Triple mRNA vaccine and application thereof

Abstract

The invention relates to an OX40L, IL-23 and IL-36 gamma triple mRNA (messenger Ribonucleic Acid) vaccine and application thereof. According to the present invention, the human OX40L mRNA sequence, the human IL-23 mRNA sequence and the human IL-36 gamma mRNA sequence in the mRNA-2752 are optimized, such that the expression level of the mRNA in the cell is significantly improved; the optimized mRNA shows a treatment effect which is superior to that of mRNA-2752 in animal experiments. The OX40L, IL-23 and IL-36 gamma triple mRNA vaccine disclosed by the invention can be used for immunotherapy of tumors, and has a remarkable clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a triple mRNA vaccine of OX40L, IL-23 and IL-36γ and applications thereof. Background Art

[0002] OX40 (CD134, TNFRSF4) was originally defined as a T cell activation marker, and was later found to be a member of the NGFR / TNFR superfamily with co-activation function. OX40 binds to its ligand OX40L (CD252, TNFSF4) to transmit co-stimulatory signals. OX40L can be expressed on antigen presenting cells (APCs), such as B cells, dendritic cells, and macrophages; in addition, it can also be induced to express in other cell types such as Langerhans cells, endothelial cells, smooth muscle cells, mast cells, and NK cells. It can be seen that the combination of OX40 and OX40L is involved in a variety of physiological reactions between T cells and lymphocytes and non-lymphocytes.

[0003] IL-36γ belongs to the IL-1 family. It acts on dendritic cells to promote their maturation and increase the secretion of related cytokines and chemokines. It can promote the proliferation of T cells and the differentiation of Th1 and Th9. Studies have found that it has anti-tumor effects. IL-23 belongs to the IL-12 family. It can not only activate dendritic cells, but also activate other immune cells such as NKT. It can not only expand and maintain Th17 cells, but also act on antigen-stimulated T cells.

[0004] mRNA-2752 is a messenger RNA (mRNA) drug developed by Moderna that encodes OX40L, IL-23, and IL-36γ and is currently in Phase 1 clinical trials. mRNA-2752 is encapsulated in lipid nanoparticles (LNP) and delivered directly to the tumor site via intratumoral injection (ITu). Preclinical data show that it has a synergistic effect when used in combination with PD-L1 inhibitors. The drug is intended to treat solid tumors and lymphomas, and by expressing these immunomodulatory factors, mRNA-2752 is expected to enhance the patient's immune response and effectively fight cancer cells.

[0005] mRNA drugs face some challenges in sequence optimization. The mRNA molecule itself is unstable and easily degraded. This can affect the effectiveness and durability of the drug. The translation efficiency of mRNA is affected by its sequence and structure. Optimizing codon usage and secondary structure can improve translation efficiency, but this requires complex calculations and experimental verification. Summary of the invention

[0006] 1. Technical issues to be solved

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides an optimized OX40L, IL-23 and IL-36γ triple mRNA vaccine and its application. By optimizing the human OX40L, human IL-23 and human IL-36γ mRNA sequences in mRNA-2752, the expression level of mRNA in cells is significantly improved. The optimized mRNA showed a therapeutic effect superior to that of mRNA-2752 in animal experiments. The OX40L, IL-23 and IL-36γ triple mRNA vaccine of the present invention can be used for the immunotherapy of tumors and has significant clinical application potential.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] In one aspect, the present invention provides a human OX40L mRNA molecule, wherein the mRNA comprises a CDS coding region encoding human OX40L, wherein the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 1-12 or a nucleotide sequence having codon degeneracy and at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO: 1-12.

[0011] In one aspect, the present invention provides a human IL-36γ mRNA molecule, wherein the mRNA comprises a CDS coding region encoding human IL-36γ, and the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 14-23 or a nucleotide sequence having codon degeneracy and at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO: 14-23.

[0012] In one aspect, the present invention provides a human IL-23 mRNA molecule, wherein the mRNA comprises a CDS coding region encoding human IL-23, wherein the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 25-34 or a nucleotide sequence having codon degeneracy and at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO: 25-34.

[0013] In one aspect, the present invention provides an mRNA pharmaceutical composition, comprising human OX40L mRNA molecules, human IL-36γ mRNA molecules and human IL-23 mRNA molecules. The mass ratio of human OX40L mRNA molecules, human IL-36γ mRNA molecules and human IL-23 mRNA molecules is preferably 1:1:1.

