HLA-a02-targeting liver cancer vaccine, and preparation method therefor and use thereof

Abstract

An HLA-A*02-targeting liver cancer vaccine, and a preparation method therefor and a use thereof. First, neoantigen targets having sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 20 are obtained by means of screening, then the neoantigen targets are modified and then used for constructing an mRNA sequence, mRNA-lipid nanoparticles are prepared by using microfluidic technology, and the active ingredients of the vaccine are encapsulated in the lipid nanoparticles, thereby being conducive to transferring a viral protein into cells, and protecting the cells from being damaged by a destructive enzyme. Provided is an mRNA tumor vaccine for an HLA-A*02:01 liver cancer patient. The mRNA tumor vaccine is an mRNA vaccine that contains an ultra-broad spectrum antigen with tumor-specific alternative splicing, and is designed strictly on the basis of the HLA typing of a patient and has high-coverage and high-immunogenic tumor neoantigens.

Description

Anti-hepatocarcinoma HLA-A02 vaccine, preparation method and application thereof

[0001] Priority claim

[0002] The present application is based on the prior application of Chinese patent application No. 2024107161101, entitled "Anti-hepatocarcinoma HLA-A*02 vaccine, preparation method and application thereof", filed on June 4, 2024, and Chinese patent application No. 2025105878203, entitled "Anti-hepatocarcinoma HLA-A*02 vaccine, preparation method and application thereof", filed on May 8, 2025. The present application incorporates the entire contents of the above-mentioned Chinese patent applications by reference. TECHNICAL FIELD

[0003] The present application relates to an anti-hepatocarcinoma HLA-A02 (HLA-A*02) vaccine, a preparation method and application thereof, and belongs to the technical field of biological medicine and vaccine. BACKGROUND

[0004] Hepatocarcinoma is a highly malignant tumor occurring in the liver. The existing anti-hepatocarcinoma therapies have reached their upper limit of efficacy, and further improving the prognosis of patients relies on new clinical diagnosis and treatment strategies. Tumor neoantigens are antigens specific to tumors and not present in normal human tissues, which have obvious advantages in tumor specificity and do not cause off-target damage to non-tumor tissues, thus having better safety.

[0005] Most of the existing tumor target sources are mutation-derived antigen peptides. They are more effective for tumors with high tumor mutation burden (TMB), such as melanoma, but the number of mutations in hepatocarcinoma is limited, and there is no obvious population distribution rule. Therefore, the number of mutation-derived antigens is limited, and the application is limited. Recent studies have found that tumor aberrant transcripts may be an effective source of neoantigens. Pre-mRNA alternative splicing (AS) is crucial in the transcription process of eukaryotes and often occurs abnormally in tumor progression. It has been found that about 50% of cancer driver mutations cause splicing errors; the polypeptides produced by abnormal transcripts can be effective candidate targets for neoantigens.

[0006] Most vaccines are dendritic cell (DC) vaccines or polypeptide vaccines, which have limited efficacy. It is difficult to ensure the activity of the transfused cells after the DC vaccine is transfused into the human body, and the natural rejection reaction of the human body causes the death of the transfused cells; the polypeptide vaccine also has corresponding shortcomings. The physicochemical properties of different sequence polypeptides differ greatly, and the process route and quality standard research are difficult.

[0007] Lack of effective antigen recognition and microenvironment immunosuppression is the main reason for failure to exert anti-tumor immunity, resulting in poor effect of single immune checkpoint inhibitor (ICI). mRNA vaccine can encode multiple antigens, enhance immune response against adaptive pathogens, and can target multiple HCC antigen epitopes in a single formulation. Importantly, the new antigen-specific T cell response generated by the vaccine has an immune memory phenotype, which can function for a long time to prevent tumor recurrence.

[0008] Therefore, nucleic acid vaccine is a promising means to avoid the above problems. Most of the vaccines currently under development are mRNA vaccines. After entering human cells through a specific delivery system, mRNA is translated into protein by human cells, and the protein expressed as a certain antigen protein possessed by the virus is recognized as a foreign antigen by antigen-presenting cells (APCs), driving the maturation of dendritic cells (DCs), and then activating B cells and T cells to produce a strong immune response, causing both humoral and cellular immune responses. mRNA vaccine breaks through the traditional immune activation mode of vaccine, and innovatively uses human cells to produce antigens to activate dual-specific immune response and form immune memory, providing more durable specific immunity.

[0009] Most of the vaccines under development by domestic pharmaceutical companies such as Simu Gen are personalized tumor vaccines. First, they do not consider the characteristics of a wide range of population, and second, they are limited by surgical samples, which need to be sequenced, calculated, and developed and produced after surgical resection, taking at least 3-6 months. During this period, patients often have difficulty in treatment due to tumor recurrence; the cost of "one person, one drug" is extremely high, and it is difficult to sell and apply on a large scale in the later stage. There is an urgent need to develop a universal tumor vaccine based on domestic sequencing data.

[0010] HLA-A*02:01 is the most frequent HLA type based on The Cancer Genome Atlas (TCGA) database data, and is the third most common HLA type in Asian populations. Studies have shown that tumor antigens presented by HLA-A*02:01 have the strongest immune activation effect. Existing TCR-T cell therapy targeting hepatocellular carcinoma antigens alpha-fetoprotein (AFP) and Glypcan-3 (GPC-3) is also for hepatocellular carcinoma patients with HLA-A*02:01.

[0011] Therefore, it is of great significance to screen high-frequency and high-immunogenicity hepatocellular carcinoma tumor neoantigens and design anti-hepatocellular carcinoma vaccines targeting HLA-A*02:01. SUMMARY

[0012] The present application aims at: in view of the more or less defects of the existing tumor vaccines (whether DC vaccine or polypeptide vaccine) or the limitation of the application thereof, and in view of the fact that there is no general tumor vaccine for hepatocellular carcinoma at present, the present application provides an anti-hepatocarcinoma vaccine and a preparation method and application thereof.

[0013] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0014] In a first aspect of the present application, a tumor neoantigen peptide is provided, which comprises a combination of one or more polypeptides with an amino acid sequence as shown in SEQ ID NO: 1-20, or a combination of one or more polypeptides with an amino acid sequence as shown in SEQ ID NO: 21-40.

