HLA-a11-targeted liver cancer vaccine, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are an HLA-A*11-targeted liver cancer mRNA vaccine, and a preparation method therefor and a use thereof. The mRNA vaccine of the present invention is an mRNA vaccine designed on the basis of the HLA-A*11:01 typing of a patient and having high-coverage and high-immunogenic tumor neoantigens, and is formed by transcribing DNA having a nucleotide sequence shown in SEQ ID NO: 32 to form an mRNA, and encapsulating the mRNA in lipid nanoparticles.

Description

Anti-hepatocarcinoma HLA-A11 vaccine, preparation method and application thereof

[0001] Priority statement

[0002] The present application is based on the prior application of Chinese patent application No. 2024107161084, entitled "Anti-hepatocarcinoma HLA-A*11 vaccine, preparation method and application thereof", filed on June 4, 2024, and Chinese patent application No. 202510587761X, entitled "Anti-hepatocarcinoma HLA-A*11 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-A11 vaccine and a construction method thereof, and belongs to the field of biological medicine and vaccine technology. BACKGROUND

[0004] The existing traditional diagnosis and treatment measures for malignant tumors in clinical practice include surgical treatment, interventional treatment, chemotherapy and targeted therapy, but the overall effect is still unsatisfactory. Most malignant tumor patients are at risk of high mortality and high recurrence rate. In recent years, with the progress of technology, tumor diagnosis and treatment has entered the era of immunotherapy. However, although immunotherapy drugs represented by ICB have significantly prolonged the survival time of patients, a considerable part of tumor patients cannot benefit from immunotherapy. For example, "cold" tumors such as hepatocellular carcinoma and pancreatic cancer, the overall response rate of patients to immunotherapy is less than 20%, which has caused great pain to patients. Innovative immunotherapy such as CAR-T and TCR-T cell therapy has shown strong anti-tumor ability in hematological tumors, but due to the lack of ideal solid tumor treatment targets and inhibitory immune microenvironment, it still lacks breakthrough progress in the treatment of solid tumors.

[0005] 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. The existing tumor target sources are mostly 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 liver cancer is limited, and there is no obvious population distribution rule. Alternative splicing (also called selective splicing) refers to the process of producing different mRNA splice isoforms from an mRNA precursor through different splicing methods (selecting different splicing site combinations), so that the final protein products will exhibit different or antagonistic functions and structural properties, or different phenotypes due to different expression levels in the same cell. In recent years, research has found that alternative splicing isoforms are also an important source of tumor neoantigens.

[0006] Tumor vaccines are introduced into antigen-presenting cells (APCs) through specific delivery systems, so that the desired tumor-specific neoantigens can be synthesized in the APC cells. The APC cells further present the specific tumor antigens to the cell surface and are recognized and activated by effector immune cells such as T cells, B cells, and NK cells, thereby specifically and continuously killing tumor cells and achieving the effect of inhibiting tumor growth. 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, and the physicochemical properties of different sequence polypeptides differ greatly, and the process route and quality standard research are difficult.

[0007] Nucleic acid vaccines are a promising means to avoid the above problems. Most of the vaccines currently under research are mRNA vaccines. After mRNA enters human cells through a specific delivery system, the human body itself translates the mRNA into a protein, and the protein expressed as a certain antigen protein possessed by a virus is recognized as a foreign antigen by antigen-presenting cells (APCs), driving the maturation of dendritic cells (DCs) and further activating B cells and T cells to produce a strong immune response, causing a dual immune response of humoral and cellular immunity. The mRNA vaccine breaks through the traditional immune activation mode of vaccines and innovatively uses the human body's own cells to produce antigens to activate dual-specific immunity and form immune memory, providing more persistent specific immunity.

[0008] Most of the domestic pharmaceutical companies such as Abobio, S Microorganism and the like develop vaccines for personalized tumor vaccines, which are not considered for the characteristics of a large population, and are limited by surgical sample vaccines, which need to be sequenced, calculated and developed and produced after surgical resection, and at least need 3-6 months, during which the patients are often difficult to treat 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 period.

