MRNA (messenger ribonucleic acid) vaccine containing double targets and application of mRNA vaccine

Abstract

The invention provides an mRNA vaccine containing double targets and application thereof, and belongs to the technical field of medical preparations, and the mRNA vaccine is obtained by transcription of a target gene; the target gene comprises 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 a sequence table, and the nucleic acid artificial sequence of the BCMA single-chain antibody is as shown in SEQ ID NO.9 in the sequence table. The CD20 single-chain antibody and the BCMA single-chain antibody serve as double targets, nucleotide sequences of the CD20 single-chain antibody and the BCMA single-chain antibody are optimized, a CAR structure is constructed and transcribed into mRNA, the mRNA is delivered into a body through lipid nanoparticles (LNP), CAR-T cells are generated and used for treating systemic lupus erythematosus, the treatment effect is improved, and meanwhile the side effects of cytotoxicity and the like caused by a traditional autologous CAR-T therapy are overcome.

Description

Technical Field

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

[0002] Systemic lupus erythematosus (SLE) is a diffuse, systemic autoimmune disease that can affect multiple organs and systems, with the skin and kidneys being the most commonly affected organs. As a highly heterogeneous autoimmune disease, its pathogenesis is complex and its etiology is unknown. Studies have shown that dysregulation of B cell activation plays a key role in the pathogenesis of SLE. Abnormally activated B cells secrete autoantibodies and cause damage to multiple organs through complement activation and antibody-dependent cell-mediated cytotoxicity (ADCC). At this stage, drugs targeting B cell targets continue to emerge.

[0003] In recent years, CAR-T cell therapy with targets such as CD19, CD20, and BCMA has produced good therapeutic effects on patients with systemic lupus erythematosus, providing a new, safe and effective treatment option for patients with systemic lupus erythematosus. CD19 and CD20 are proteins expressed on the surface of B cells, which play an important role in the development and function of B cells. BCMA is a member of the tumor necrosis factor receptor superfamily. Both are highly expressed in SLE, so they have become important targets for the treatment of systemic lupus erythematosus.

[0004] At present, the treatment of systemic lupus erythematosus is mainly 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. It is determined that the dual-target cell therapy group has a more efficient in vivo anti-tumor activity than the single-target therapy group. A paper published in the New England Journal of Medicine showed the good and lasting efficacy and safety of CAR-T therapy in immune thrombocytopenia of systemic lupus erythematosus, 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 methods use CAR-T cells in 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 returning them to the patient, 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 an adjuvant treatment. The entire process is complicated and time-consuming, so the cost is very expensive and the price is extremely high. Moreover, once the in vitro reprogrammed CAR-T cell therapy is introduced into the human body, it can exist in the human body for months or even years, and may have multiple side effects for specific diseases.

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

[0007] There are no reports of therapeutic mRNA vaccines for systemic lupus erythematosus in the prior art. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides an mRNA vaccine containing dual targets and its application to achieve the following invention objectives: the mRNA vaccine containing dual targets has high transfection efficiency for T cells and has obvious therapeutic effect on systemic lupus erythematosus.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions: A dual-target mRNA vaccine, wherein the mRNA vaccine is transcribed from a target gene; the target gene comprises a CD20 single-chain antibody and a BCMA single-chain antibody; the artificial nucleic acid sequence of the CD20 single-chain antibody is shown in SEQ ID NO.6 in the sequence listing, and the artificial nucleic acid sequence of the BCMA single-chain antibody is shown in SEQ ID NO.9 in the sequence listing.

[0010] The target gene is obtained by serially connecting the following modules: 5′-UTR, guide, CD20 single-chain antibody, linker, BCMA single-chain antibody, CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, and 3′-UTR.

[0011] The artificial nucleic acid sequence of the target gene is shown as SEQ ID NO.2 in the sequence list.

[0012] The mRNA vaccine is an LNP-encapsulated mRNA vaccine.