[0015] Preferably, the tumor neoantigen peptide consists of polypeptides with an amino acid sequence as shown in SEQ ID NO: 21-40.

[0016] In a second aspect of the present application, a nucleic acid / polynucleotide is provided, which encodes a combination of polypeptides with an amino acid sequence as shown in SEQ ID NO: 1-20, or which encodes a combination of polypeptides with an amino acid sequence as shown in SEQ ID NO: 21-40.

[0017] In some preferred embodiments, the combination of polypeptides is concatenated by linkers from polypeptides with an amino acid sequence as shown in SEQ ID NO: 1-20, or the combination of polypeptides is concatenated by linkers from polypeptides with an amino acid sequence as shown in SEQ ID NO: 21-40.

[0018] Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 41 or SEQ ID NO: 42.

[0019] In some preferred embodiments, the nucleic acid encodes a polypeptide concatenated by SEQ ID NO: 21-SEQ ID NO: 40 in sequence.

[0020] Preferably, the nucleic acid encodes a polypeptide as shown in SEQ ID NO: 43 or a variant thereof; the variant is a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 43, and retains the function of the polypeptide as shown in SEQ ID NO: 43.

[0021] In some preferred embodiments, the nucleic acid is DNA;

[0022] Preferably, the DNA has a sequence as set forth in SEQ ID NO: 44 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44.

[0023] In some preferred embodiments, the nucleic acid is RNA, for example, mRNA;

[0024] Preferably, the mRNA has a transcription sequence of a DNA sequence as set forth in SEQ ID NO: 44 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44.

[0025] Preferably, when the nucleic acid is mRNA, the 5’ end of the mRNA comprises a 5’ cap structure (Cap) and / or a 5’ untranslated region (UTR); and / or, the 3’ end of the mRNA comprises a 3’ UTR and / or a 3’ poly(A) tail.

[0026] Preferably, the poly(A) tail is cytosine (C) modified; and / or, the 5’ cap structure (Cap) is Cap1 m6AG modified.

[0027] In a third aspect of the present application, there is provided a recombinant expression vector comprising the nucleic acid of the second aspect or a transcribed nucleic acid thereof;

[0028] Preferably, the backbone of the recombinant expression vector is a plasmid or a virus, for example, pUC57 or a modified plasmid thereof.

[0029] In some preferred embodiments, the plasmid backbone comprises a T7 promoter sequence, a 5’ end UTR region, a 3’ end UTR region, a 5’ end cap structure, and a 3’ end poly(A) tail.

[0030] In a fourth aspect of the present application, there is provided a composition comprising (1) the nucleic acid as described above, or the recombinant expression vector as described above, and (2) a delivery vehicle;

[0031] Preferably, the delivery vehicle comprises a liposome; and / or, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0032] In some preferred embodiments, the lipid nanoparticle comprises a cationic lipid and a non-cationic lipid; wherein, the cationic lipid can be, for example, ionizable cationic lipids (ICLs).

[0033] Preferably, the ionizable cationic lipids (ICLs) comprise DLin-MC3-DMA (MC3), SM-102 and ALC-0315; and / or, the non-cationic lipids comprise phospholipids.

[0034] In some preferred embodiments, the delivery vehicle further comprises cholesterol;

[0035] Preferably, the delivery vehicle comprises ionizable cationic lipids (ICLs), phospholipids and cholesterol.

[0036] Preferably, the nucleic acid is mRNA, and is encapsulated in the delivery vehicle.

[0037] In a fifth aspect, the present application provides a pharmaceutical composition comprising the composition of the fourth aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0038] In a sixth aspect, the present application provides a vaccine comprising the nucleic acid as described above, the recombinant expression vector as described above, the composition as described above and / or the pharmaceutical composition as described above, and a pharmaceutically acceptable adjuvant.

[0039] Preferably, the vaccine is a protein vaccine;

[0040] and / or, the vaccine is a nucleic acid vaccine;

[0041] and / or, the vaccine is a DNA vaccine or an mRNA vaccine.

[0042] In a seventh aspect, the present application provides use of the tumor neoantigen peptide as described above, the nucleotide as described above, the recombinant expression vector as described above, the composition as described above, the pharmaceutical composition as described above and / or the vaccine as described above in the preparation of a medicament for preventing and / or treating liver cancer.

[0043] Preferably, the liver cancer is HLA-A*02 type liver cancer;

[0044] More preferably, the liver cancer is HLA-A*02:01 type liver cancer.

[0045] In some preferred embodiments, the medicament is a vaccine;

[0046] Preferably, the vaccine is a protein vaccine; and / or, the vaccine is a nucleic acid vaccine; and / or, the vaccine is a DNA vaccine or an mRNA vaccine.

[0047] In an eighth aspect, the present application provides a method for preparing an anti-liver cancer mRNA vaccine, comprising the following steps:

[0048] Step 1: cloning the polypeptide with the sequence as shown in SEQ ID NO: 21-40 into a vector in series through a linker for plasmid synthesis;

[0049] Step 2: synthesizing mRNA in vitro from the synthesized plasmid containing the sequence of the series of antigens;

[0050] Step 3: modifying the synthesized mRNA in vitro, including 3' end modification and 5' end modification;

[0051] Step 4: preparing mRNA-lipid nanoparticles by using microfluidic technology;

[0052] Preferably, the amino acid sequence of the linker in step 1 is as shown in SEQ ID NO: 41 or SEQ ID NO: 42.

[0053] In a ninth aspect, the present application provides a method for preventing and / or treating liver cancer, i.e. administering an effective amount of the nucleotide of the second aspect, the recombinant expression vector of the third aspect, the composition of the fourth aspect, the pharmaceutical composition of the fifth aspect, and / or the vaccine of the sixth aspect to a subject in need.

[0054] Preferably, the method further comprises administering a second therapeutic agent to the subject in need.

[0055] More preferably, the second therapeutic agent is selected from immune checkpoint inhibitors, and the immune checkpoint inhibitors are selected from any one or more of PD-1 antibodies, PD-L1 antibodies and CTLA-4 antibodies.

[0056] Preferably, the liver cancer further comprises HLA-A*02:07, HLA-A*02:06, HLA-A*02:03, HLA-A*02:10, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:08, HLA-A*02:09, HLA-A*02:011, but is not limited to the above-mentioned types.