[0009] Importantly, tumor antigen presentation depends on the patient's HLA typing. HLA-A*11:01 is the highest frequency of HLA typing in Asians, which is significantly different from the proportion of HLA typing in Caucasians abroad. According to the data investigation, the HLA-A*11:01 typing reaches 20.893%. Therefore, it is of great significance to develop an mRNA tumor vaccine for HLA-A*11:01 hepatocellular carcinoma typing, which can adapt to most hepatocellular carcinoma patients.

[0010] In summary, the existing treatment drugs and treatment methods for hepatocellular carcinoma have reached the upper limit, and there is an urgent need to develop new hepatocellular carcinoma treatment drugs and treatment methods. SUMMARY

[0011] The purpose of the present application is: in view of the deficiencies of the prior art, the present application provides an anti-hepatocellular carcinoma HLA-A*11 type vaccine and a construction method thereof.

[0012] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0013] In a first aspect of the present application, a tumor neoantigen peptide is provided, which comprises a combination of one or more polypeptides with amino acid sequences as shown in SEQ ID NO: 1-14, or a combination of one or more polypeptides with amino acid sequences as shown in SEQ ID NO: 15-28.

[0014] Preferably, the tumor neoantigen peptide consists of polypeptides with amino acid sequences as shown in SEQ ID NO: 15-28.

[0015] In a second aspect of the present application, a nucleic acid is provided, which encodes a combination of polypeptides with amino acid sequences as shown in SEQ ID NO: 1-14, or which encodes a combination of polypeptides with amino acid sequences as shown in SEQ ID NO: 15-28.

[0016] In some preferred embodiments, the combination of polypeptides is a combination of polypeptides with amino acid sequences as shown in SEQ ID NO: 1-14 linked by a linker, or the combination of polypeptides is a combination of polypeptides with amino acid sequences as shown in SEQ ID NO: 15-28 linked by a linker.

[0017] Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 29 or SEQ ID NO: 30.

[0018] In some preferred embodiments, the nucleic acid encodes a polypeptide sequentially linked by SEQ ID NO: 15-28.

[0019] Preferably, the nucleic acid encodes a polypeptide as shown in SEQ ID NO: 31 or a variant thereof; the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 31 and retains the function of the polypeptide as shown in SEQ ID NO: 31.

[0020] In some preferred embodiments, the nucleic acid is DNA.

[0021] Preferably, the DNA has a sequence as shown in SEQ ID NO: 32 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 32.

[0022] In some preferred embodiments, the nucleic acid is RNA, for example, mRNA.

[0023] Preferably, the mRNA has a transcription sequence of a DNA sequence as shown in SEQ ID NO: 32 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 32.

[0024] 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.

[0025] Preferably, the poly(A) tail is modified with cytosine (C); and / or, the 5' cap structure (Cap) is modified with Cap1 m6AG.

[0026] 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.

[0027] Preferably, the backbone of the recombinant expression vector is a plasmid or a virus, such as pUC57 or a modified plasmid thereof.

[0028] In some preferred embodiments, 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.

[0029] 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.

[0030] Preferably, the delivery vehicle comprises a liposome; and / or, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0031] 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).

[0032] 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.

[0033] In some preferred embodiments, the delivery vehicle further comprises a cholesterol.

[0034] Preferably, the delivery vehicle comprises an ionizable cationic lipid (ICL), a phospholipid, and a cholesterol.

[0035] Preferably, the nucleic acid is an mRNA and is encapsulated in the delivery vehicle.

[0036] In a fifth aspect of the present application, there is provided a pharmaceutical composition comprising the composition of the fourth aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0037] In a sixth aspect of the present application, there is provided 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.

[0038] Preferably, the vaccine is a protein vaccine.

[0039] and / or, the vaccine is a nucleic acid vaccine.

[0040] And / or, the vaccine is a DNA vaccine or an mRNA vaccine.

[0041] In a seventh aspect of the present application, the tumor neoantigen peptide, the nucleic acid, the recombinant expression vector, the composition, the pharmaceutical composition and / or the vaccine are used for preparing a medicament for preventing and / or treating liver cancer.

[0042] Preferably, the liver cancer is HLA-A*11 type liver cancer.

[0043] More preferably, the liver cancer is HLA-A*11:01 type liver cancer.

