A targeting sequence element for enhancing antigen presentation of mRNA tumor vaccines and its application

Abstract

The present invention discloses a targeting sequence element for enhancing antigen presentation of mRNA tumor vaccine and its application. The targeting sequence element includes a MITD element, and the mRNA sequence of the MITD element includes any one of the sequences shown in SEQ ID NO.18 to SEQ ID NO.21 or a combination of at least two of them. The present invention designs and screens specific targeting sequence elements, especially MITD elements; and designs different MITD molecules and mutants thereof, directly detects the presentation amount of antigen epitopes bound to HLA I molecules through in vitro cell flow cytometry experiments, verifies the tumor vaccine anti-tumor efficacy through in vivo animal experiments, and the obtained nucleic acid construct can enhance the presentation effect of antigen epitopes expressed by tumor vaccines on HLA class I molecules, and improve the anti-tumor effect of tumor vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a targeting sequence element for enhancing antigen presentation of mRNA tumor vaccines and an application thereof. Background Art

[0002] As an important tumor immunotherapy, tumor vaccines have shown good therapeutic effects in clinical trials for different types of cancer. Tumor vaccines activate or enhance the body's own immune system to kill tumor cells to prevent further development of cancer. Tumor vaccines can be divided into many types, including cell vaccines, DNA vaccines, mRNA vaccines, peptide vaccines, dendritic cell vaccines and nanovaccines. Tumor mRNA vaccines are cancer treatment drugs based on mRNA vaccine technology. They translate mRNA molecules into functional proteins in the body to directly regulate the growth of tumor cells, or activate anti-tumor specific immune effects to attack cancer cells. In recent years, tumor treatment and vaccine development based on mRNA technology have made rapid progress. However, in-depth research is still needed in the sequence design of mRNA vaccines, mRNA translation efficiency, antigen presentation efficiency, cellular immune response, etc., in order to further improve the effect of tumor vaccines.

[0003] mRNA tumor vaccines are designed with tumor antigens as targets, so that the expressed tumor antigen epitopes are recognized by the major histocompatibility complex (MHC) in antigen-presenting cells (APCs), promoting antigen presentation on the APC cell surface and activating anti-tumor T lymphocytes in the body to kill tumor cells. The binding and presentation of tumor antigen epitopes to MHC molecules is a key step in the function of tumor vaccines. The properties, processing and presentation of tumor antigen epitopes affect the antigen-specific CD8 + and CD4 + T cell response: only T cells activated by APC cells can kill tumor cells. The presentation of MHC class I molecule antigen epitopes is a relatively inefficient process. The low density of antigen epitopes / MHC complexes on the surface of APC cells will cause inefficient activation of T cells, showing a weaker cellular immune response. Therefore, improving the presentation efficiency of MHC molecule antigens is of great significance for the development of tumor vaccines.

[0004] Signal sequences on vaccines, including signal peptides, can help tumor antigen epitopes efficiently bind to and present MHC molecules. If the transport domain of human leukocyte antigen (HLA) (MHC class I trafficking signal, MITD) is present at the C-terminus of tumor vaccines, it can improve the efficiency of antigen epitope binding and presentation to MHC molecules. However, there is still a lack of research on which signal peptide and MITD can achieve the best effect.

[0005] At present, the common strategy for antigen presentation by MHC molecules is to co-deliver MHC molecules with antigens or to target antigens to MHC molecules to enhance presentation efficiency. The theoretical basis of these strategies is that the terminal sequence of the intracellular domain of transmembrane proteins contains a signal sequence based on tyrosine or leucine, which is connected to the antigen sequence to form a protein complex, mediating the translocation of the expressed antigen into the subcellular space, and assisting the efficient binding and presentation of tumor antigen epitopes to MHC molecules. The MITD of human leukocyte antigen regulates its circulation between different subcellular organelles, and connects to the C-terminus of tumor vaccine antigen protein, which can improve the binding and presentation efficiency of antigen epitopes to MHC molecules. Although MHC molecules can enhance antigen presentation, there is still a lack of research on how to screen out targeting signals with high matching with tumor antigens, improve antigen presentation efficiency, and enhance antigen immunogenicity. Summary of the invention

[0006] In view of the shortcomings of the prior art and actual needs, the present invention provides a targeting sequence element for enhancing antigen presentation of mRNA tumor vaccines and its application, by targeting HLA Different mutation combinations in the molecular sequences enhance the antigen presentation effect of tumor mRNA vaccines.

[0007] In the process of studying the targeting signal sequence for improving antigen epitope presentation, the present invention discovered a targeting sequence that significantly improves the efficiency of antigen presentation, as well as its optimal combination with the antigen sequence, and preliminarily proved that it can achieve good anti-tumor effects as a tumor vaccine element, which shows that it has application prospects in the research of tumor prevention and treatment drugs.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a targeting sequence element for an mRNA tumor vaccine, wherein the targeting sequence element comprises a MITD element, and the mRNA sequence of the MITD element comprises any one of the sequences shown in SEQ ID NO.18 to SEQ ID NO.21 or a combination of at least two of them.