[0014] In one aspect, the present invention provides a triple mRNA vaccine, comprising a pharmaceutical composition and a drug delivery vector.

[0015] In one aspect, the present invention provides a method for preparing a triple mRNA vaccine, comprising the following steps:

[0016] (1) Dissolving a cationic lipid, an auxiliary phospholipid, cholesterol and a pegylated phospholipid in an organic solvent, ethanol, to obtain a mixture A of lipid nanoparticles; wherein the cationic lipid is DLin-MC3-DMA, the auxiliary phospholipid is DSPC, and the pegylated phospholipid is DMG-PEG2000.

[0017] (2) diluting the mRNA with a sodium acetate solution to obtain a mixture B;

[0018] (3) Mixture A and mixture B are mixed by a microfluidic device to prepare a lipid nanoparticle composition, and dialyzed in PBS.

[0019] In one aspect, the present invention provides the use of the above-mentioned pharmaceutical composition, the above-mentioned triple mRNA vaccine or the triple mRNA vaccine prepared according to the above-mentioned preparation method in the preparation of a drug for treating tumors. The use also includes administering an effective amount of an immune checkpoint inhibitor to a subject.

[0020] In one embodiment, the mRNA molecule further comprises a 5′UTR and a 3′UTR, wherein the 5′UTR preferably has a nucleotide sequence as shown in SEQ ID NO:36, and the 3′UTR preferably has a nucleotide sequence as shown in SEQ ID NO:37. The mRNA molecule further comprises a flag tag and Poly(A), wherein the flag tag is preferably Flag tag x2, and the Poly(A) is preferably

[0021] In one embodiment, the above mRNA may be modified to improve the stability and translation efficiency of the mRNA. The modification of mRNA includes: N6-methyladenosine (m6A), 5-methylcytosine (m5C), 1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), 4-acetylcytosine (ac4C), etc. Preferably, the modification of mRNA is to replace all uridine (U) in the CDS sequence with N1-methyl-pseudouridine (N1-Methyl-Pseudouridine, m1Ψ) bases.

[0022] In one embodiment, the drug delivery carrier is selected from liposome nanoparticles (LNP), cationic lipid complexes, polymer nanoparticles, protamine nanoparticles, polypeptide nanoparticles, polypeptide nanoparticles, viral vectors, inorganic nanoparticles, cell penetrating peptides or self-assembling RNA-lipid complexes. The LNP comprises cationic lipids, auxiliary phospholipids, cholesterol and PEGylated phospholipids.

[0023] In one embodiment, the preparation method of the lipid nanoparticle composition (mRNA-LNP) comprises the following steps:

[0024] (1) dissolving DLin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 in an organic solvent ethanol in a certain molar ratio to obtain a mixture A with a lipid nanoparticle concentration of 25 mM; the molar ratio is 45-55:8-12:35-45:1-2, preferably 50:10:38.5:1.5;

[0025] (2) diluting 200 μg of expressed mRNA to 0.35 mg / ml with a 100 mM sodium acetate solution at pH 4 to obtain a mixture B;

[0026] (3) Mixture A and mixture B were mixed using a microfluidic device to prepare a lipid nanoparticle composition (mRNA-LNP), and dialyzed in PBS.

[0027] In one embodiment, the tumor comprises head and neck squamous cell carcinoma, triple negative breast cancer, bladder cancer, non-small cell lung cancer, melanoma, non-Hodgkin's lymphoma.

[0028] In one embodiment, the immune checkpoint inhibitor includes a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist. The PD-1 antagonist is an antibody or an antigen binding portion thereof that specifically binds to PD-1, and the PD-L1 antagonist is an antibody or an antigen binding portion thereof that specifically binds to PD-1. The CTLA-4 antagonist is an antibody or an antigen binding portion thereof that specifically binds to CTLA-4. The PD-1 antagonist is selected from nivolumab, pembrolizumab, and pilizumab, and the PD-L1 antagonist is selected from durvalumab, avelumab, and atezolizumab, and the CTLA-4 antagonists are ipilimumab and tremelimumab.