[0044] More preferably, the liver cancer can also be HLA-A11:02, HLA-A11:03, HLA-A11:04, HLA-A11:05, HLA-A11:06, HLA-A11:07, HLA-A11:08, HLA-A11:09, HLA-A11:10, HLA-A11:11, HLA-A11:12, or HLA-A11:100 type.

[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 of the present application, a preparation method of an anti-liver cancer mRNA vaccine is provided, comprising the following steps:

[0048] Step 1: polypeptides with sequences as shown in SEQ ID NO: 15-SEQ ID NO: 28 are connected by linkers, and then cloned into a vector for plasmid synthesis;

[0049] Step 2: the synthesized plasmid containing the tandem antigen sequence is subjected to in vitro transcription to synthesize mRNA;

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

[0051] Step 4: mRNA-lipid nanoparticles are prepared by using microfluidic technology.

[0052] Preferably, the amino acid sequence of the linker in step 1 is as shown in SEQ ID NO: 29 or SEQ ID NO: 30.

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

[0054] Preferably, the liver cancer is of a patient with HLA-A*11 type.

[0055] More preferably, the liver cancer is of a patient with HLA-A*11:01 type.

[0056] Preferably, the method further comprises administering to the subject in need thereof a second therapeutic agent.

[0057] More 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.

[0058] Preferably, the liver cancer can further comprise HLA-A11:02, HLA-A11:03, HLA-A11:04, HLA-A11:05, HLA-A11:06, HLA-A11:07, HLA-A11:08, HLA-A11:09, HLA-A11:10, HLA-A11:11, HLA-A11:12, HLA-A11:13, HLA-A11:14 HLA-A11:15, HLA-A11:16, HLA-A11:17, HLA-A11:100, HLA-A11:101, or HLA-A11:109 type, but is not limited to the above-mentioned types.

[0059] 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*11:01 type, and the administration of the vaccine after inoculation of human liver cancer cells can significantly reduce the tumor volume and increase CD8 + T cell infiltration.

[0060] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred embodiments of the present application.

[0061] The reagents and raw materials used in the present application are commercially available.

[0062] Compared with the prior art, the present application has the following beneficial effects:

[0063] (1) The A11 type antigen screened by the application can have strong affinity with multiple HLA-A*11 subtypes (including HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:07 and HLA-A*11:10);

[0064] The application provides an mRNA tumor vaccine for HLA-A*11:01 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 subtype of the patient; and since the proportion of HLA-A*11:01 subtype of Chinese people is as high as 20.893%, the mRNA tumor vaccine for HLA-A*11:01 liver cancer subtype provided by the application has important significance, which can be adapted to most Chinese liver cancer patients, has clinical application value and good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0065] Fig. 1 is a schematic diagram of the screening strategy of HLA-A*1101 potential antigens;

[0066] Fig. 2 is a heat map of the affinity of the HLA-A*11 type antigens screened by the application with multiple HLA-A*11 subtypes;

[0067] Fig. 3 is a microscopic image of T cell activation after in vitro addition of HLA-A*1101 antigen peptide stimulation;

[0068] Fig. 4 is the detection of 14 antigen peptide specific T cell immune responses by ELISpot method, wherein A is the IFN-γ spot count statistics of 20 antigen peptides identified by IFN-γ ELISPOT method in three PMBC samples, B is the IFN-γ spot map of 14 antigen peptides in P13 sample; neg is DMSO negative control, and pos is PHA (Phytohemagglutinin) positive control;

[0069] Fig. 5 is the detection of the activation level of 14 antigen peptide specific T cells by flow cytometry, wherein A is the flow cytometry detection results of CD3 + ; CD4 + ; 4-1BB + cell subgroups, and B is the flow cytometry detection results of CD3 + ; CD8 + ; 4-1BB + cell subgroups.

