively ligated to construct a recombinant plasmid, wherein the optimized nucleic acid artificial sequences were used for ScFv(CD20) and ScFv(BCMA).

[0046] Specifically as follows:

[0047] The nucleic acid artificial sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14 were successively ligated. The ligated sequence, as shown in SEQ ID NO.2 in the sequence listing, was used to obtain the PVAX1-CAR-2 recombinant plasmid according to the above method. The plasmid was diluted to 2 μg / μL and stored at -80 °C for later use.

[0048] The obtained PVAX1-CAR-1 recombinant plasmid and PVAX1-CAR-2 recombinant plasmid were respectively subjected to enzymatic digestion experiments and purification to obtain linearized DNA fragments. Transcription was completed under the action of T7 polymerase, and further capping and tailing were carried out using commercial reagents. Finally, the mRNA of PVAX1-CAR-1 was purified using the MEGAclear kit to obtain purified mRNA, named CAR-mRNA-1 (abbreviated as mRNA-1), and the mRNA of PVAX1-CAR-2 was purified to obtain purified mRNA, named CAR-mRNA-2 (abbreviated as mRNA-2), and their concentrations were both adjusted to 1.0 μg / μL.

[0049] Example 2 Preparation and Characterization of LNP for Packaging mRNA

[0050] 1. Preparation of Ethanol Solution of Liposome Mixture

[0051] 3 mg of ionizable lipid DLin-MC3-DMA, 1.37 mg of cholesterol, 0.35 mg of DMG-PEG2000, 0.74 mg of DSPC, and 0.16 mg of DSPE-PEG-maleimide were dissolved in 1 mL of ethanol to obtain an ethanol solution of the liposome mixture.

[0052] 2. Preparation of Aqueous mRNA Solution

[0053] Dissolve the two purified mRNAs prepared in Example 1 in a citrate buffer, add PBS, and pipette 50 times to mix evenly, obtaining an aqueous solution of mRNA-1 and an aqueous solution of mRNA-2 respectively. The volume ratio of the purified mRNA to the citrate buffer (10 mM, pH = 4) and PBS is 1:1250:2500.

[0054] 3. Synthesis of LNP / mRNA Vaccine

[0055] Using a nanoparticle synthesis system (Ignite), mix an ethanol solution of the liposome mixture with the aqueous mRNA solution at a ratio of 1:3 (volume ratio) to synthesize LNP particles; dialyze the obtained LNP particles in a PBS solution (pH 7.4) for 24 h. After dialysis, a solution containing LNP is obtained, which is concentrated using an Amicon ultrafiltration filter and passed through a 0.22 μm filter membrane twice. Adjust the concentration of mRNA to 2 μg / mL and store it at -20 °C for later use to obtain the LNP / mRNA vaccine.

[0056] Prepare the LNP / mRNA vaccines from the aqueous solutions of mRNA-1 and mRNA-2 respectively using the above method, and label them as LNP / mRNA-1 vaccine and LNP / mRNA-2 vaccine.

[0057] Replace the aqueous mRNA solution with an equal amount of DEPC water, and label the prepared vaccine as LNP / blank vaccine.

[0058] Example 3 Characterization and Analysis of LNP / mRNA Nanoparticles

[0059] Measure the size, dispersion index, and encapsulation efficiency of the nanoparticles in each group of vaccines prepared in Example 2.

[0060] Analyze the size and dispersion index (PDI) of the nanoparticles in the LNP-mRNA vaccine using dynamic light scattering (DLS), and measure the encapsulation efficiency of the nanoparticles using Quant-iT™ RiboGreen® RNA Reagent and Kit (purchased from Invitrogen).

[0061] The results show that the size of the nanoparticles in the LNP / mRNA-1 vaccine is 81 - 108 nm, the dispersion index PDI is 0.126, and the encapsulation efficiency is 91.2%; the size of the nanoparticles in the LNP / mRNA-2 vaccine is 83 - 110 nm, the dispersion index PDI is 0.141, and the encapsulation efficiency is 92.9%; the size of the nanoparticles in the LNP / blank vaccine is 88 - 114 nm, the dispersion index is 0.139, and the encapsulation efficiency is 89.8%.

[0062] Example 4: Cytotoxicity and in vitro transfection experiments of LNP / mRNA-1 vaccine and LNP / mRNA-2 vaccine in T cells

[0063] (1)Preparation of activated T cells

[0064] Take 50 mL of peripheral blood and use TBD sample density separation solution (purchased from Tianjin Haoyang Huake Biotechnology) to separate and obtain PMBC. After inducing and culturing with DMEM medium (purchased from CORNING) containing 1000 IU / mL recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical) for 24 h, add recombinant IL-2 (purchased from Shenyang Sansheng Pharmaceutical) at a final concentration of 1000 IU / mL, OKT-3 at a final concentration of 50 ng / mL, and 5 vol% (final concentration) of the patient's autologous plasma, and continue to culture for 24 h. Double the volume of the medium every two days and culture until the 14th day. Use flow cytometry to detect the positive expression rates of CD3+ and CD56+ in T cells (CD3-FITC, CD16 / CD56-PE antibodies are purchased from BECKMAN, A07735). The results show that the positive rate of CD3+ > 90% and the double-positive rate of CD3+CD56+ > 20%, indicating successful induction of T cells.

[0065] (2)Cytotoxicity experiment

[0066] Inoculate the activated T cells into a 96-well plate (5×10 3 cells / well), and after culturing overnight, add LNP / mRNA-1 vaccine (5.5 ng / well in terms of mRNA) and LNP / mRNA-2 vaccine (5.5 ng / well in terms of mRNA) respectively, and co-incubate for 0 h, 6 h, 12 h, 18 h, 24 h to detect the viability of T cells. The results are as Figure 1 shown.