[0057] The present application also provides a humanized mouse model for in vivo verification of the immune effect of the vaccine as described above, wherein the model reconstitutes a human immune system of HLA-A*02:01 type, and the administration of the vaccine after inoculation of human liver cancer cells can significantly reduce the tumor volume and increase the CD8 + T cell infiltration.

[0058] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, i.e. to obtain each preferred example of the present application.

[0059] The reagents and raw materials used in the present application are commercially available.

[0060] Compared with the prior art, the present application has the following beneficial effects:

[0061] (1) The HLA-A*02:01 type antigen screened in the present application can simultaneously target the remaining HLA-A02 types (HLA-A*02:03, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, etc.). The data detection results show that the screened new antigen exhibits high affinity to the above-mentioned HLA-A02 remaining types, and thus is suitable for most HLA-A*02 populations.

[0062] (2) The present application provides an mRNA tumor vaccine for HLA-A*0201 liver cancer patients, which is a super-large range and broad-spectrum antigen containing tumor mutations, gene fusions and alternative splicing, and is an mRNA vaccine of high coverage and high immunogenicity of tumor neoantigens designed according to the HLA typing of the patient.

[0063] (3) The mRNA vaccine of the present application has good transfection efficiency in vivo and in vitro, and has good targeting property. Animal in vivo experiments show that it has good anti-tumor effect, and shows good clinical application value and prospect. BRIEF DESCRIPTION OF DRAWINGS

[0064] Fig. 1 is a schematic diagram of the screening strategy of HLA-A*0201 potential antigens;

[0065] Fig. 2 is the ELISpot result of T cells activated by candidate antigens in PBMC for screening IFN-γ;

[0066] Fig. 3 is a heat map of the HLA affinity of the screened 02 type antigens and multiple HLA-A*02 types;

[0067] Fig. 4 is the detection of T cell immune response specific to 20 antigen peptides by ELISpot method, wherein A is the statistical result of IFN-γ spot number of 20 antigen peptides identified by IFN-γ ELISPOT method in three PMBC samples, B is the IFN-γ spot map of 20 antigen peptides in P05 sample; neg is DMSO negative control, and pos is PHA (Phytohemagglutinin) positive control;

[0068] Fig. 5 is the detection of the activation level of T cells specific to 20 antigen peptides by flow cytometry, wherein A is the flow cytometry detection result of CD3 + ; CD4 + ; 4-1BB + cell subpopulation, and B is the flow cytometry detection result of CD3 + ; CD8 +4-1BB + Flow cytometry results of cell subpopulations.

[0069] Figure 6 is the amino acid sequence of A02; the sequence highlighted in color (yellow) is the extended amino acid sequence; the bold + single underline represents the core sequence of the neoantigen target; the blue + double underline represents the amino acid sequence of the linker;

[0070] Figure 7 is the ORF sequence of A02; the sequence highlighted in color (yellow) is the DNA sequence corresponding to the extended amino acid sequence; the blue + double underline represents the DNA sequence of the linker;

[0071] Figure 8 is the plasmid map of the pUC57 vector used to carry the HLA-A*0201 antigen sequence plasmid;

[0072] Figure 9 is the composition of the mRNA structure element constructed in the embodiment of the present application;

[0073] Figure 10 is a fluorescence photograph of HLA-A*0201 cells SNU-398 transfected with eGFP-mRNA vaccine;

[0074] Figure 11 is a flow cytometry result graph of HLA-A*0201 cells SNU-398 transfected with eGFP-mRNA vaccine;

[0075] Figure 12 is a live imaging photograph of mice injected with eGFP-mRNA vaccine 24 hours later; from left to right are the physiological saline injection group, the tail vein injection group and the abdominal injection group of mice images;

[0076] Figure 13 is a fluorescence imaging photograph of the internal organs of mice injected with eGFP-mRNA vaccine 24 hours later, the left side is the tail vein injection group, and the right side is the abdominal injection group;

[0077] Figure 14 is a fluorescence and light microscope image of liver tissue sections of mice 48 hours after injection of physiological saline and eGFP-mRNA vaccine;

[0078] Figure 15 is a photograph of the tumor body of the mouse after tumor formation;

[0079] Figure 16 is a tumor body line graph of the mRNA vaccine inoculated tumor-bearing mice and the control group, wherein vector represents the blank mRNA control group inoculated;

[0080] Figure 17 is a hematoxylin-eosin staining photograph of the sections of the spleen, brain, lung, kidney, heart and liver of mice after using A02 type mRNA tumor vaccine. DETAILED DESCRIPTION

[0081] In order to make the present application more obvious and easy to understand, the preferred embodiments are described in detail below with the help of the accompanying drawings.

[0082] The terms of the present application are defined as follows unless otherwise indicated.

[0083] In the present application, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can include modified amino acids. The terms also encompass amino acid polymers that have been modified by intervention, e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with labels. For example, also included are polypeptides containing one or more analogs of an amino acid (e.g., an unnatural amino acid such as homoarginine, ornithine, p-acetylphenylalanine, D-amino acids, and sarcosine), and other modifications known in the art.

[0084] The term "polypeptide" refers to proteins and peptides of any size, structure, or function. Polypeptides include encoded nucleic acid / polynucleotide products, natural polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be a monomer or a multimeric complex, such as a dimer, trimer, or tetramer. They can also include single- or multi-chained polypeptides. Disulfide linkages are most commonly found in multi-chained polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. In some embodiments, a "polypeptide" can be less than or equal to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0085] The term "signal peptide" includes the N-terminal 15-60 amino acids of a protein that are typically required for translocation across the membrane in the secretory pathway, and thus, are ubiquitous in eukaryotes and prokaryotes for controlling entry of most proteins into the secretory pathway. Signal peptides generally include three regions: an N-terminal region of varying length, often including positively charged amino acids; a hydrophobic region; and a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein directs the ribosome to the rough endoplasmic reticulum (ER) membrane and transports the growing peptide chain across the membrane for processing, after which the signal peptide is cleaved from the precursor protein. Signal peptides can also facilitate localization of a protein to the cell membrane. However, signal peptides are not responsible for the final destination of the mature protein. Secretory proteins without additional address tags in their sequence are by default secreted into the external environment.