[0070] Figure 6 is the amino acid sequence of A11; the sequence highlighted in color (yellow) is the extended amino acid sequence; the bold + single underline indicates the core sequence of the neoantigen target; the blue + double underline indicates the amino acid sequence of the linker;

[0071] Figure 7 is the ORF sequence of A11; the sequence highlighted in color (yellow) is the DNA sequence corresponding to the extended amino acid sequence; the blue + double underline indicates the DNA sequence of the linker;

[0072] Figure 8 is a pUC57 plasmid vector map for loading the HLA-A*1101 antigen sequence plasmid;

[0073] Figure 9 is a fluorescence image of HLA-A*11:01 cell huh7 transfected with eGFP-mRNA vaccine;

[0074] Figure 10 is a flow cytometry result graph of HLA-A*11:01 cell huh7 transfected with eGFP-mRNA vaccine;

[0075] Figure 11 is a live imaging image of mice injected with eGFP-mRNA vaccine;

[0076] Figure 12 is a tumor body line graph of tumor-bearing mice inoculated with mRNA vaccine and control group, wherein vector represents the blank mRNA control group inoculated;

[0077] Figure 13 is a hematoxylin-eosin staining image of the spleen, brain, lung, kidney, heart and liver of mice after using A11 type mRNA tumor vaccine. DETAILED DESCRIPTION

[0078] 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.

[0079] Unless otherwise specified, the terms of the present application are defined as follows.

[0080] In the present application, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can include modified amino acids. These terms also include amino acid polymers that have been modified in any way; for example, by disulfide formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, the definition also includes polypeptides containing one or more analogs of an amino acid (for example, unnatural amino acids such as homoarginine, ornithine, p-acetylphenylalanine, D-amino acids and sarcosine), as well as other modifications known in the art.

[0081] 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-chain polypeptides. Disulfide linkages are most commonly found in multi-chain 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.

[0082] The term "signal peptide" includes the N-terminal 15-60 amino acids of a protein that are typically required for translocation across the membrane of the secretory pathway, and thus, are universally controlled in both eukaryotes and prokaryotes for 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. Secreted proteins without additional address tags in their sequence are by default secreted into the external environment.

[0083] The term "sequence optimization" refers to a process or series of processes by which bases in a reference nucleic acid / polynucleotide sequence are replaced with alternative bases to produce a nucleic acid / polynucleotide sequence with improved properties, e.g., improved protein expression or reduced immunogenicity. In general, 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.

[0084] In the context of sequence optimization, the term "codon substitution" refers to the substitution of one codon for another in a reference nucleic acid / polynucleotide sequence. A codon can be substituted in a reference nucleic acid / polynucleotide sequence, for example, by chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to a "substitution" or "replacement" in a certain position of a nucleic acid / polynucleotide sequence, such as an mRNA, or in a certain region or subsequence of a nucleic acid / polynucleotide sequence, such as an mRNA, refers to the substitution 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, which when expressed, produces a polypeptide or protein.

[0085] The term "nucleic acid / polynucleotide" includes any compound and / or substance composed of nucleotides. These polymers are commonly referred to as nucleic acids / polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), ene nucleic acid (ENA), cyclohexyl nucleic acid (CeNA), or mixtures or combinations thereof. "Polynucleotide" includes triple-stranded, double-stranded, and single- stranded DNA or RNA. It also includes polynucleotides that are modified, e.g., by alkylations, and / or by capping, as well as 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.

[0086] 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 certain 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.

[0087] 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 flanked by regulatory elements capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered, including promoters and polyadenylation signals. The coding sequence can further include a sequence encoding a signal peptide.

[0088] Further, at each end of the ORF (Open Reading Frame) of an mRNA, there is a region of non-translated sequence, referred to as the 5' UTR and the 3' UTR, respectively. The "5' untranslated region" (5' UTR) refers to the region of an mRNA that is directly upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript that is translated by the ribosome) and that does not encode a polypeptide. The "3' untranslated region" (3' UTR) refers to the region of an mRNA that is directly downstream (i.e., 3') of the stop codon (i.e., the codon in the mRNA transcript that signals termination of translation) and 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)), ends with a stop codon (e.g., TAA, TAG, or TGA), and codes for a polypeptide. Untranslated regions cannot be translated into amino acids, but can bind RNA binding proteins, thereby modulating the degradation and translational 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.

[0089] A "polyA" or "poly(A)" is a region of an mRNA that is downstream, e.g., directly downstream (i.e., 3'), of the 3' UTR and comprises 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), the function of a poly(A) tail is to protect the mRNA from enzymatic degradation, e.g., in the cytoplasm, and to aid in transcription termination, mRNA export from the nucleus, and translation.