[0029] (III) Beneficial effects

[0030] The present invention provides a triple mRNA vaccine of OX40L, IL-23 and IL-36γ and its application. Compared with the prior art, it has the following beneficial effects:

[0031] Using the independently developed deep learning-based mRNA sequence design method, the codon frequency, degeneracy and rarity; GC content and Gibbs free energy of the sequence; and secondary structure of the sequence and other parameters were comprehensively evaluated, and the corresponding mRNA sequences were designed for the amino acid sequences of human OX40L, human IL-23 and human IL-36γ. The expression level of the optimized mRNA in cells was significantly higher than that of mRNA-2752, and it showed a better therapeutic effect in animal experiments. The OX40L, IL-23 and IL-36γ triple mRNA vaccine of the present invention can be used for tumor immunotherapy and has significant clinical application potential.

[0032] (IV) Terms and Definitions

[0033] As used herein, "nucleotide" refers to a nucleoside containing a phosphate group. Nucleotides are generally molecules composed of three parts: a five-carbon sugar (ribose or deoxyribose), a phosphate group, and a nitrogenous base (purine or pyrimidine).

[0034] "mRNA (messenger RNA)" as described herein refers to a single-stranded nucleic acid molecule that is transcribed from a DNA template, carries genetic information, and directs protein synthesis on ribosomes in the cytoplasm. The structure of mRNA generally includes a 5' cap structure (5' cap), a 5' untranslated region (5'-UTR), a coding region (open reading frame, ORF), a 3' untranslated region (3'-UTR) and / or a polyadenylic acid tail (Poly-A tail).

[0035] The "CDS coding region" described herein refers to the portion of the mRNA molecule that is actually translated into protein. The CDS starts with the start codon (usually AUG) and ends with the stop codon (such as UAA, UAG or UGA). It is located between the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR). The CDS consists of exons and contains all the nucleotide sequences that encode proteins.

[0036] The "codon degeneracy" described herein refers to the phenomenon that multiple different codons in the genetic code can encode the same amino acid.

[0037] "Identity" as used herein refers to the degree of similarity between two or more biomacromolecule sequences (such as DNA, RNA or protein sequences). When comparing two sequences, sequence identity is quantified by determining the ratio of sequences with the same components at the same position.

[0038] "Modification" as used herein refers to a change in the state or structure of a molecule. Modification can be performed in a variety of ways, including chemical, structural and functional modifications. For example, an mRNA molecule can be modified by introducing non-natural nucleosides and / or nucleotides, such as modifications of natural ribonucleotides A, U, G and C.

[0039] "Drug delivery carrier" as described herein refers to a system for safely and effectively delivering mRNA drugs to target cells or tissues. The types of drug delivery carriers generally include liposome nanoparticles (LNP), cationic lipid complexes, polymer nanoparticles, protamine nanoparticles, polypeptide nanoparticles, polypeptide nanoparticles, viral vectors, inorganic nanoparticles, cell penetrating peptides or self-assembling RNA-lipid complexes.

[0040] The "liposome nanoparticle (LNP)" described in this article is a nanoscale delivery system composed of lipid molecules and is widely used in drug and gene delivery. LNPs are composed of multiple lipid bilayers and can encapsulate and protect drug molecules such as mRNA, siRNA, DNA, etc. Lipid nanoparticles (LNPs) are usually composed of four main types of lipids: ionizable cationic lipids, phospholipids, cholesterol or its derivatives, and polyethylene glycol (PEG)-lipids. The size of liposome nanoparticles (LNPs) is usually between 50 and 200 nanometers. The size of LNPs can be controlled by adjusting its composition and preparation method to optimize its delivery efficiency and biodistribution.

[0041] The "mRNA vaccine" described in this article refers to a vaccine developed based on messenger RNA (mRNA) technology. Its working principle is to introduce mRNA encoding a specific antigen protein into human cells, so that the cells themselves produce the antigen protein, thereby stimulating the immune system to produce an immune response against the antigen. The structure of the mRNA vaccine mainly includes the following key parts: mRNA sequence, delivery vector and excipients such as stabilizers and buffers.

[0042] As used herein, an "effective amount" refers to a dose of a drug that produces a desired or beneficial effect, such as the results achieved in clinical treatment. Therefore, an "effective amount" may vary depending on the context of the specific application. For example, in cancer treatment, an effective amount of a drug refers to a dose that achieves the desired therapeutic effect compared to not using the drug.

[0043] The "immune checkpoints" described in this article refer to a class of molecules that regulate T cell activity in the immune system. These checkpoints maintain immune balance by regulating the recognition of T cell receptors (TCRs) and antigens. Immune checkpoints can be divided into two categories: co-stimulation and co-inhibition, which promote or inhibit immune responses, respectively. Common immune checkpoints include: PD-1 / PD-L1, CTLA-4, TIM-3, LAG-3, TIGIT, B7-H3, VISTA, CD200R, CD47.