[0010] Preferably, the mRNA sequence of the MITD element comprises the sequence shown in SEQ ID NO.21.

[0011] SEQ ID NO.18:

[0012] AUCGUGGGCAUUGUUGCUGGCCUGGCUGUCCUAGCAGUUGUGGUCAUCGGAGCUGUGGUCGCUACUGUGAUGUGUAGGAGGAAGAGCUCAGGUGGAAAAGGAGGGAGCUACUCUCAGGCUGCGUCCAGCGACAGUGCCCAGGGCUCUGAUGUGUCUCUCACAGCUUGA.

[0013] SEQ ID NO.19:

[0014] AUCGUGGGCAUUGUUGCUGGCCUGGCUGUCCUAGCAGUUGUGGUCAUCGGAGCUGUGGUCGCUACUGUGAUGUGUAGGAGGAAGAGCUCAGGUGGAAAAGGAGGGAGCUACUCUCAGGCUGCGUCCAGCGACAGUGCCCAGGGCUCUCUGGUGUCUCUCACAGCUUGA.

[0015] SEQ ID NO.20:

[0016] AUCGUGGGCAUUGUUGCUGGCCUGGCUGUCCUAGCAGUUGUGGUCAUCGGAGCUGUGGUCGCUACUGUGAUGUGUAGGAGGAAGAGCUCAGGUGGAAAAGGAGGGAGCUACUCUCAGGCUGCGUCCAGCGACAGUGCCCAGGGCUCUGAUGUGGGCCUCACAGCUUGA.

[0017] SEQ ID NO.21:

[0018] AUCGUGGGCAUUGUUGCUGGCCUGGCUGUCCUAGCAGUUGUGGUCAUCGGAGCUGUGGUCGCUACUGUGAUGUGUAGGAGGAAGAGCUCAGGUGGAAAAGGAGGGAGCUACUCUCAGGCUGCGUCCAGCGACAGUGCCCAGGGCUCUCUGGUGGGCCUCACAGCUUGA.

[0019] The present invention designs and screens specific targeting sequence elements, especially MITD elements; and designs different MITD molecules and mutants thereof, directly detects the presentation amount of antigen epitopes bound to HLA I molecules through in vitro cell flow cytometry experiments, verifies the tumor-suppressing efficacy of tumor vaccines through in vivo animal experiments, and the obtained nucleic acid construct can enhance the presentation effect of antigen epitopes expressed by tumor vaccines on HLA class I molecules, and improve the anti-tumor effect of tumor vaccines.

[0020] Preferably, the amino acid sequence encoded by the MITD element includes any one of the sequences shown in SEQ ID NO.26 to SEQ ID NO.29 or a combination of at least two of them.

[0021] Preferably, the amino acid sequence encoded by the MITD element includes the sequence shown in SEQ ID NO.29.

[0022] SEQ ID NO.26:

[0023] IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA.

[0024] SEQ ID NO.27:

[0025] IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSLVSLTA.

[0026] SEQ ID NO.28:

[0027] IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVGLTA.

[0028] SEQ ID NO.29:

[0029] IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSLVGLTA.

[0030] In a second aspect, the present invention provides a MITD nucleic acid molecule of an mRNA tumor vaccine, wherein the DNA sequence of the MITD nucleic acid molecule comprises any one of the sequences shown in SEQ ID NO.8 to SEQ ID NO.11 or a combination of at least two of them.

[0031] Preferably, the DNA sequence of the MITD nucleic acid molecule comprises the sequence shown in SEQ ID NO.11.

[0032] SEQ ID NO.8:

[0033] ATCGTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGTGGTCATCGGAGCTGTGGTCGCTACTGTGATGTGTAGGAGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTCAGGCTGCGTCCAGCGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGA。

[0034] SEQ ID NO.9:

[0035] ATCGTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGTGGTCATCGGAGCTGTGGTCGCTACTGTGATGTGTAGGAGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTCAGGCTGCGTCCAGCGACAGTGCCCAGGGCTCTCTGGTGTCTCTCACAGCTTGA。

[0036] SEQ ID NO.10:

[0037] ATCGTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGTGGTCATCGGAGCTGTGGTCGCTACTGTGATGTGTAGGAGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTCAGGCTGCGTCCAGCGACAGTGCCCAGGGCTCTGATGTGGGCCTCACAGCTTGA。

[0038] SEQ ID NO.11:

[0039] ATCGTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGTGGTCATCGGAGCTGTGGTCGCTACTGTGATGTGTAGGAGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTCAGGCTGCGTCCAGCGACAGTGCCCAGGGCTCTCTGGTGGGCCTCACAGCTTGA。

[0040] In a third aspect, the present invention provides a nucleic acid construct, wherein the structure of the nucleic acid construct from 5' to 3' includes: a T7 promoter, a 5' UTR, a signal peptide (tumor vaccine N-terminal signal peptide, signal peptides, SP), a first linker (Linker1), a target gene, a second linker (Linker2), the MITD nucleic acid molecule described in the second aspect, a 3' UTR and Poly (A).