[0086] The term "sequence optimization" refers to a process or series of processes by which a base in a reference nucleic acid / polynucleotide sequence is replaced with an alternative base, thereby producing a nucleic acid / polynucleotide sequence with improved properties, e.g., improved protein expression or reduced immunogenicity. Generally, the goal of sequence optimization is to produce a synonymous nucleic acid / polynucleotide sequence that encodes the same polypeptide sequence as the reference nucleic acid / polynucleotide sequence. Thus, in a polypeptide encoded by a codon-optimized nucleic acid / polynucleotide sequence, there is no substitution of amino acids relative to the polypeptide encoded by the reference nucleic acid / polynucleotide sequence.

[0087] In the context of sequence optimization, the term "codon substitution" refers to the replacement of a codon in a reference nucleic acid / polynucleotide sequence with another codon. A codon can be replaced in a reference nucleic acid / polynucleotide sequence, e.g., by chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to a "replacement" or "substitution" in a position of a nucleic acid / polynucleotide sequence, such as an mRNA, or in a region or subsequence of a nucleic acid / polynucleotide sequence, such as an mRNA, refers to the replacement of a codon at that position or region. As used herein, the term "coding region" refers to an open reading frame (ORF) in a nucleic acid / polynucleotide that, when expressed, produces a polypeptide or protein.

[0088] The term "nucleic acid / polynucleotide" includes any compound and / or substance composed of nucleotide polymers. These polymers are often referred to as nucleic acid / polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), ene nucleic acids (ENAs), cyclohexene nucleic acids (CeNAs), or mixtures or combinations thereof. "Nucleic acid / polynucleotide" includes triple-stranded, double-stranded, and single- stranded DNA or RNA. It also includes modified, e.g., by alkylation, and / or by capping, and unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxynucleotides (containing 2-deoxy-D- ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA.

[0089] In particular aspects, “nucleic acid / polynucleotide” includes mRNA. In one aspect, the mRNA is a synthetic mRNA. In certain aspects, the synthetic mRNA includes at least one non-natural base. In certain aspects, all of a class of nucleobases are replaced with non-natural bases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with non-natural bases, e.g., 5-methoxyuridine). In certain aspects, the polynucleotide (e.g., synthetic RNA or synthetic DNA) includes only natural bases, i.e., A (adenosine), G (guanosine), C (cytosine), and T (thymidine) in the case of synthetic DNA, or A, C, G, and U (uridine) in the case of synthetic RNA.

[0090] The term “nucleic acid / polynucleotide sequence encodes” refers to the coding sequence of a nucleic acid / polynucleotide (e.g., mRNA or DNA molecule) that encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered. The coding sequence can further include a sequence encoding a signal peptide.

[0091] Further, at each end of the ORF (Open Reading Frame) of an mRNA is a non-translated region, referred to as the 5' UTR and the 3' UTR, respectively. The “5' untranslated region” (5' UTR) refers to the region of mRNA directly upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript to be translated by the ribosome) that does not encode a polypeptide. The “3' untranslated region” (3' UTR) refers to the region of mRNA directly downstream (i.e., 3') of the stop codon (i.e., the codon in the mRNA transcript that signals the termination of translation) that does not encode a polypeptide. An “open reading frame” is a continuous stretch of DNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide. Untranslated regions cannot be translated into amino acids, but can bind RNA binding proteins, thereby regulating the degradation and translation efficiency of the mRNA product. The 5'-UTR or 3' UTR can be homologous or heterologous to the open reading frame in the nucleic acid / polynucleotide. Multiple 5'-UTRs or 3' UTRs can be included in the flanking regions, which can be the same or different sequences.

[0092] "polyA" or "poly(A)" is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), of the 3' UTR, comprising multiple consecutive adenosine monophosphates. A polyA tail can comprise 10 to 300 adenosine monophosphates. For example, a polyA tail can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates. In a relevant biological context (e.g., in a cell, in vivo), a poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, mRNA export from the nucleus, and translation.

[0093] The term "cationic lipid" has its ordinary meaning in the art and can refer to a lipid comprising one or more positively charged groups. As used herein, "positively charged group" refers to a chemical group that carries a positive electronic charge, e.g., monovalent (+1), divalent (+2), trivalent (+3), and the like. Examples of positively charged groups include amine groups, ammonium groups, pyridine groups, guanidine groups, and imidazole groups. In certain embodiments, an ionizable lipid molecule can include an amine group and can be referred to as an ionizable amino lipid. In the present invention, cationic lipids include, but are not limited to, DLin-MC3-DMA (MC3), SM-102, and ALC-0315.

[0094] Herein, "lipid nanoparticle" or "LNP" is used for the delivery of mRNA. In some embodiments, an LNP essentially comprises (i) at least one cationic lipid; (ii) a phospholipid, a neutral lipid selected from any one or more combinations of DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, e.g., cholesterol.

[0095] As used herein, the term "synthetic" refers to produced, prepared, and / or manufactured by hand. Synthesis of polynucleotides or other molecules can be chemical or enzymatic. As used herein, "expression" of a nucleic acid sequence refers to translation of a polynucleotide, such as mRNA, into a polypeptide or protein and / or post-translational modification of a polypeptide or protein. Methods of transfection include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.

[0096] The term "transcription" refers to the process of producing mRNA (e.g., an mRNA sequence or template) from DNA (e.g., a DNA template or sequence). "Transfection" refers to the introduction of a polynucleotide (e.g., foreign nucleic acid) into a cell, where the polynucleotide encodes a polypeptide that is expressed (e.g., mRNA) or a polypeptide that modulates cellular function (e.g., siRNA, miRNA). Transfection can occur in vitro, ex vivo, or in vivo, for example. "Modification" refers to the alteration of any substance, compound, or molecule in some way. A molecule can undergo a series of modifications, and each modified molecule can serve as the "unmodified" starting molecule for a subsequent modification.

[0097] The term "variant" or "mutant" includes natural variants (such as polymorphisms, isomers, etc.) and artificial variants, in which at least one amino acid residue in a native or starting sequence (such as a wild-type sequence) has been removed and a different amino acid inserted at the same position. These variants can be described as "replacement variants". The replacement can be single, i.e. only one amino acid in the molecule is replaced, or multiple, i.e. two or more amino acids in the same molecule are replaced. If an amino acid is inserted or deleted, the resulting variant will be an "insertion variant" or a "deletion variant", respectively.