[0044] The "immune checkpoint inhibitors" described herein refer to siRNA, dsRNA, miRNA, shRNA, gene editing systems, and / or antibodies, antigen-binding fragments or other small molecule drugs targeting immune checkpoints. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 This is the quality control chart of IVT mRNA.

[0047] Figure 2 This is a graph showing the screening results for the expression level of human OX40L mRNA.

[0048] Figure 3 This is a graph showing the screening results for the expression level of human IL-36γ mRNA.

[0049] Figure 4 This is a diagram showing the screening results for the expression level of human IL-23 mRNA.

[0050] Figure 5 This is a graph showing the particle size, particle size distribution and zeta potential measurement results of lipid nanoparticles (LNP).

[0051] Figure 6 This is a graph showing the drug encapsulation efficiency determination results of lipid nanoparticles (LNP).

[0052] Figure 7 This is the result of flow cytometry detection of CD3 and CD4 one week after PBMC injection.

[0053] Figure 8 This is the survival result graph of the tumor mouse model after treatment with mRNA-LNP. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In order to better understand the technical solution, the technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0056] Example 1: mRNA sequence design

[0057] Two main optimization strategies were employed for in vitro screening and selection of mRNAs, including coding region and UTR optimization approaches.

[0058] For the coding region, the focus was on improving translation efficiency and mRNA and protein stability. The protein expression level and duration of different mRNA sequences were detected in the HEK293T cell line. Tag label selection and its position (Flag tag x2, C-terminus), fixed with recommended UTR, PolyA (A100), and m1ψ bases. Synthesize linearized template and 100μg mRNA, transfect HEK293T, and detect the expression of the target protein. Evaluate the protein expression level of the screened mRNA after transfection in HEK 293T, and obtain the effect of CDS design sequence on protein expression.

[0059] For the UTR screening part, a combination of 5'UTR (5′-CBT3) and 3'UTR (3′-CBT4) was selected. The tag label selection and its position (Flag tag x2, C-terminus) were fixed, and the recommended CDS, PolyA (A100), and m1ψ bases were fixed. The linearized template was synthesized and transfected with 100 μg of mRNA in the HEK 293T cell line, and protein expression screening was performed at 24 hours and 48 hours. The selection criteria for the optimal UTR are: a. The peak concentration of protein expression at 24 hours b. The concentration of protein expression at 48 hours.

[0060] The optimal UTR combination was combined with the selected coding region sequence to complete the mRNA, and the final sequence was determined based on the Western blot results for mRNA-LNP preparation.

[0061] Based on the target amino acid sequence (XH-h40L), 12 human OX40L mRNA CDS coding region sequences (SEQ ID NO: 1-12) were designed. The full-length mRNA structure includes: 5'UTR (SEQ ID NO: 36), CDS, 3'UTR (SEQ ID NO: 37), flagtag and Poly (A100).

[0062] Based on the target amino acid sequence (XH-h36γ), 10 human IL-36γ mRNA sequences (SEQ ID NO: 14-23) were designed. The full-length mRNA structure includes: 5′UTR (SEQ ID NO: 36), CDS, 3′UTR (SEQ ID NO: 37), flag tag and Poly (A100).

[0063] Based on the target amino acid sequence (XH-h23), 10 human IL-23 mRNA sequences (SEQ ID NO: 25-34) were designed. The full-length mRNA structure includes: 5'UTR (SEQ ID NO: 36), CDS, 3'UTR (SEQ ID NO: 37), flag tag and Poly (A100).

[0064] XH-WTh40L-AG (SEQ ID NO: 13), XH-WTh36γ-AG (SEQ ID NO: 24), and XH-WTh23-AG (SEQ ID NO: 35) are the full-length sequences of mRNA-2752 (including 5'UTR, CDS, and 3'UTR).