[0041] It can be understood that the nucleic acid construct of the present invention includes but is not limited to a linker, a signal peptide, etc., and other applicable nucleic acid molecules in the art are within the protection scope of the present invention.

[0042] Preferably, the mRNA sequence of the signal peptide includes the sequence shown as SEQ ID NO.13; the DNA sequence of the signal peptide includes the sequence shown as SEQ ID NO.3.

[0043] SEQ ID NO.13:

[0044] AUGCGGGUCACGGCGCCCCGAACCGUCCUCCUGCUGCUCUGGGGGGCAGUGGCCCUGACCGAGACCUGGGCC.

[0045] SEQ ID NO.3:

[0046] ATGCGGGTCACGGCGCCCCGAACCGTCCTCCTGCTGCTCTGGGGGGCAGTGGCCCTGACCGAGACCTGGGCC.

[0047] Preferably, the target gene includes an OVA antigen epitope; the mRNA sequence of the OVA antigen epitope includes the sequence shown in SEQ ID NO.15; and the DNA sequence of the OVA antigen epitope includes the sequence shown in SEQ ID NO.5.

[0048] SEQ ID NO. 15: AGUAUAAUCAACUUUGAAAAACUG.

[0049] SEQ ID NO. 5: AGTATAATCAACTTTGAAAAACTG.

[0050] Preferably, the 5'UTR includes human α-globin 5'UTR; the mRNA sequence of human α-globin 5'UTR includes the sequence shown in SEQ ID NO.12; and the DNA sequence of human α-globin 5'UTR includes the sequence shown in SEQ ID NO.2.

[0051] SEQ ID NO.12:

[0052] AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC.

[0053] SEQ ID NO.2:

[0054] AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC.

[0055] Preferably, the 3'UTR includes human β-globin 3'UTR; the mRNA sequence of human β-globin 3'UTR includes the sequence shown in SEQ ID NO.17; and the DNA sequence of human β-globin 3'UTR includes the sequence shown in SEQ ID NO.7.

[0056] SEQ ID NO.17:

[0057] GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA.

[0058] SEQ ID NO.7:

[0059] GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA.

[0060] Preferably, the mRNA sequence of the first connector includes the sequence shown as SEQ ID NO.14; the DNA sequence of the first connector includes the sequence shown as SEQ ID NO.4.

[0061] SEQ ID NO. 14: GGCAGCGGCGGCAGC.

[0062] SEQ ID NO. 4: GGCAGCGGCGGCAGC.

[0063] Preferably, the mRNA sequence of the second connector includes the sequence shown as SEQ ID NO.16; the DNA sequence of the second connector includes the sequence shown as SEQ ID NO.6.

[0064] SEQ ID NO. 16: GGCGGCGGCGGCAGCGGC.

[0065] SEQ ID NO. 6: GGCGGCGGCGGCAGCGGC.

[0066] Preferably, the DNA sequence of the T7 promoter includes the sequence shown in SEQ ID NO.1.

[0067] SEQ ID NO. 1: TAATACGACTCACTATA.

[0068] Preferably, the length of Poly(A) is 90-120 nt.

[0069] Preferably, the protein encoded by the nucleic acid construct comprises: a signal peptide, an OVA antigen epitope, a first linker and a second linker.

[0070] Preferably, the amino acid sequence of the signal peptide includes the sequence shown in SEQ ID NO.22.

[0071] SEQ ID NO. 22: MRVTAPRTVLLLLWGAVALTETWA.

[0072] Preferably, the amino acid sequence of the OVA antigen epitope includes the sequence shown in SEQ ID NO.24.

[0073] SEQ ID NO.24: SIINFEKL.

[0074] Preferably, the amino acid sequence of the first connector includes the sequence shown in SEQ ID NO.23.

[0075] SEQ ID NO.23:GSGGS.

[0076] Preferably, the amino acid sequence of the second connector includes the sequence shown in SEQ ID NO.25.

[0077] SEQ ID NO.25: GGGGSG.

[0078] In a fourth aspect, the present invention provides use of the targeting sequence element of the mRNA tumor vaccine described in the first aspect, the MITD nucleic acid molecule described in the second aspect, or the nucleic acid construct described in the third aspect in the preparation of an mRNA tumor vaccine.

[0079] In a fifth aspect, the present invention provides an mRNA tumor vaccine, wherein the mRNA tumor vaccine comprises an mRNA sequence and a pharmaceutically acceptable carrier;

[0080] The C-terminal targeting sequence element of the mRNA sequence includes the targeting sequence element of the mRNA tumor vaccine described in the first aspect.