[0098] As used herein, the term "vaccine" is a biological preparation that primes the immune system to produce a response to a certain agent or antigen, typically an infectious pathogen or a part thereof, in a non-infectious or non-pathogenic form, into the human or animal body. Once the immune system is primed, upon later exposure of the immune system to the pathogen, a rapid and strong immune response is generated that eliminates the pathogen before it can multiply and infect enough cells in the host organism to cause symptoms of disease. The agent or antigen used to prime the immune system can be the entire organism, which is less infectious, i.e. a so-called attenuated organism, or in some cases a component of the organism, such as a carbohydrate, protein or peptide representing various structural components of the organism. Thus, according to the present application, the vaccine can be a protein vaccine or a nucleic acid vaccine. The nucleic acid vaccine can be a DNA vaccine or an mRNA vaccine. When a protein vaccine, the vaccine can be encoded by a polynucleotide or mRNA of the present application.

[0099] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" a cancer can refer to inhibiting survival, growth, and / or spread of a tumor. Treatment can be performed on a subject who does not exhibit symptoms of a disease, disorder, and / or condition and / or on a subject who exhibits only early symptoms of a disease, disorder, and / or condition, in order to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0100] In certain embodiments, the therapeutic and / or prophylactic measures is an mRNA. The mRNA can encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size, can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0101] As used herein, "and / or" shall be taken to mean either one or both of the items specifically recited. For example, a list of "A and / or B" means A alone, B alone, or A and B together. Likewise, a list of "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together. As used herein, "and / or" shall be taken to mean either one or both of the items specifically recited. For example, a list of "A and / or B" means A alone, B alone, or A and B together. Likewise, a list of "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.

[0102] It should also be noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. When used, the terms "comprising" and "including" also include the terms "consisting essentially of" and "consisting of". When a composition is described as containing, including, or comprising certain components, it can be intended that the composition also consist essentially of, or consist of, the recited components. Likewise, when a method or process is described as containing, including, or comprising certain steps, it can be intended that the method or process consist essentially of, or consist of, the recited steps. Furthermore, it is intended that the inclusion in the specification of an integrating declaration for an element or step also permits said element or step to be an optional element or step. Furthermore, it is understood that the order of steps or order for performing certain actions is immaterial so long as the present application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0103] The term "identity" refers to the similarity or correspondence between the sequences of two or more polypeptides or nucleic acids / polynucleotides, as determined by comparing the sequences. The "percentage of identity" applicable to polypeptide or nucleic acid / polynucleotide sequences is defined as the percentage of residues in an amino acid or nucleic acid / polynucleotide sequence that are identical with the residues in another amino acid sequence or nucleic acid / polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for aligning are well known in the art. Identity is dependent on the calculation of the percentage of identity, but can vary in value due to the introduction of gaps and penalties in the calculation.

[0104] "Identity" is determined by one of skill in the art using alignment tools and specific parameters. Such alignment tools include those in the BLAST suite (Stephen F. Altschul, et al. (1997), with specific parameter settings well known to one of skill in the art.

[0105] If other polypeptide or nucleic acid / polynucleotide molecules have a certain degree of identity with the polypeptide or nucleic acid / polynucleotide molecules herein, they also fall within the scope of the present application. Specifically, a variant of other nucleic acid / polynucleotide or polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a specific nucleic acid / polynucleotide or polypeptide protected by the present application, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity on the basis of core sequence or amino acid residue identity, which can be considered equivalent or identical to the specific polypeptide or nucleic acid / polynucleotide molecule protected by the present application.

[0106] The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the instructions of the commodity; the materials, reagents, etc. used, if not specially specified, are all conventional commercially available goods.

[0107] Examples

[0108] (I) Screening for liver cancer HLA-A*02:01 neoantigen target (schematic diagram of screening strategy is shown in Figure 1):

[0109] 1. Through the largest liver cancer multi-omics sequencing queue in China, the WES and RNA-seq sequencing data of HLA-A*02:01 liver cancer patients were selected for potential antigen prediction;

[0110] 2. The source of potential antigens considers tumor mutations, gene fusions, and variable splicing, etc. which may produce tumor neoantigens.

[0111] 3. Antigen mining: Mutect was used for mutation prediction algorithm (Reference: Cibulskis K, Lawrence MS, Carter SL, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol. 2013; 31(3):213-219. doi: 10.1038 / nbt.2514.); STARfusion was used for gene fusion prediction algorithm (Reference: Haas BJ, Dobin A, Li B, Stransky N, Pochet N, Regev A. Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods. Genome Biol. 2019; 20(1):213); rMATS was used for alternative splicing prediction algorithm (Reference: Shen S, Park JW, Lu ZX, et al. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc Natl Acad Sci U S A. 2014; 111(51):E5593-E5601. doi: 10.1073 / pnas.1419161111).

[0112] 4. The results of ELISpot for screening candidate antigens to activate T cells in PBMC for IFN-γ are shown in Figure 2, the HLA-A*02 type HLA affinity heat map of the screened 02 type antigens is shown in Figure 3, and the final screened HLA-A*02:01 neoantigen target core sequence is shown in Table 1 SEQ ID NO: 1-20:

[0113] Table 1 HLA-A*02:01 neoantigen target sequence

[0114] 5. ELISpot method for detecting antigen peptide-specific T cell immune response

[0115] First, PBMCs from three HLA-typed healthy individuals (P05, P29, P33) were isolated. Then, the 20 antigen peptides (SEQ ID NO: 1-20) were incubated with the PBMCs for 24 hours in a 37°C incubator. If the immune cells produced specific immune responses to the antigen peptides, they would secrete cytokine IFN-γ, and each responding T cell would form a spot. Finally, the level of specific T cell response activated by the 20 antigen peptides was evaluated by counting the number of IFN-γ positive spots.

[0116] The detection results are shown in FIGS. 4A and 4B. Although differences in the immune response levels of PBMCs from different sources were observed, high levels of immune responses (more than 200 spots in at least one PBMC) were produced by all the 20 antigen peptides.