[0065] Table 1 Designed mRNA and corresponding sequences

[0066] CDS Sequence Number mRNA CDS Sequence Number mRNA SEQ ID NO: 1 XH-h40L-HGC1 SEQ ID NO: 18 XH-h36γ-LGC1 SEQ ID NO: 2 XH-h40L-HGC2 SEQ ID NO: 19 XH-h36γ-LGC2 SEQ ID NO: 3 XH-h40L-HGC3 SEQ ID NO: 20 XH-h36γ-LGC3 SEQ ID NO: 4 XH-h40L-HGC4 SEQ ID NO: 21 XH-h36γ-MGC1 SEQ ID NO: 5 XH-h40L-LGC1 SEQ ID NO: 22 XH-h36γ-MGC2 SEQ ID NO: 6 XH-h40L-LGC2 SEQ ID NO: 23 XH-h36γ-MGC3 SEQ ID NO: 7 XH-h40L-LGC3 SEQ ID NO: 25 XH-h23-HGC1 SEQ ID NO: 8 XH-h40L-MGC1 SEQ ID NO: 26 XH-h23-HGC2 SEQ ID NO: 9 XH-h40L-MGC2 SEQ ID NO: 27 XH-h23-HGC3 SEQ ID NO: 10 XH-h40L-MGC3 SEQ ID NO: 28 XH-h23-HGC4 SEQ ID NO: 11 XH-WTh40L-s1 SEQ ID NO: 29 XH-h23-LGC1 SEQ ID NO: 12 XH-WTh40L-s2 SEQ ID NO: 30 XH-h23-LGC2 SEQ ID NO: 14 XH-h36γ-HGC1 SEQ ID NO: 31 XH-h23-LGC3 SEQ ID NO: 15 XH-h36γ-HGC2 SEQ ID NO: 32 XH-h23-MGC1 SEQ ID NO: 16 XH-h36γ-HGC3 SEQ ID NO: 33 XH-h23-MGC2 SEQ ID NO: 17 XH-h36γ-HGC4 SEQ ID NO: 34 XH-h23-MGC3

[0067] Example 2: Preparation of mRNA

[0068] 1. DNA template linearization

[0069] (1) Linearize the plasmid DNA template by PCR and quality control the DNA template by agarose gel electrophoresis.

[0070] Table 2 PCR reaction system

[0071] Components volume Components volume 2x Reaction Buffer 25μL DNA Polymerase 1μL dNTP Mix 1μL Plasmid DNA 3μL CBT3-F 2μL RNase-free Water 16μL CBT4-R 2μL Total 50μL

[0072] Table 3 PCR program

[0073] program Temperature ℃ time Number of cycles Pre-denaturation 95 3min 1 transsexual 95 15s 35 annealing 60 15s 35 extend 72 1min / kb 35 Complete extension 72 5min 1 Hold 4 ∞ 1

[0074] (2) Purify the linear DNA template.

[0075] ① Add an appropriate amount of DNA binding buffer to the PCR reaction solution and mix thoroughly;

[0076] ② Transfer the mixed sample to the purification centrifuge column, centrifuge at 10000g for 30s, and discard the waste liquid;

[0078] ③ Wash twice with washing buffer, transfer the purification centrifuge column to a new centrifuge tube, and add the nuclease-free

[0079] Let the water stand at room temperature for 3 minutes;

[0080] ④ Place the sample column in a centrifuge and centrifuge at 10,000 x g for 30 seconds. The resulting solution is the linear DNA product; ⑤ Detect DNA concentration and purity;

[0081] ⑥Quality control of DNA template by agarose gel electrophoresis.

[0082] 2. Preparation of mRNA by IVT

[0083] The linearized DNA was used as a template and T7 promoter was used for in vitro transcription to obtain mRNA, and the mRNA was quality-controlled by agarose gel electrophoresis.

[0084] In vitro transcription (IVT) reactions were performed using the NEB HiScribe T7 in vitro transcription kit (New England Biolabs, Ipswich, MA). Following the instructions, 100 mM NTPs (ATP, GTP, CTP, and UTP nucleotides, or chemically modified counterparts) were thawed and mixed together to prepare a 2.5X NTP mix. A 50 μL IVT reaction included approximately 2.5 μL DNA template, 10X IVT reaction buffer, NTP mix, and 5 μL T7 RNA polymerase mix. The reaction mixture was incubated at 37°C for 2 h, diluted with 700 μL nuclease-free water, and then 10X DNase I buffer and 20 μL RNase-free DNase I were added and incubated at 37°C for 15 min to degrade the DNA template. The synthesized RNA was purified by RNeasy Maxi columns (Qiagen, Hilden, Germany), eluted with RNase-free water, and quantified by NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

[0085] Table 4 IVT reaction system

[0086] Components volume Components volume 10x Reaction buffer 2.5μL Cap1 AG 2μL ATP 2.5μL DNA template 2.5μL CTP 2.5μL T7 RNA Polymerase 5μL GTP 2.5μL RNase-Free Water Up to 50μL N1-methyl-pseudoUTP 2.5μL

[0087] program:

[0088] ① After thorough mixing, incubate at 37℃ for 2h;

[0089] ②After incubation, add DNaseI and DNaseI buffer and incubate at 37℃ for 15min.