[0081] Preferably, the N-terminus of the mRNA sequence has a signal peptide.

[0082] Preferably, the DNA sequence of the signal peptide is shown as SEQ ID NO.3; or the mRNA sequence of the signal peptide is shown as SEQ ID NO.13; or the amino acid sequence of the signal peptide is shown as SEQ ID NO.22.

[0083] SEQ ID NO.3:

[0084] ATGCGGGTCACGGCGCCCCGAACCGTCCTCCTGCTGCTCTGGGGGGCAGTGGCCCTGACCGAGACCTGGGCC.

[0085] SEQ ID NO.13:

[0086] AUGCGGGUCACGGCGCCCCGAACCGUCCUCCUGCUGCUCUGGGGGGCAGUGGCCCUGACCGAGACCUGGGCC.

[0087] SEQ ID NO. 22: MRVTAPRTVLLLLWGAVALTETWA.

[0088] Preferably, the pharmaceutically acceptable carrier comprises lipid nanoparticles.

[0089] In a sixth aspect, the present invention provides the use of the mRNA tumor vaccine described in the fifth aspect in the preparation of a drug for preventing and / or treating tumors.

[0090] In a seventh aspect, the present invention provides use of the nucleic acid construct described in the third aspect in preparing mRNA in vitro.

[0091] Compared with the prior art, the present invention has the following beneficial effects:

[0092] The present invention designs and screens specific targeting sequence elements, especially MITD elements; and designs different MITD molecules and mutants thereof, directly detects the presentation amount of antigen epitopes bound to HLA I molecules through in vitro cell flow cytometry experiments, verifies the tumor-suppressing efficacy of tumor vaccines through in vivo animal experiments, and the obtained nucleic acid construct can enhance the presentation effect of antigen epitopes expressed by tumor vaccines on HLA class I molecules, and improve the anti-tumor effect of tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 The graph shows the average fluorescence intensity of antigen presentation complexes mediated by different mRNA lipid nanoparticles at the cellular level;

[0094] Figure 2 This is a graph showing the changes in tumor volume in mice after immunization with different mRNA vaccines;

[0095] Figure 3 This is a graph showing the weight changes of mice after immunization with different mRNA vaccines;

[0096] Figure 4 This is a test diagram of antigen presentation mediated by different mRNA vaccines in mice;

[0097] Figure 5 This is a graph showing the results of detecting specific cytotoxic T lymphocytes (CTL) mediated by different mRNA vaccines in the spleen of mice;

[0098] Figure 6 This is a graph showing the results of specific CTL detection mediated by different mRNA vaccines in the lymph nodes of mice. DETAILED DESCRIPTION

[0099] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0100] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0101] The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, usually according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through regular commercial channels.

[0102] Example 1

[0103] Construction of template plasmid containing MITD element.

[0104] Targeting the transmembrane domain and C-terminal domain of MITD, this example constructs 308-362 Element sequence (named MITD 308-362-WT) or its mutant sequence vector. This step was entrusted to Nanjing GenScript Biotechnology Co., Ltd. for gene synthesis and cloning. The gene fragment was cloned into the pVAX1 vector to obtain a successfully constructed circular plasmid and glycerol strain. D357 and S359 were selected as mutation sites. 308-362 -WT mutated to MITD 308-362 -M1 (including D357L mutation), MITD 308-362 -M2 (including S359G mutation), MITD 308-362 -M3 (containing D357L and S359G mutations).

[0105] OVA 257-264 (sequence SIINFEKL) as the model antigen epitope, the mRNA transcription template DNA sequence from 5' to 3' is composed of T7 promoter, human α-globin 5' UTR, signal peptide SP, Linker 1, OVA 257-264 Antigenic epitope (named OVA 257-264 -WT), Linker 2, MITD 308-362 -WT or its mutant sequence, human β-globin 3'UTR and Poly(A) (including 110A) element, and their corresponding mRNA sequences were named OVA-MITD-WT-mRNA, OVA-MITD-M1-mRNA, OVA-MITD-M2-mRNA and OVA-MITD-M3-mRNA, respectively.

[0106] Without MITD 308-362 -WT sequence was used as a control (named OVA-mRNA), and the transcription template DNA sequence from 5' to 3' consisted of T7 promoter, human α-globin 5' UTR, signal peptide SP, Linker 1, OVA 257-264 -WT, human β-globin 3'UTR and Poly(A) (including 110A) elements are connected. The mRNA sequence nomenclature is shown in Table 1, the DNA sequence of each element is shown in Table 2, the RNA sequence of each element is shown in Table 3, and the amino acid sequence of each element is shown in Table 4.