[0117] The above results show that the 20 antigen peptides (SEQ ID NO: 1-20) screened by the present application can all induce strong specific T cell immune responses.

[0118] 6. Flow detection of the activation level of antigen peptide-specific T cells

[0119] PBMCs from one HLA-typed healthy individual were selected for detection. First, the 20 antigen peptides (SEQ ID NO: 1-20) screened above were incubated with the PBMCs for 48 hours in a 37°C incubator. After pre-cooled PBS was added for washing, a mixture of CD3, CD4, CD8, and 4-1BB antibodies was added to label the cells (4°C, dark for 30 minutes). CD3 is a surface marker of T cells. CD4 and CD8 are surface markers of helper T cells and cytotoxic T cells, respectively. 4-1BB is a surface marker representing T cell activation. Based on the CD3 positivity, the activation level of helper T cells was evaluated by detecting the CD4 + ; 4-1BB + cell subpopulation. The activation level of cytotoxic T cells was evaluated by detecting the CD8 + ; 4-1BB + cell subpopulation. The detection results are shown in FIGS. 5A and 5B. The 20 antigen peptides can all increase the expression level of 4-1BB of T cells (at least in one T cell, the expression level of 4-1BB is higher than that of the control sample).

[0120] The above results show that the 20 antigen peptides (SEQ ID NO: 1-20) screened by the present application can all increase the activation level of antigen peptide-specific T cells.

[0121] (ii) Construction of mRNA sequences of the tandem neoantigen targets

[0122] 1. The HLA-A*02:01 neoantigen targets (SEQ ID NO: 1-20) screened above are modified to obtain extended amino acid sequences (SEQ ID NO: 21-40) with physiological activity, the extended amino acid sequences (SEQ ID NO: 21-40) are connected by linkers, the amino acid sequence of the linker is: GGSGGGGSGG (SEQ ID NO: 41) or GGSLGGGGSG (SEQ ID NO: 42), the amino acid sequence after connection is added with a secretion signal peptide tPA and a 6*His epitope tag at the N- and C-termini, respectively, to obtain a recombinant polypeptide, the sequence of which is shown in SEQ ID NO: 43 (Figure 6), further, without changing the amino acid sequence, according to the codon bias of E. coli, the codon is optimized and modified as an ORF of mRNA, the ORF sequence is shown in SEQ ID NO: 44 (Figure 7); the ORF sequence is cloned into a modified pUC57 vector (the plasmid map of which is shown in Figure 8) to synthesize a plasmid, and this step is entrusted to GenScript to complete. In this step, pUC57 is selected as the plasmid vector, and the antigen coding sequence of the mRNA vaccine and the DNA expression element are introduced by conventional molecular biology means.

[0123] 2. The plasmid containing the antigen sequence of interest is amplified and purified using a polymerase to obtain an in vitro transcription template; under the action of a polymerase, the purified product is used as a transcription template to synthesize mRNA by in vitro transcription, and finally the mRNA stock solution is obtained after purification and filtration steps. The obtained mRNA sequence is composed of five key structural elements: 5' cap structure (Cap), 5' untranslated region (UTR), open reading frame (ORF), 3' untranslated region (UTR) and poly(A) tail, as shown in Figure 9.

[0124] 3. The generated mRNA is modified in vitro, including 3' end modification and 5' end modification. Among them, the polyA tail is modified with cytosine (C); the 5' cap is modified with Cap1m6AG.

[0125] (Three) Lipid nanoparticles (LNP):

[0126] The composition of LNP includes ionizable cationic lipids, phospholipids, and cholesterol.

[0127] Ionizable cationic lipids (ICLs), including DLin-MC3-DMA (MC3), SM-102 and ALC-0315 are all monoamine-based lipids. ALC-0315, MC3 and SM-102 are three ionizable lipids that can be used in humans, which can be assembled in a specific ratio under acidic conditions to form ICLs. Cationic liposomes can bind to negatively charged mRNA through electrostatic interactions and encapsulate them based on their hydrophobicity, forming mRNA-loaded lipid nanoparticles (LNPs).

[0128] (iv) Preparation of mRNA-lipid nanoparticles (mRNA-LNP) using microfluidic mixing

[0129] 1. Most of the existing published formulations recommend a mRNA: LNP ratio of 1:6, but in actual preparation, about 25% of phospholipids are lost during mixing, encapsulation and ultrafiltration, so the N / P ratio used in this application is 8. A higher N / P ratio can significantly reduce the particle size increase caused by ultrafiltration and dialysis, and improve the utilization rate of mRNA.

[0130] 2. Calculate the required RNA concentration according to N / P = 8, FRR = 3: the average molecular weight of the bases of RNA is 324, and each reduced base carries 1 phosphate, so the phosphorus content in RNA is 3.09 nmol / pg. The average molecular weight of DNA is calculated as 318, and the phosphorus content of DNA is 3.14 nmol / pg. When calculating the nitrogen-phosphorus ratio, only the number of nitrogen atoms in the main lipids is calculated, so there are 0.5 moles of N in each mole of mixed lipids. Each mM of total lipids can carry

[0131] 3. Prepare mRNA-citric acid buffer: Use ultrapure water to prepare 100 mM citric acid monohydrate (molecular weight: 210.14, weigh 1.05 g) and sodium citrate dihydrate (molecular weight: 294.10, weigh 1.47 g) solution each 50 mL. Take 33.0 mL of citric acid solution and 17.0 mL of sodium citrate solution, mix, then add DEPC (diethypyrocarbonate), stand for 30 minutes, then remove DEPC under high pressure, sterilize, then use DEPC water to constant volume to 100 mL to get 50 mM pH = 4 citric acid buffer. After determining the concentration of mRNA, dilute it to the required concentration using the citric acid buffer according to the lipid concentration.

[0132] 4. Formulation of lipid solution and encapsulation by microfluidic device: this step is commissioned by stemiRNA Therapeutics. By microfluidic device, the lipid solution and mRNA solution can be mixed thoroughly in a mixer, and LNP with uniform particle size can be formed rapidly. Since the lipid is dissolved in ethanol and the nucleic acid is dissolved in an acidic buffer, dialysis or ultrafiltration is required to remove residual ethanol and replace the solution system with a neutral buffer.