[0013] The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, and DSPE-PEG-maleimide, with a mass ratio of (2.9-3.1): (1.35-1.39): (0.34-0.36): (0.72-0.76): (0.15-0.18).

[0014] The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, and DSPE-PEG-maleimide, with a mass ratio of 3:1.37:0.35:0.74:0.16.

[0015] The use of the mRNA vaccine in the preparation of a drug for treating systemic lupus erythematosus.

[0016] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention uses CD20 single-chain antibody and BCMA single-chain antibody as dual targets and optimizes the nucleotide sequences of the two to construct a CAR structure that is transcribed into mRNA, which is delivered into the body by lipid nanoparticles (LNP) to generate CAR-T cells for the treatment of systemic lupus erythematosus, thereby improving the therapeutic effect while overcoming the side effects such as cytotoxicity brought about by traditional autologous CAR-T therapy.

[0017] The mRNA vaccine of the present invention has high transfection efficiency for T cells, can prolong the survival period of systemic lupus erythematosus, improve its survival rate, and reduce the values ​​of systemic lupus erythematosus-related indicators in its serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a bar graph for evaluating the toxicity of LNP / mRNA-1 vaccine and LNP / mRNA-2 vaccine to T cells; Figure 2 Flow cytometry graphs showing the expression rates of CD20 and BCMA in T cells transfected with LNP / mRNA-1 vaccine; A is a flow cytometric graph of the expression rate of CD20 in T cells transfected with LNP / mRNA-1 vaccine; B is a flow cytometric graph of the expression rate of BCMA in T cells transfected with LNP / mRNA-1 vaccine; Figure 3 Flow cytometry graphs showing the expression rates of CD20 and BCMA in T cells transfected with LNP / mRNA-2 vaccine; A is a flow cytometric graph of the expression rate of CD20 in T cells transfected with LNP / mRNA-2 vaccine; B is a flow cytometric graph of the expression rate of BCMA in T cells transfected with LNP / mRNA-2 vaccine; Figure 4 is a line graph of the survival rate and survival period of mice in Example 5; Figure 5 is a bar graph of the systemic lupus erythematosus-related indicators in mice in Example 6; Among them, A is a bar graph of IgG content in mouse serum; B is a bar graph of IgM content in mouse serum; C is a bar graph of anti-dsDNA content in mouse serum; D is a bar graph of IL-6 content in mouse serum. DETAILED DESCRIPTION

[0019] Example 1 Construction of mRNA containing CAR (CD20-BCMA) structure The target gene modules and their artificial nucleic acid sequences are as follows: (1) 5′-UTR, the artificial nucleic acid sequence of which is shown in SEQ ID NO.3 in the sequence listing; (2) a guide, the artificial nucleic acid sequence of which is shown as SEQ ID NO.4 in the sequence listing; (3) ScFv (CD20), the artificial nucleic acid sequence before optimization is shown as SEQ ID NO.5 in the sequence listing; the artificial nucleic acid sequence after optimization is shown as SEQ ID NO.6 in the sequence listing; (4) a linker, whose artificial nucleic acid sequence is shown in SEQ ID NO.7 in the sequence listing; (5) ScFv (BCMA), the artificial nucleic acid sequence before optimization is shown as SEQ ID NO.8 in the sequence listing; the artificial nucleic acid sequence after optimization is shown as SEQ ID NO.9 in the sequence listing; (6) CD8 Hinge region, the artificial nucleic acid sequence of which is shown as SEQ ID NO.10 in the sequence listing; (7) CD28 transmembrane region, whose artificial nucleic acid sequence is shown as SEQ ID NO.11 in the sequence list; (8) CD28-4-1BB co-stimulatory region, whose artificial nucleic acid sequence is shown in SEQ ID NO.12 in the sequence list; (9) CD3ζ intracellular region, whose artificial nucleic acid sequence is shown in SEQ ID NO.13 in the sequence listing; (10) 3′-UTR, whose artificial nucleic acid sequence is shown as SEQ ID NO.14 in the sequence listing.