[0107] Table 1

[0108]

[0109] Table 2

[0110]

[0111] Table 3

[0112]

[0113] Table 4

[0114]

[0115] Example 2

[0116] Preparation of linear mRNA by in vitro transcription

[0117] 1. Preparation of linearized plasmid

[0118] The glycerol bacteria were inoculated into LB medium (containing 50 mg / mL kanamycin) and cultured at 37 °C. The overnight culture solution was collected and the plasmid was extracted using an endotoxin-free plasmid extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The detailed steps are as follows:

[0119] (a) Add 2.5 mL of equilibration solution to the adsorption column (place the adsorption column in a 50 mL collection tube), centrifuge at 8000 rpm for 2 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0120] (b) Take 100 mL of overnight culture medium and add it to a centrifuge tube. Centrifuge at 8000 rpm for 3 min at 25 °C to collect the bacteria and remove the supernatant as much as possible.

[0121] (c) Add 8 mL of solution P1 and vortex to thoroughly resuspend the precipitate.

[0122] (d) Add 8 mL of solution P2 to the centrifuge tube. Immediately and gently invert the tube eight times to fully lyse the bacteria. Incubate the tube at 25°C for 5 min.

[0123] (e) Add 8 mL of solution P4 to the centrifuge tube and gently invert it upside down 6 times to mix thoroughly until white dispersed flocculent precipitates appear in the solution. Then place it at 25 °C for 10 min. Centrifuge at 8000 rpm for 10 min to allow the white precipitate to centrifuge to the bottom of the tube. Pour all the solution carefully into the filter and slowly push the handle to filter. Collect the filtrate in a clean 50 mL tube.

[0124] (f) Add 0.3 times the volume of isopropanol to the filtrate, mix by inversion, and transfer to the adsorption column. Centrifuge at 8000 rpm for 2 min at 25°C, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0125] (g) Add 10 mL of rinse solution to the adsorption column, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0126] (h) Repeat step (g).

[0127] (i) Add 3 mL of anhydrous ethanol to the adsorption column and centrifuge at 8000 rpm for 2 min at 25 °C. Discard the waste liquid.

[0128] (j) Place the adsorption column back into the collection tube and centrifuge at 8000 rpm for 5 min to remove the remaining rinse solution in the adsorption column.

[0129] (k) Place the adsorption column in a clean 50 mL collection tube, add 1 mL of enzyme-free water to the middle of the adsorption membrane, place at 25 °C for 5 min, and then centrifuge at 8000 rpm for 2 min at 25 °C. Transfer all the eluate in the 50 mL centrifuge tube to a clean 1.5 mL centrifuge tube and store at -20 °C.

[0130] 2. Plasmid digestion

[0131] The plasmid was digested with Bsa I (purchased from Nanjing Novozymes Biotech Co., Ltd.).

[0132] The enzyme digestion system is as shown in Table 5:

[0133] Table 5

[0134]

[0135] Enzyme digestion conditions: 37 ℃ for 3 h.

[0136] The digestion product was purified using a DNA column purification kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The detailed steps are as follows:

[0137] (a) Add 500 μL of equilibration solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0138] (b) Add 5 times the volume of the enzyme digestion reaction solution and mix thoroughly.

[0139] (c) Add the solution obtained in the previous step to the adsorption column and place it at 25°C for 2 min. Centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.

[0140] (d) Add 600 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and place the adsorption column in the collection tube.

[0141] (e) Repeat step (d).

[0142] (f) Place the centrifugal adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min to remove as much rinse solution as possible. Place the adsorption column at 25°C for several minutes to dry thoroughly.

[0143] (g) Take out the adsorption column and place it in a clean centrifuge tube. Add 30 μL of enzyme-free water to the middle of the adsorption membrane. Incubate at 25 °C for 2 min. Centrifuge at 12,000 rpm for 2 min to collect the DNA solution.

[0144] 3. Preparation of linear mRNA by in vitro transcription

[0145] (1) In vitro transcription and synthesis of mRNA

[0146] The obtained linear plasmid was used as a template to synthesize mRNA using a T7 in vitro transcription kit (T7 High Yield RNA Synthesis Kit, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.). The specific reaction system is as shown in Table 6:

[0147] Table 6

[0148]

[0149] Gently mix the components with a pipette, collect them by brief centrifugation, and incubate them at 37°C for 2 h; then add 1.0 μL DNase I, mix well, and incubate at 37°C for 20 min to digest the linear DNA template.

[0150] (2) Linear mRNA purification

[0151] The transcription product obtained above was purified using lithium chloride precipitation method.

[0152] (a) Add 7.5 M lithium chloride solution (final concentration 2.8 M) to the transcript and incubate at -20 °C for 2 h.

[0153] (b) Centrifuge at 12,000 rpm for 15 min, remove the supernatant, add 500 μL 70% ethanol for washing, centrifuge at 12,000 rpm for 5 min, and repeat this step once.

[0154] (c) Thoroughly remove the 70% ethanol solution and air dry, then add 100 μL of enzyme-free water to dissolve the mRNA.

[0155] (d) Determination of mRNA concentration and integrity.