[0090] Purification of mRNA:

[0091] ① Add 2 volumes of binding buffer and 3 volumes of anhydrous ethanol to the solution after IVT reaction and mix thoroughly;

[0092] ② Transfer the sample to the purification column, centrifuge at 13000g for 1 min, and discard the waste liquid;

[0093] ③ Add 500 μL washing buffer, centrifuge at 13000 g for 1 min, discard the waste liquid, and repeat this step twice;

[0094] ④ Transfer the purification column to a new centrifuge tube and elute the product with nuclease-free water;

[0095] ⑤Stand at room temperature for 5 minutes, and centrifuge at 13000g for 1 minute to obtain the mRNA product;

[0096] ⑥ Detection of mRNA concentration

[0097] ⑦Quality control of mRNA by agarose gel electrophoresis.

[0098] Using the NEB enzymatic capping method, the mRNA was denatured at 65°C for 5 minutes and then rapidly cooled to release the secondary conformation. 100 μg of denatured mRNA, 10 μL of 1X capping buffer, 5 μL of 10 mM GTP, 5 μL of 4 mM SAM, 5 μL of 10 U / mL vaccinia virus capping enzyme, and 5 μL of mRNA cap 2 / -O-methyltransferase (50 U / mL) were mixed and adjusted to a final volume of 100 μL with nuclease-free water and incubated at 37°C for 1 hour. The capped mRNA after the reaction was purified using an RNeasy column, eluted with nuclease-free water, and quantified using a NanoDrop spectrophotometer. The purity of the mRNA was confirmed by denaturing urea polyacrylamide gel electrophoresis.

[0099] The experimental results showed that the mRNA bands in each lane were clear and no mixed bands appeared, indicating that the prepared mRNA had qualified purity and met the requirements of subsequent experiments (see Figure 1 ).

[0100] Example 3: Screening of mRNA expression

[0101] HEK293T cells were used for cell transfection experiments. A 24-well plate was selected, and 7*10^4 cells were plated in each well. The cells were cultured in an incubator at 37°C and 5% CO2. 500μL of culture medium was added to each well, and 3 replicates were cultured for 24h. Then, Lipofetamine transfection reagent was prepared: RNAiMAX: 5μL+50μL opti-MEM, mixed, and allowed to stand for 5min; mRNA transfection reagent was prepared: 1μg mRNA+50μL opti-MEM, mixed; the last two reagents were fully blown and mixed, and allowed to stand for 15min; 100μL / well of transfection reagent was added; after 48h of culture, the cells were collected for lysis and 100μL of lysis solution was added for treatment. The protein concentration was determined by the BCA method, and the Western blot experiment was performed. The cell body sample loading amount was 2μg, and the antibody was added for incubation. After incubation, the ECL colorimetric solution was used for development and exposure imaging was performed on the developer. Data statistics and analysis,All statistical graphs were drawn using Graphpad Prism software.

[0102] The specific steps for Western blot detection of protein expression are as follows:

[0103] ① Load 10 μL of culture medium supernatant and perform SDS-PAGE protein electrophoresis;

[0104] ②After electrophoresis, use iBlot 2 for semi-dry transfer;

[0105] ③ Use 5% skim milk to block for 1 hour;

[0106] ④Incubate with primary antibody at 4°C overnight;

[0107] ⑤ Wash the protein membrane with PBST, 15 min / time, 3 times;

[0108] ⑥ Protect from light and incubate the secondary antibody at room temperature for 2 hours;

[0109] ⑦ Wash the protein membrane with PBST, 15 min / time, 3 times;

[0110] ⑧Protein imaging in a protein imager.

[0111] The experimental results showed that the expression levels of human OX40L mRNA after sequence optimization were better than those of the control XH-WTh40L-AG, among which XH-WTh40L-MGC2 was the human OX40L mRNA with the highest expression level screened (see Figure 2 ). XH-WTh36γ-HGC4 had the highest expression level in the human IL-36γ mRNA sequence screening (see Figure 3 ). XH-WTh23-HGC2 had the highest expression level in the human IL-23 mRNA sequence screening (see Figure 4 ).