[0020] A. Construction of recombinant plasmid PVAX1-CAR-1 The artificial nucleic acid sequences of 5′-UTR, guide, ScFv (CD20), linker, ScFv (BCMA), CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, and 3′-UTR were connected in sequence to construct a recombinant plasmid, among which ScFv (CD20) and ScFv (BCMA) used the artificial nucleic acid sequences before optimization.

[0021] The details are as follows: 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 connected in sequence, and the connected sequence was shown in SEQ ID NO.1 in the sequence table. Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the entire expression frame 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), and the linearized DNA fragments were obtained by gel cutting and recovery, and the PVAX1-CAR-1 expression vector was formed by overnight connection at 16°C. The above-mentioned PVAX1-CAR-1 expression vector was transformed into E. coli (DH5α), and the positive clones were picked for PCR identification. Then the plasmid was extracted and sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing. If the sequencing identification was correct, 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 use.

[0022] B. Construction of recombinant plasmid PVAX1-CAR-2 The artificial nucleic acid sequences of 5′-UTR, guide, ScFv (CD20), linker, ScFv (BCMA), CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, and 3′-UTR were connected in sequence to construct a recombinant plasmid, among which ScFv (CD20) and ScFv (BCMA) used optimized artificial nucleic acid sequences.

[0023] The details are as follows: The artificial nucleic acid 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 connected in sequence. The connected sequence is shown in SEQ ID NO.2 in the sequence table. According to the above method, the PVAX1-CAR-2 recombinant plasmid was obtained, diluted to 2 μg / μL, and stored at -80°C for use.

[0024] The obtained PVAX1-CAR-1 recombinant plasmid and PVAX1-CAR-2 recombinant plasmid were subjected to restriction digestion experiments and purification to obtain linearized DNA fragments, which were transcribed under the action of T7 polymerase, and further capped and tailed using commercial reagents. Finally, the PVAX1-CAR-1 mRNA was purified using the MEGAclear kit to obtain the purified mRNA, which was named CAR-mRNA-1 (referred to as mRNA-1), and the PVAX1-CAR-2 mRNA was purified to obtain the purified mRNA, which was named CAR-mRNA-2 (referred to as mRNA-2). The concentrations of both were adjusted to 1.0 μg / μL.

[0025] Example 2 Preparation and Characterization of LNPs Packaging mRNA 1. Preparation of ethanol solution of liposome mixture 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 a liposome mixture.

[0026] 2. Preparation of mRNA aqueous solution The two purified mRNAs prepared in Example 1 were dissolved in citric acid buffer, PBS was added, pipetted 50 times, and mixed to obtain mRNA-1 aqueous solution and mRNA-2 aqueous solution, respectively. The volume ratio of purified mRNA to citric acid buffer (10 mM, pH = 4) and PBS was 1:1250:2500.

[0027] 3. Synthesis of LNP / mRNA vaccines Using a nanoparticle synthesis system (Ignite), the ethanol solution of the liposome mixture and the mRNA aqueous solution were mixed in a ratio of 1:3 (volume ratio) to synthesize LNP particles; the obtained LNP particles were dialyzed in a PBS solution (pH 7.4) for 24 hours to obtain a solution containing LNP, which was concentrated using an Amicon ultracentrifugal filter and passed through a 0.22μm filter membrane twice. The concentration of mRNA was adjusted to 2μg / mL and stored at -20°C for use to obtain an LNP / mRNA vaccine.

[0028] The mRNA-1 aqueous solution and the mRNA-2 aqueous solution were used to prepare LNP / mRNA vaccines respectively using the above method, and were labeled as LNP / mRNA-1 vaccine and LNP / mRNA-2 vaccine.

[0029] The mRNA aqueous solution was replaced with an equal amount of DEPC water, and the prepared vaccine was labeled as LNP / blank vaccine.

[0030] Example 3 Characterization and Analysis of LNP / mRNA Nanoparticles The size, dispersion index and encapsulation efficiency of the nanoparticles of each group of vaccines prepared in Example 2 were measured.