[0133] 5. After the preparation is completed, the mRNA-LNP is used for in vitro transfection efficiency verification, in vivo infection efficiency verification, and in vivo liver targeting verification.

[0134] (1) In vitro transfection efficiency verification of mRNA-LNP

[0135] In order to verify the transfection efficiency, LNP encapsulating eGFP-mRNA is used to transfect HLA-A*0201 cells SNU-398, and the results are shown in Figures 10-11: Figure 10, upper left, is the cell morphology observed under a 100x light microscope; upper right, is the cell transfection efficiency observed under a 100x fluorescence microscope; lower left, is the cell morphology observed under a 200x light microscope; lower right, is the cell transfection efficiency observed under a 200x fluorescence microscope; it can be seen that after 24h, the LNP containing eGFP-mRNA can enter the cells and express, and the green fluorescence is mainly expressed in the cytoplasm. Figure 11 is the flow detection of the proportion of eGFP positive cells, it can be seen that compared with the negative cells, the positive rate of HLA-A*0201 cells SNU-398 transfected by LNP containing eGFP-mRNA is more than 90%.

[0136] (2) In vivo transfection efficiency verification of mRNA-LNP

[0137] Figure 12 is the live imaging picture of mice 24h after injection of eGFP-mRNA vaccine; Figure 13 is the fluorescence imaging picture of internal organs of mice 24h after injection of eGFP-mRNA vaccine; Figure 14 is the fluorescence and light microscope images of liver tissue sections of mice 48h after injection of physiological saline and eGFP-mRNA vaccine; the results show that the mRNA of the present application targets and accumulates in the liver after being injected into the animal body.

[0138] The above results show that the mRNA of the present application has good in vitro and in vivo transfection efficiency, and good targeting.

[0139] 6. Animal in vivo experiment

[0140] Construction of HLA-A*02:01 type subcutaneous tumor mouse model: the animal model uses NZGL (NOD-Prkdc em26Cd52 Il2rg em26Cd22Gpt-GM-SCF-IL3) mice, CD34 + The stem cells were injected into the reconstituted mice to reconstruct the immune system. Peripheral blood was taken at 8 weeks for detection, and the detection results were as follows: hCD45 > 30%, hCD3 > 25%, and the mouse was qualified. At 10 weeks, the mouse was inoculated with a tumor (SNU-398 HLA-A*02:01 tumor cell line conforming to HLA), and regular detection was performed. After the mouse tumor grew stably, the experiment was performed, and the tumor photos after the mouse was tumorized are shown in Fig. 15.

[0141] The above constructed HLA-A*02:01 type subcutaneous tumor mouse model was divided into two groups, one group was inoculated with the constructed 02 type mRNA tumor vaccine (mRNA-LNP) of the application, and the other group was inoculated with blank mRNA as a control group (vector group). The tumor growth in the mouse was detected at 1-31 days after inoculation, and the tumor volume measurement results of the mouse are shown in Fig. 16. It can be seen from the figure that the mRNA-LNP of the application has the effect of continuously inhibiting the growth of hepatocellular carcinoma tumor, and has great clinical application prospect.

[0142] Fig. 17 is a slice and HE staining of the spleen, brain, lung, kidney, heart and liver of the mouse after the mouse was killed at the observation endpoint after using the A02 tumor vaccine. It can be seen that the mouse spleen is infiltrated with a large number of lymphocytes, and other important physiological organs have no obvious increase in immune infiltration. This result proves that the A02 tumor vaccine has good tumor targeting and no obvious damage to other organs, and has good safety.

[0143] Therefore, a patient clinically diagnosed as having hepatocellular carcinoma or a patient whose postoperative pathology confirms hepatocellular carcinoma after surgical resection, peripheral blood or tumor tissue detection is HLA-A*0201 type, or HLA-A*02:07 type, HLA-A*02:06 type, HLA-A*02:03 type, HLA-A*02:10 type, HLA-A*02:04 type, HLA-A*02:05 type, HLA-A*02:06 type, HLA-A*02:08 type, HLA-A*02:09 type, HLA-A*02:011 type and the like, can use the mRNA vaccine of the application for treatment; the mRNA vaccine of the application is a freeze-dried powder, which is dissolved and administered by injection when used. The injection methods include but are not limited to subcutaneous injection, intramuscular injection, intravenous injection and intraperitoneal injection. The mRNA vaccine of the application can be used alone or in combination with PD-1 and / or PD-L1 monoclonal antibodies or other anti-tumor drugs.

[0144] Among them, the mRNA vaccine combined treatment strategy is a new antigen mRNA vaccine combined treatment strategy proposed according to multi-color immunofluorescence combined with pathological histology deep learning technology to accurately judge the immune microenvironment type. The scheme can activate the body to produce specific T cell response to the neoantigen, and break the tumor edge 'barrier', increase T cell infiltration, remove immune suppression, and improve the immune microenvironment to achieve the effect of synergistic treatment. The application prospect of the scheme is broad, and it is expected to become one of the main directions of future HCC immunotherapy.

[0145] The above is only a preferred embodiment of the present application, not any form and substantial limitation of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application.

Claims

1. A tumor neoantigen peptide, characterized in that, The tumor neoantigen peptide comprises a combination of one or more of polypeptides with amino acid sequences as shown in SEQ ID NO: 1-20, or a combination of one or more of polypeptides with amino acid sequences as shown in SEQ ID NO: 21-40.

2. A nucleic acid, characterized in that, The nucleic acid encodes a combination of polypeptides with amino acid sequences as shown in SEQ ID NO: 1-20, or the nucleic acid encodes a combination of polypeptides with amino acid sequences as shown in SEQ ID NO: 21-40.

3. The nucleic acid of claim 2, wherein The combination of polypeptides is in series by linkers from polypeptides with amino acid sequences as shown in SEQ ID NO: 1-20, or the combination of polypeptides is in series by linkers from polypeptides with amino acid sequences as shown in SEQ ID NO: 21-40. Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 41 or SEQ ID NO:

42.

4. The nucleic acid of claim 2 or 3, wherein The nucleic acid encodes a polypeptide in series from SEQ ID NO: 21-40; Preferably, the nucleic acid encodes a polypeptide as shown in SEQ ID NO: 43 or a variant thereof; the variant is a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 43, and retains the function of the polypeptide as shown in SEQ ID NO:

43.