[0156] Example 3

[0157] Preparation of mRNA-loaded lipid nanoparticles.

[0158] Cationic lipids, DSPC, cholesterol, and DMG-PEG2000 were weighed separately, dissolved in anhydrous ethanol, and prepared into a solution with a concentration of 10 mg / mL. The carrier mixed solution was prepared at a molar ratio of 48:10:40:2 as the organic phase. mRNA was dissolved in 50 mM citric acid buffer (pH 4.0) and diluted to prepare a 0.15 mg / mL solution as the aqueous phase. 2 mL of organic phase and 7 mL of aqueous phase were drawn out with a syringe, the organic phase was placed in the left pump, and the aqueous phase was placed in the right pump, and the LNP intermediate solution was prepared in the microfluidic device. 9 times the volume of PBS solution was added for dilution, and ultrafiltration and concentration were performed on the ultrafiltration device. The pore size of the ultrafiltration membrane was 50 KD. When it was concentrated to about 10 mL, 4 times the volume of PBS solution was added for dilution until the concentrated volume was less than 5 mL and the ultrafiltration was stopped.

[0159] The above method was used to prepare lipid nanoparticles encapsulating OVA-mRNA, OVA-MITD-WT-mRNA, OVA-MITD-M1-mRNA, OVA-MITD-M2-mRNA, OVA-MITD-M3-mRNA obtained in Example 2 and blank lipid nanoparticles without mRNA.

[0160] Example 4

[0161] Flow cytometry was used to detect the intracellular antigen presentation ability.

[0162] Mouse bone marrow-derived macrophages (BMDCs) were obtained by culturing from mouse bone marrow precursors and cultured in vitro with the cytokine GM-CSF (20 ng / mL). BMDCs were plated one day in advance at a concentration of 2 × 10 5 / well were inoculated in a 24-well plate, and the lipid nanoparticles loaded with OVA-mRNA, OVA-MITD-WT-mRNA, OVA-MITD-M1-mRNA, OVA-MITD-M2-mRNA, and OVA-MITD-M3-mRNA prepared in Example 3 were used for cell transfection and cultured for 24 h.

[0163] Take the cultured cells, centrifuge them, wash them with PBS solution, and resuspend the cells with 100 μL PBS. OVA presented by H2Kb 257-264 The complex (H2Kb-OVA) formed after the antigen peptide (SIINFEKL) was incubated with H2Kb-OVA antibody, CD11c antibody and CD45 antibody. After the antibody incubation, PBS buffer was added to wash the cells, and then the cells were resuspended in PBS buffer and detected by flow cytometry.

[0164] Example 5

[0165] Evaluation of the antitumor effect of mRNA lipid nanoparticles in vivo.

[0166] The OVA-mRNA group, OVA-MITD-WT-mRNA group, OVA-MITD-M1-mRNA group, OVA-MITD-M2-mRNA group, OVA-MITD-M3-mRNA group and blank LNP group prepared in Example 3 were used for mouse experiments, wherein the mRNA content in the lipid nanoparticle sample was 0.1 mg / mL. C57BL / 6 mice were randomly divided into 5 groups to investigate the anti-tumor effect of mRNA lipid nanoparticles in immunizing tumor-bearing mice.

[0167] Testing and result analysis:

[0168] 1. Flow cytometry to detect intracellular antigen presentation

[0169] The prepared lipid nanoparticles of blank LNP group, OVA-mRNA group, OVA-MITD-WT-mRNA group, OVA-MITD-M1-mRNA group, OVA-MITD-M2-mRNA group, and OVA-MITD-M3-mRNA group were used in cell experiments, and the presentation of OVA by MHC class I molecules on the surface of BMDC was detected by flow cytometry. 257-264 The results of the mean fluorescence intensity (MFI) of the antigen presentation complexes mediated by different mRNA lipid nanoparticles at the cellular level are shown in Table 7.

[0170] Table 7

[0171]

[0172] The results of antigen presentation flow cytometry mediated by different mRNA lipid nanoparticles at the cellular level are as follows Figure 1 As shown, compared with OVA-mRNA, the addition of MITD 308-362 mRNA lipid nanoparticles containing these components can mediate OVA at the cellular level 257-264 The average fluorescence intensity of the antigen peptide complex was relatively enhanced, and OVA-MITD-M3-mRNA had the best effect, which was superior to OVA-MITD-M2-mRNA, OVA-MITD-M1-mRNA and OVA-MITD-WT-mRNA. 257-264 Antigen presentation effect, and MITD 308-362 -M3 has a stronger antigen presentation effect on tumor vaccines than MITD 308-362 -M2, MITD 308-362 -M1, and the amino acid mutation sequence is better than MITD 308-362 -WT.