[0031] Dynamic light scattering (DLS) was used to analyze the size and dispersion index (PDI) of the nanoparticles of the LNP-mRNA vaccine, and Quant-iT™ RiboGreen® RNA Reagent and Kit (purchased from Invitorgen) was used to measure the encapsulation efficiency of the nanoparticles.

[0032] The results showed that the size of nanoparticles in the LNP / mRNA-1 vaccine was 81-108nm, the dispersion index PDI was 0.126, and the encapsulation efficiency was 91.2%; the size of nanoparticles in the LNP / mRNA-2 vaccine was 83-110nm, the dispersion index PDI was 0.141, and the encapsulation efficiency was 92.9%; the size of nanoparticles in the LNP / blank vaccine was 88-114nm, the dispersion index was 0.139, and the encapsulation efficiency was 89.8%.

[0033] Example 4 Cytotoxicity and in vitro transfection experiments of LNP / mRNA-1 and LNP / mRNA-2 vaccines in T cells (1) Preparation of activated T cells 50 mL of peripheral blood was taken and PMBC was isolated using TBD sample density separation fluid (purchased from Tianjin Haoyang Huake Biological). After induction culture for 24 h with DMEM (purchased from CORNING) containing 1000 IU / mL recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical), 1000 IU / mL (final concentration) of recombinant IL-2 (purchased from Shenyang Sansheng Pharmaceutical), 50 ng / mL (final concentration) of OKT-3 and 5 vol% (final concentration) of autologous plasma of patients were added to induce and continue culture for 24 h. The liquid was doubled every two days and cultured until the 14th day. The positive expression rate of CD3+ and CD56+ in T cells was detected by flow cytometry (CD3-FITC, CD16 / CD56-PE antibodies were purchased from BECKMAN, A07735). The results showed that the CD3+ positive rate was >90% and the CD3+CD56+ double positive rate was >20%, which was considered to be successful induction of T cells.

[0034] (2) Cytotoxicity assay Activated T cells were seeded in 96-well plates (5×10 3 After overnight culture, 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) were added, and T cell activity was detected at 0 h, 6 h, 12 h, 18 h, and 24 h. The results are shown in the figure. Figure 1 shown.

[0035] Depend on Figure 1 It can be seen that both LNP / mRNA-1 vaccine and LNP / mRNA-2 vaccine have no obvious cytotoxicity and have good safety.

[0036] (3) In vitro transfection experiment Activated T cells were seeded in 24-well plates (6 × 10 4 The cells were divided into three groups 24 hours later and LNP / mRNA-1 vaccine (55 ng / well, calculated as mRNA) and LNP / mRNA-2 vaccine (55 ng / well, calculated as mRNA) were added respectively. After 24 hours of incubation, the cells were collected and the transfection efficiency was detected by TROP2 flow cytometry antibody (purchased from Abcam, catalog number: ab214488). The results are shown in the figure. Figure 2 and Figure 3 shown.

[0037] Depend on Figure 2 and Figure 3It can be seen that the expression rate of CD20 in T cells transfected with LNP / mRNA-1 vaccine is 36.7%, and the expression rate of BCMA is 38.2%. The expression rate of CD20 in T cells transfected with LNP / mRNA-2 vaccine is 60.4%, and the expression rate of BCMA is 62.2%, indicating that the vaccine prepared in the present application successfully obtains CAR-T cells after transfecting T cells, and the vaccine prepared after sequence optimization has higher efficiency in transfecting T cells.