[0112] Example 3: Preparation of lipid nanoparticles (LNP)

[0113] The mRNAs XH-h23-HGC2, XH-h36γ-HGC4 and XH-h40L-MGC2 with the highest expression levels obtained by screening in Example 2 were selected to prepare LNP-mRNA together.

[0114] (1) A cationic lipid, an auxiliary phospholipid, cholesterol and a pegylated phospholipid are dissolved in an organic solvent ethanol at a molar ratio of 50:10:38.5:1.5 to obtain a mixture A of lipid nanoparticles with a concentration of 25 mM; wherein the cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-hepttriacontica-6,9,28,31-tetraen-19-yl lipid, referred to as DLin-MC3-DMA; the auxiliary phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); the cholesterol is used as a stabilizer to increase the stability of the lipid nanoparticle composition; and the pegylated phospholipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

[0115] (2) diluting 200 μg of expressed mRNA to 0.35 mg / ml with a 100 mM sodium acetate solution at pH 4 to obtain a mixture B;

[0116] (3) Mixture A and mixture B were mixed by a microfluidic device (Precision NanoSystems, Vancouver, BC, Canada) to prepare a lipid nanoparticle composition (mRNA-LNP), and dialyzed in phosphate buffered saline (PBS), wherein the microfluidic device parameters were: total flow rate 12 ml / min, water phase / ethanol phase (volume ratio) = 3 / 1, temperature was room temperature; the dialysis cassette was purchased from Beijing Novi Biotechnology Co., Ltd., and the dialysis steps and name were Slide-A-Lyzer TM , Dialysis Cassettes, 3.5K MWCO dialysis cassettes follow the same operating instructions.

[0117] (4) Particle size distribution of LNPs and qualitative determination of drug-loaded LNPs. The size and zeta potential of LNPs were measured using a dynamic light scattering (DLS) nanoparticle size analyzer Zetasizer (Malvern, USA).

[0118] (5) Determination of mRNA encapsulation efficiency (EE%) by RiboGreen assay (Invitrogen, USA). First, the amount of RNA free outside the LNP particles in the prepared mRNA LNP solution was detected, and then Triton-100 was added to destroy the LNP structure, releasing the mRNA encapsulated by the LNP into the external solution, and the total amount of RNA in the solution was detected. The difference between the two was calculated to obtain the encapsulation efficiency, that is, the amount of RNA encapsulated inside the LNP particles.

[0119] The experimental results show that the prepared LNP-mRNA has uniform morphology and is stable and not prone to aggregation (see Figure 5 ), the drug encapsulation efficiency reached 88.27% (see Figure 6 ).

[0120] Example 4: Therapeutic effect of LNP-mRNA on mouse model

[0121] (1) Six-week-old female NOG tumor-bearing mice were used for subsequent experiments after one week of adaptive feeding.

[0122] (2) Immune reconstruction: Inject 2*10^6 PBMC cells into the tail vein and wait for 1 week. After 1 week, collect blood from the eye sockets and measure CD3, 4, and 8 by flow cytometry to check the effect of immune reconstruction.

[0123] Flow cytometry experiment process:

[0124] 100 μL orbital blood was collected from each mouse and placed in a centrifuge tube (with heparin). 500 μL red blood cell lysis buffer was added to each tube for 10 min, centrifuged, supernatant removed, 1 mL PBS added, resuspended, centrifuged, repeated twice, supernatant removed, 300 μL PBS added to each tube, 2 μL CD3, CD4, CD8 antibodies were added respectively, incubated for 20 min, and detected by flow cytometry, with APC channel for CD3, FITC channel for CD4, and PE channel for CD8.

[0125] Flow cytometry detection combination: CD45 FITC / CD3 APC-Cy7 / CD4 BV421 / CD8 PerCP-Cy5.5 / Live-Dead Zombie Aqua.

[0126] One week after PBMC injection, CD3 and CD4 flow cytometry analysis confirmed that PBMC humanization was successful (see Figure 7 ).

[0127] (3) Modeling and grouping

[0128] Tumor cell line: FaDu

[0129] Tumor model: Subcutaneous tumors were formed in immune reconstructed mice, 0.5*10^6 / mouse, and the tumor reached about 100mm 3 Enter treatment.

[0130] Experimental groups: treatment group A (control) (n=5); treatment group B (mRNA-2752LNP prepared by the method of Example 3) (n=5); treatment group C (LNP-mRNA prepared in Example 3, i.e., XH-h23-HGC2, XH-h36γ-HGC4 and XH-h40L-MGC2 LNP) (n=5).