[0173] 2. Effect of mRNA lipid nanoparticles on tumor size in mice

[0174] The density of E.G7-OVA tumor cell suspension was adjusted to 2×10 6 cells / mL, 100 μL was drawn and injected subcutaneously into the upper right armpit of C57BL / 6 mice, and mice were immunized intravenously on the 6th day after inoculation, and immunized once every 2 days for a total of 3 times. The dosage of each mouse was 10 μg mRNA (the same volume was given to the LNP group). On the day of the first administration (recorded as day 0), the tumor size of tumor-bearing mice was measured with an electronic vernier caliper, and then recorded once every 2 days, and the tumor growth volume and time curve of tumor-bearing mice was drawn.

[0175] The results of the in vivo anti-tumor effects of mRNA lipid nanoparticles are as follows:

[0176] Detection of MITD in tumor-bearing mice inoculated with 308-362 The mRNA vaccine of the components was used to treat tumors. The vaccine was injected intravenously three times after tumor bearing. The changes in tumor volume were measured to compare the differences in the tumor inhibition effect of the vaccine. The results of the changes in tumor volume of mice after immunization with different mRNA vaccines are shown in Table 8.

[0177] Table 8

[0178]

[0179] like Figure 2 As shown, containing MITD 308-362 After treatment with the mRNA vaccine of the components, the tumor volume of mice was significantly reduced compared with the control group, showing a good tumor inhibition effect. Moreover, OVA-MITD-M3-mRNA had the best effect, which was better than OVA-MITD-M2-mRNA, OVA-MITD-M1-mRNA and OVA-mRNA.

[0180] 3. Effects of mRNA lipid nanoparticles on mouse body weight

[0181] To preliminarily evaluate the safety of the drug administration, the weight of tumor-bearing mice was weighed using an electronic balance on the day of the first administration, and then recorded every 2 days. The weight-time curve of the tumor-bearing mice was plotted to evaluate the safety of the mRNA vaccine, and a curve of the weight and time of the tumor-bearing mice was plotted.

[0182] The in vivo safety results of mRNA lipid nanoparticles detected by changes in mouse body weight are shown in Table 9.

[0183] Table 9

[0184]

[0185] like Figure 3 As shown, during the entire treatment process, there was no significant weight loss in each group of mice, indicating that each mRNA vaccine had no obvious toxic side effects and had good safety for in vivo administration.

[0186] 4. mRNA lipid nanoparticles mediated antigen presentation in vivo

[0187] After the above immunization experiment, peripheral blood cells were collected from the mouse tail. OVA presented by H2Kb 257-264 The complex (H2Kb-OVA) formed after the antigen peptide (SIINFEKL) was incubated with H2Kb-OVA antibody, CD45 antibody and CD11c antibody. After the antibody incubation, PBS buffer was added to wash the cells, and then the cells were resuspended in PBS buffer and detected by flow cytometry. The results of antigen presentation mediated by different mRNA vaccines in mice are shown in Table 10.

[0188] Table 10

[0189]

[0190] Results of antigen presentation assay mediated by mRNA lipid nanoparticles in vivo Figure 4 As shown, compared with OVA-mRNA, the addition of MITD 308-362 The mRNA lipid nanoparticles of the components can enhance the antigen presentation mediated in vivo, and OVA-MITD-M3-mRNA has the best effect, which is better than OVA-MITD-M2-mRNA, OVA-MITD-M1-mRNA and OVA-MITD-WT-mRNA. 257-264 Antigen presentation effect, and MITD 308-362 -M3 has a stronger antigen presentation effect on tumor vaccines than MITD 308-362 -M2, MITD 308-362 -M1, and the amino acid mutation sequence is better than MITD 308-362 -WT.

[0191] 5. mRNA lipid nanoparticles mediate the production of specific cytotoxic T lymphocytes (CTL) in vivo

[0192] All mice were killed and dissected, and lymph nodes and spleens were collected and filtered to obtain single-cell suspensions. After centrifugation at 400 g, red blood cell lysis buffer was added, lysed and centrifuged at room temperature at 25, and the supernatant was discarded. OVA presented by H2Kb 257-264The complex (H2Kb-OVA) formed after the antigen peptide (SIINFEKL) was incubated with different protein antibodies (H2Kb-OVA antibody, CD45 antibody, CD11c antibody, CD3ε antibody, CD4 antibody, CD8 antibody). After the antibody incubation, PBS buffer was added to wash the cells, and then the cells were resuspended in PBS buffer and detected by flow cytometry. The results of specific CTL detection mediated by different mRNA vaccines in mouse spleen are shown in Table 11.

[0193] Table 11

[0194]

[0195] The results of specific CTL detection mediated by different mRNA vaccines in lymph nodes of mice are shown in Table 12.