[0067] As Figure 1 can be seen, neither the LNP / mRNA-1 vaccine nor the LNP / mRNA-2 vaccine has obvious cytotoxicity and has good safety.

[0068] (3)In vitro transfection experiment

[0069] Inoculate the activated T cells into a 24-well plate (6×10 4 cells / well). After 24 h, divide them into three groups and add LNP / mRNA-1 vaccine (55 ng / well in terms of mRNA) and LNP / mRNA-2 vaccine (55 ng / well in terms of mRNA) respectively. After co-incubating for 24 h, collect the cells and use the TROP2 flow antibody (purchased from Abcam, product number: ab214488) to detect the transfection efficiency. The results are as Figure 2 and Figure 3 shown.

[0070] From Figure 2 and Figure 3 it can be seen that the expression rates of CD20 and BCMA in T cells transfected with the LNP / mRNA-1 vaccine are 36.7% and 38.2% respectively, and the expression rates of CD20 and BCMA in T cells transfected with the LNP / mRNA-2 vaccine are 60.4% and 62.2% respectively. This shows that the vaccine prepared in this application successfully obtains CAR-T cells after transfecting T cells, and the vaccine prepared after sequence optimization has a higher efficiency of transfecting T cells.

[0071] Example 5 Observation of Animal Survival Period

[0072] Purchase 5-6-month-old NZB / W F1 mice (Beijing ChengTian Biotechnology Co., Ltd.), randomly divide them into 3 groups, with 10 mice in each group, which are respectively:

[0073] Experimental Group A: Inject 200 μL of LNP / blank vaccine;

[0074] Experimental Group B: Inject 200 μL of LNP / mRNA-1 vaccine;

[0075] Experimental Group C: Inject 200 μL of LNP / mRNA-2 vaccine;

[0076] After injection, observe and record the survival status of experimental animals every day. The results are as Figure 4 shown. The survival period of the mice in Experimental Group A is much lower than that in Experimental Group B and Experimental Group C, and all the mice in Experimental Group A finally died on the 120th day; 6 mice in Experimental Group B died on the 111th day, and 4 mice survived, with a survival rate of 40%; 4 mice in Experimental Group C died on the 117th day, and 6 mice survived, with a survival rate of 60%. The survival period of the mice in Experimental Group C is longer than that in Experimental Group B, and the survival rate is higher, indicating that the LNP / mRNA-2 vaccine prepared after sequence optimization of ScFv(CD20) and ScFv(BCMA) has a significant effect on the treatment of systemic lupus erythematosus mice, greatly prolonging the survival cycle of mice and improving the survival rate of mice.

[0077] Example 6 Detection of Systemic Lupus Erythematosus-Related Indicators

[0078] Randomly divide 5-6-month-old NZB / W F1 mice into 3 groups, with 10 mice in each group,

[0079] Experimental Group A: Inject 300 μL of LNP / blank vaccine;

[0080] Experimental Group B: Inject 300 μL of LNP / mRNA-1 vaccine;

[0081] Experimental Group C: Inject 300 μL of LNP / mRNA-2 vaccine;

[0082] On the 15th day after vaccination, the peripheral blood of the surviving mice in each group was obtained and placed in an anticoagulant tube. After standing at room temperature for one hour, it was centrifuged at 1500 g for 10 min. The supernatant was taken and placed in a clean centrifuge tube. The levels of immunoglobulins, anti-double-stranded DNA antibodies and inflammatory factors in the mouse serum were detected using IgG, IgM, anti-dsDNA, and IL-6 ELISA kits (all purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.). The results are shown in Table 1 and Figure 5 as follows.

[0083] The results showed that the contents of immunoglobulin IgG, IgM, anti-dsDNA and IL-6 in the model mice of experimental group A were much higher than those of the other two groups. All indicators of experimental group B and experimental group C were significantly down-regulated, indicating that the prepared vaccine had a good therapeutic effect. After treating the diseased mice with the LNP / mRNA-2 vaccine (experimental group C) prepared by optimizing the ScFv(CD20) and ScFv(BCMA) sequences, the contents of immunoglobulin IgG, IgM, anti-dsDNA and IL-6 in their bodies were most significantly down-regulated, indicating that the vaccine prepared by optimizing the antibody sequence in this application had a significant improvement effect on the treatment of systemic lupus erythematosus.

[0084] Table 1

[0085]

Claims

1. A dual-target mRNA vaccine, characterized in that: The mRNA vaccine is transcribed from a target gene; the target gene includes a CD20 single-chain antibody and a BCMA single-chain antibody; the coding gene of the CD20 single-chain antibody is as shown in SEQ ID NO.6 in the sequence listing, and the coding gene of the BCMA single-chain antibody is as shown in SEQ ID NO.9 in the sequence listing; The target gene is obtained by sequentially connecting the following modules: 5′–UTR, leader, CD20 single-chain antibody coding gene, linker, BCMA single-chain antibody coding gene, CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, 3′–UTR; The mRNA vaccine is an LNP-encapsulated mRNA vaccine.

2. The vaccine according to claim 1, wherein: The nucleic acid sequence of the target gene is as shown in SEQ ID NO.2 in the sequence listing.

3. The vaccine according to claim 2, characterized in that: The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, DSPE-PEG-maleimide, and the mass ratio is (2.9-3.1):(1.35-1.39):(0.34-0.36):(0.72-0.76):(0.15-0.18).

4. The vaccine according to claim 3, characterized in that: The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, DSPE-PEG-maleimide, and the mass ratio is 3:1.37:0.35:0.74:0.

16.

5. Use of the vaccine according to any one of claims 1-4 in the preparation of a medicament for treating systemic lupus erythematosus.

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