[0131] Treatment plan: The mice were treated with drugs starting from Day 1 until they died naturally, and their body weights were recorded after death. LNP-mRNA was injected intratumorally once a week, 5ug / mouse / time.

[0132] (4) Basic monitoring

[0133] Data collection: Tumor volume and mouse body weight were measured regularly (every 3 days).

[0134] The experimental results showed that the optimized mRNA can improve the survival rate of tumor-bearing mice, and the survival rate and survival results (therapeutic effect) are better than those of mRNA-2752 (see Figure 8 ).

[0135] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A human OX40L mRNA molecule, characterized in that The mRNA comprises a CDS coding region encoding human OX40L, wherein the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 1-12 or a nucleotide sequence having codon degeneracy and at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1-12.

2. A human IL-36γ mRNA molecule, characterized in that: The mRNA comprises a CDS coding region encoding human IL-36γ, wherein the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 14-23 or a nucleotide sequence having codon degeneracy and at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 14-23.

3. A human IL-23 mRNA molecule, characterized in that The mRNA comprises a CDS coding region encoding human IL-23, wherein the CDS coding region is selected from the nucleotide sequence shown in SEQ ID NO: 25-34 or a nucleotide sequence having codon degeneracy and at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 25-34.

4. An mRNA pharmaceutical composition, characterized in that The mRNA pharmaceutical composition comprises the human OX40L mRNA molecule of claim 1 , the human IL-36γ mRNA molecule of claim 2 , and the human IL-23 mRNA molecule of claim 3 .

5. The human OX40L mRNA molecule according to claim 1, the human IL-36γ mRNA molecule according to claim 2, the human IL-23 mRNA molecule according to claim 3 or the pharmaceutical composition according to claim 4, characterized in that: The mRNA molecule further comprises a 5′UTR and a 3′UTR, wherein the 5′UTR preferably has a nucleotide sequence as shown in SEQ ID NO:36, and the 3′UTR preferably has a nucleotide sequence as shown in SEQ ID NO:

37.

6. The human OX40L mRNA molecule according to claim 1, the human IL-36γ mRNA molecule according to claim 2, the human IL-23 mRNA molecule according to claim 3 or the pharmaceutical composition according to claim 4, characterized in that: The mRNA is modified with N6-methyladenosine (m6A), 5-methylcytosine (m5C), 1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ) or 4-acetylcytosine (ac4C); preferably, the modification of the mRNA is to replace all uridine (U) in the CDS sequence with N1-methyl-pseudouridine (N1-Methyl-Pseudouridine, m1Ψ) bases.

7. A triple mRNA vaccine, characterized in that: The vaccine comprises the pharmaceutical composition according to any one of claims 4 to 6 and a drug delivery carrier, wherein the drug delivery carrier is selected from liposome nanoparticles (LNP), cationic lipid complexes, polymer nanoparticles, protamine nanoparticles, polypeptide nanoparticles, polypeptide nanoparticles, viral vectors, inorganic nanoparticles, cell penetrating peptides or self-assembled RNA-lipid complexes.

8. The method for preparing the triple mRNA vaccine according to claim 7, characterized in that: The preparation method comprises the following steps: (1) dissolving a cationic lipid, an auxiliary phospholipid, cholesterol and a pegylated phospholipid in an organic solvent ethanol at a certain molar ratio to obtain a mixture A of lipid nanoparticles; wherein the cationic lipid is DLin-MC3-DMA, the auxiliary phospholipid is DSPC, and the pegylated phospholipid is DMG-PEG2000; (2) diluting the mRNA with a sodium acetate solution to obtain a mixture B; (3) Mixture A and mixture B are mixed by a microfluidic device to prepare a lipid nanoparticle composition, and dialyzed in PBS.

9. Use of the pharmaceutical composition according to any one of claims 4 to 6, the triple mRNA vaccine according to claim 7, or the triple mRNA vaccine prepared by the preparation method according to claim 8 in the preparation of a drug for treating tumors, characterized in that: The tumors include head and neck squamous cell carcinoma, triple-negative breast cancer, bladder cancer, non-small cell lung cancer, melanoma, and non-Hodgkin's lymphoma.

10. The use according to claim 9, characterized in that: The use also includes administering an effective amount of an immune checkpoint inhibitor to the subject.

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