[0038] Example 5 Animal survival observation 5-6 month old NZB / W F1 mice were purchased (Beijing Chengtian Biotechnology Co., Ltd.) and randomly divided into 3 groups, 10 mice in each group, namely: Experimental group A: injection of 200 μL LNP / blank vaccine; Experimental group B: injection of 200 μL LNP / mRNA-1 vaccine; Experimental group C: injection of 200 μL LNP / mRNA-2 vaccine; The survival status of the experimental animals was observed and recorded every day after injection. Figure 4 As shown, the survival period of mice in experimental group A was much lower than those in experimental group B and experimental group C, and all the mice in experimental group A died on the 120th day; 6 mice in experimental group B died on the 111th day, and 4 survived, with a survival rate of 40%; 4 mice in experimental group C died on the 117th day, and 6 survived, with a survival rate of 60%. The survival period of mice in experimental group C was greater than that in experimental group B, and the survival rate was higher, indicating that the LNP / mRNA-2 vaccine prepared after optimization of ScFv (CD20) and ScFv (BCMA) sequences has a significant effect on the treatment of systemic lupus erythematosus mice, greatly prolongs the survival period of mice, and improves the survival rate of mice.

[0039] Example 6 Detection of systemic lupus erythematosus related indicators 5-6 month old NZB / W F1 mice were randomly divided into 3 groups, 10 mice in each group. Experimental group A: injection of 300 μL LNP / blank vaccine; Experimental group B: injection of 300 μL LNP / mRNA-1 vaccine; Experimental group C: injection of 300 μL LNP / mRNA-2 vaccine; On the 15th day after vaccination, peripheral blood of surviving mice in each group was obtained in anticoagulant tubes, and after being placed at room temperature for one hour, the blood was centrifuged at 1500 g for 10 min. The supernatant was placed in a clean centrifuge tube, and IgG, IgM, anti-dsDNA, and IL-6 ELISA kits (all purchased from Shanghai ELISA Biotechnology Co., Ltd.) were used to detect immunoglobulins, anti-double-stranded DNA antibody levels, and inflammatory factors in mouse serum. The results are shown in Tables 1 and Figure 5 shown.

[0040] The results showed that the levels of immunoglobulins IgG, IgM, anti-dsDNA and IL-6 in the model mice in experimental group A were much higher than those in the other two groups. All indicators of experimental groups B and experimental groups C were significantly reduced, indicating that the prepared vaccine has a good therapeutic effect. In addition, after the LNP / mRNA-2 vaccine (experimental group C) prepared after ScFv (CD20) and ScFv (BCMA) sequence optimization was used to treat the sick mice, the levels of immunoglobulins IgG, IgM, anti-dsDNA and IL-6 in their bodies were most significantly reduced, indicating that the vaccine prepared by antibody sequence optimization in this application has a significant improvement effect on the treatment of systemic lupus erythematosus.

[0041] Table 1

Claims

1. An mRNA vaccine containing dual targets, characterized in that: The mRNA vaccine is transcribed from the target gene; the target gene includes CD20 single-chain antibody and BCMA single-chain antibody; the artificial nucleic acid sequence of the CD20 single-chain antibody is shown in SEQ ID NO.6 in the sequence list, and the artificial nucleic acid sequence of the BCMA single-chain antibody is shown in SEQ ID NO.9 in the sequence list.

2. The mRNA vaccine containing dual targets according to claim 1, characterized in that: The target gene is obtained by serially connecting the following modules: 5′-UTR, guide, CD20 single-chain antibody, linker, BCMA single-chain antibody, CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, and 3′-UTR.

3. The mRNA vaccine containing dual targets according to claim 1, characterized in that: The artificial nucleic acid sequence of the target gene is shown as SEQ ID NO.2 in the sequence list.

4. The mRNA vaccine containing dual targets according to claim 1, characterized in that: The mRNA vaccine is an LNP-encapsulated mRNA vaccine.

5. The mRNA vaccine containing dual targets according to claim 4, characterized in that: The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, and DSPE-PEG-maleimide, with a mass ratio of (2.9-3.1): (1.35-1.39): (0.34-0.36): (0.72-0.76): (0.15-0.18).

6. The mRNA vaccine containing dual targets according to claim 5, characterized in that: The LNP includes ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, and DSPE-PEG-maleimide, with a mass ratio of 3:1.37:0.35:0.74:0.

16.

7. Use of the mRNA vaccine according to any one of claims 1 to 6 in the preparation of a medicament for treating systemic lupus erythematosus.

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