5. The nucleic acid of claim 4, wherein The nucleic acid is DNA; Preferably, the DNA has a sequence as shown in SEQ ID NO: 44 or with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

44.

6. The nucleic acid of claim 4, wherein The nucleic acid is RNA, for example, mRNA; Preferably, the mRNA has a transcription sequence of a DNA sequence as shown in SEQ ID NO: 44 or with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

44.

7. The nucleic acid of claim 6, wherein When the nucleic acid is mRNA, the 5' end of the mRNA comprises a 5' cap structure (Cap) and / or a 5' untranslated region (UTR); and / or, the 3' end of the mRNA comprises a 3' UTR and / or a 3' poly(A) tail.

8. The nucleic acid of claim 7, wherein The poly A tail is modified with cytosine (C); and / or, the 5' cap structure (Cap) is modified with Cap1 m6AG.

9. A recombinant expression vector, characterized in that, It comprises the nucleic acid of any one of claims 2-8 or a transcribed nucleic acid thereof; Preferably, the backbone of the recombinant expression vector is a plasmid or a virus, for example, pUC57 or a modified plasmid thereof.

10. The recombinant expression vector of claim 9, wherein, The plasmid backbone comprises a T7 promoter sequence, a 5' UTR region, a 3' UTR region, a 5' terminal cap structure, and a 3' terminal PolyA tail.

11. A composition characterized in that, It comprises (1) the nucleic acid according to any one of claims 2-8, or the recombinant expression vector according to any one of claims 9-10, and (2) a delivery vector; Preferably, the delivery vector comprises a liposome; and / or, the delivery vector comprises a lipid nanoparticle (LNP).

12. The composition of claim 11, wherein The lipid nanoparticle comprises a cationic lipid and a non-cationic lipid; wherein the cationic lipid may be, for example, ionizable cationic lipids (ICLs); Preferably, the ionizable cationic lipids (ICLs) comprise DLin-MC3-DMA (MC3), SM-102, and ALC-0315; and / or, the non-cationic lipid comprises a phospholipid.

13. The composition of claim 12, wherein The delivery vector further comprises cholesterol; Preferably, the delivery vector comprises ionizable cationic lipids (ICLs), a phospholipid, and cholesterol.

14. The composition according to any one of claims 11 to 13, wherein The nucleic acid is mRNA, and is encapsulated in the delivery vector.

15. A pharmaceutical composition comprising, It comprises the composition according to any one of claims 11-14, and optionally a pharmaceutically acceptable carrier and / or adjuvant.

16. A vaccine comprising a polynucleotide of claim 1. It comprises the nucleic acid according to any one of claims 2-8, the recombinant expression vector according to any one of claims 9-10, the composition according to any one of claims 11-14, and / or the pharmaceutical composition according to claim 15, and a pharmaceutically acceptable adjuvant.

17. The vaccine as described in claim 16, characterized in that, The vaccine is a protein vaccine; and / or, the vaccine is a nucleic acid vaccine; and / or, the vaccine is a DNA vaccine or an mRNA vaccine.

18. Use of the tumor neoantigen peptide according to claim 1, the nucleic acid according to any one of claims 2-8, the recombinant expression vector according to any one of claims 9-10, the composition according to any one of claims 11-14, the pharmaceutical composition according to claim 15, and / or the vaccine according to any one of claims 16-17 in the preparation of a medicament for preventing and / or treating liver cancer; Preferably, the liver cancer is HLA-A*02 type liver cancer; More preferably, the liver cancer is HLA-A*02:01 type, HLA-A*02:07 type, HLA-A*02:06 type, HLA-A*02:03 type, HLA-A*02:10 type, HLA-A*02:04 type, HLA-A*02:05 type, HLA-A*02:06 type, HLA-A*02:08 type, HLA-A*02:09 type, HLA-A*02:011 type, etc.

19. The use of claim 19, wherein, The medicament is a vaccine; Preferably, the vaccine is a protein vaccine; and / or, the vaccine is a nucleic acid vaccine; and / or, the vaccine is a DNA vaccine or an mRNA vaccine.

20. A method for preparing an anti-liver cancer mRNA vaccine, characterized in that, The method comprises the following steps: Step 1: cloning the polypeptide with the sequence shown in SEQ ID NO: 21-SEQ ID NO: 40 in series through a linker into a vector for plasmid synthesis; Step 2: synthesizing mRNA by in vitro transcription of the synthesized plasmid containing the tandem antigen sequence; Step 3: modifying the synthesized mRNA in vitro, including 3' end modification and 5' end modification; Step 4: preparing mRNA-lipid nanoparticles by microfluidic technology; Preferably, the amino acid sequence of the linker in step 1 is as shown in SEQ ID NO: 41 or SEQ ID NO:

42.

21. A method for preventing and / or treating liver cancer, i.e. administering an effective amount of the nucleic acid of the second aspect, the recombinant expression vector of the third aspect, the composition of the fourth aspect, the pharmaceutical composition of the fifth aspect, and / or the vaccine of the sixth aspect to a subject in need thereof; Preferably, the liver cancer is HLA-A*02 type liver cancer. More preferably, the liver cancer is HLA-A*02:01 type.

22. The method of claim 21, wherein, The method further comprises administering a second therapeutic agent to a subject in need thereof; Preferably, the second therapeutic agent is selected from immune checkpoint inhibitors, which are selected from any one or more of PD-1 antibodies, PD-L1 antibodies, and CTLA-4 antibodies.

23. The use of claim 18 or the method of any one of claims 21-22, wherein, The liver cancer also includes HLA-A*02:07 type, HLA-A*02:06 type, HLA-A*02:03 type, HLA-A*02:10 type, HLA-A*02:04 type, HLA-A*02:05 type, HLA-A*02:06 type, HLA-A*02:08 type, HLA-A*02:09 type, or HLA-A*02:011 type, but is not limited to the above-mentioned types.

24. A humanized mouse model for in vivo validation of the immunization effect of the vaccine of claim 16. The model reconstructs the HLA-A*02:01 type human immune system, and after inoculating human liver cancer cells, the vaccine can significantly reduce the tumor volume and increase CD8 + T cell infiltration.

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