[0196] Table 12

[0197]

[0198] To further verify MITD 308-362 The potential of mRNA vaccines prepared with optimized sequences was tested for the production of specific CTLs in mice after vaccination. Figure 5 Results of specific CTL detection mediated by mRNA lipid nanoparticles in mouse spleen. Figure 6 The results of specific CTL detection mediated by mRNA lipid nanoparticles in mouse lymph nodes. The experimental results showed that compared with OVA-mRNA, the addition of MITD 308-362 All the mRNA lipid nanoparticles of the components can enhance the activation effect of mouse CTL, and OVA-MITD-M3-mRNA has the best effect, which is superior to OVA-MITD-M2-mRNA, OVA-MITD-M1-mRNA and OVA-MITD-WT-mRNA.

[0199] In summary, the present invention designs and screens specific targeting sequence elements, especially MITD elements; and designs different MITD molecules and mutants thereof, directly detects the presentation amount of antigen epitopes bound to HLA I molecules through in vitro cell flow cytometry experiments, verifies the tumor-suppressing efficacy of tumor vaccines through in vivo animal experiments, and the obtained nucleic acid construct can enhance the presentation effect of antigen epitopes expressed by tumor vaccines on HLA class I molecules, and improve the anti-tumor effect of tumor vaccines.

[0200] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A targeting sequence element for an mRNA tumor vaccine, characterized in that: The targeting sequence element is a MITD element, and the mRNA sequence of the MITD element is any one of the sequences shown in SEQ ID NO.19 to SEQ ID NO.21 or a combination of at least two of them.

2. The targeting sequence element according to claim 1, characterized in that The amino acid sequence encoded by the MITD element is any one of the sequences shown in SEQ ID NO.27 to SEQ ID NO.29, or a combination of at least two of them.

3. A MITD nucleic acid molecule for an mRNA tumor vaccine, characterized in that: The DNA sequence of the MITD nucleic acid molecule is any one of the sequences shown in SEQ ID NO.9 to SEQ ID NO.11 or a combination of at least two of them.

4. A nucleic acid construct, characterized in that The structure of the nucleic acid construct from 5' to 3' is: T7 promoter, 5' UTR, signal peptide, first connector, target gene, second connector, MITD nucleic acid molecule according to claim 3, 3' UTR and Poly (A); The mRNA sequence of the signal peptide is the sequence shown in SEQ ID NO.13; the DNA sequence of the signal peptide is the sequence shown in SEQ ID NO.3; The target gene includes an OVA antigen epitope; the mRNA sequence of the OVA antigen epitope is the sequence shown in SEQ ID NO.15; the DNA sequence of the OVA antigen epitope is the sequence shown in SEQ ID NO.5; The 5'UTR includes human α-globin 5'UTR; the mRNA sequence of the human α-globin 5'UTR is the sequence shown in SEQ ID NO.12; the DNA sequence of the human α-globin 5'UTR is the sequence shown in SEQ ID NO.2; The 3'UTR includes human β-globin 3'UTR; the mRNA sequence of the human β-globin 3'UTR is the sequence shown in SEQ ID NO.17; the DNA sequence of the human β-globin 3'UTR is the sequence shown in SEQ ID NO.7; The mRNA sequence of the first connector is the sequence shown in SEQ ID NO.14; the DNA sequence of the first connector is the sequence shown in SEQ ID NO.4; The mRNA sequence of the second connector is the sequence shown in SEQ ID NO.16; the DNA sequence of the second connector is the sequence shown in SEQ ID NO.6; The DNA sequence of the T7 promoter is the sequence shown in SEQ ID NO.1; The length of the Poly(A) is 90~120 nt.

5. The nucleic acid construct according to claim 4, characterized in that The protein encoded by the nucleic acid construct includes: a signal peptide, an OVA antigen epitope, a first connector and a second connector; The amino acid sequence of the signal peptide is the sequence shown in SEQ ID NO.22; The amino acid sequence of the OVA antigen epitope is the sequence shown in SEQ ID NO.24; The amino acid sequence of the first connector is the sequence shown in SEQ ID NO.23; The amino acid sequence of the second connector is the sequence shown in SEQ ID NO.

25.

6. Use of the targeting sequence element of the mRNA tumor vaccine according to claim 1 or 2, the MITD nucleic acid molecule according to claim 3, or the nucleic acid construct according to claim 4 or 5 in the preparation of an mRNA tumor vaccine.

7. An mRNA tumor vaccine, characterized in that: The mRNA tumor vaccine comprises an mRNA sequence and a pharmaceutically acceptable carrier; The C-terminal targeting sequence element of the mRNA sequence includes the targeting sequence element of the mRNA tumor vaccine according to claim 1 or 2.

8. The mRNA tumor vaccine according to claim 7, characterized in that The N-terminus of the mRNA sequence has a signal peptide; The DNA sequence of the signal peptide is shown in SEQ ID NO.3; or the mRNA sequence of the signal peptide is shown in SEQ ID NO.13; or the amino acid sequence of the signal peptide is shown in SEQ ID NO.22; The pharmaceutically acceptable carrier includes lipid nanoparticles.

9. Use of the nucleic acid construct according to claim 4 or 5 in preparing mRNA in vitro.

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