[0090] The term "cationic lipid" has its ordinary meaning in the art and can refer to a lipid that comprises one or more positively charged groups. As used herein, a "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 comprise an amine group and can be referred to as an ionizable amino lipid. In the present application, cationic lipids include, but are not limited to, DLin-MC3-DMA (MC3), SM-102, and ALC-0315.

[0091] Herein, “lipid nanoparticle” or “LNP” is used for the delivery of mRNA. In some embodiments, the 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.

[0092] As used herein, the term “synthesis” refers to production, preparation, and / or manufacture by the hand of man. 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 (e.g., 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.

[0093] The term “transcription” refers to a process of producing mRNA (e.g., mRNA sequence or template) from DNA (e.g., DNA template or sequence). “Transfection” refers to the introduction of a polynucleotide (e.g., exogenous nucleic acid) into a cell, where the polynucleotide encodes a polypeptide that is expressed (e.g., mRNA) or a polypeptide that modulates cell function (e.g., siRNA, miRNA). For example, transfection can occur in vitro, ex vivo, or in vivo. “Modification” refers to changing 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.

[0094] The term “variant” or “mutant” includes natural variants (e.g., polymorphisms, isomers, etc.) and artificial variants, where at least one amino acid residue in a native or starting sequence (e.g., wild-type sequence) has been removed and a different amino acid inserted at the same position. These variants can be described as “substitutional variants.” The substitution 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 a “insertional variant” or “deletional variant,” respectively.

[0095] As used herein, the term "vaccine" is a biological preparation that stimulates the immune system in response to a specific agent or antigen, usually a pathogenic infectious agent or a portion thereof, in a non-infectious or non-pathogenic form, into the human or animal body. Once the immune system is stimulated, the immune system, upon later exposure to the pathogenic agent, produces a rapid and strong immune response that destroys the pathogenic agent before it can multiply and infect enough cells in the host organism to cause disease symptoms. The agent or antigen used to stimulate the immune system can be the entire organism, i.e., a so-called attenuated organism, which is less infectious, or in some cases a component of the organism, such as a carbohydrate, protein, or peptide that represents various structural components of the organism. Thus, in accordance with 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 the polynucleotide or mRNA of the present application.

[0096] 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.

[0097] In certain embodiments, the therapeutic and / or prophylactic measure 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 and 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.

[0098] As used herein, "and / or" shall be taken to mean and include one or the other or both of the items so conjoined, abiding by the normal usage of that term where still possible. As used herein, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both "A and B," "A or B," "A," (alone), and "B," (alone). Likewise, the term "and / or" or "one or more of' as used in a phrase such as "A, B, and / or C" or "one or more of A, B, and C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0099] It is also to 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 having, including, or comprising specific ingredients, it is intended to be equivalent to a composition that is substantially similar to the specific ingredients. Likewise, when a method or process is described as having, including, or comprising specific steps, it is intended to be equivalent to a method or process that is substantially similar to the specific steps. Furthermore, it is intended that the recitations of a step or action in a certain order are not limited to that order. Further, two or more steps or actions can be conducted simultaneously.

[0100] The term "identity" refers to the similarity or correspondence between the sequences of two or more polypeptides or nucleic acids / polynucleotides, determined by comparing the sequences. The "percent identity" for 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 the other 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 alignment are well known in the art. Identity depends on the calculation of percent identity, but can vary in value depending on the introduction of gaps and penalties for gaps in the calculation.

[0101] "Identity" is determined by those skilled 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 those skilled in the art.

[0102] Other polypeptide or nucleic acid / polynucleotide molecules that have a certain degree of identity to the polypeptide or nucleic acid / polynucleotide molecules herein are also within the scope of the present invention. Specifically, variants of other nucleic acid / polynucleotide or polypeptide molecules that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity on a core sequence or amino acid residue basis, to a specific nucleic acid / polynucleotide or polypeptide molecule protected by the present invention, can be considered equivalent or identical to the specific polypeptide or nucleic acid / polynucleotide molecule protected by the present invention.

[0103] The experimental methods in the following examples are selected according to the conventional methods and conditions, or according to the commercial instructions, unless otherwise specified; the materials, reagents, etc. used are conventional commercial products, unless otherwise specified.

[0104] Examples

[0105] (I) Screening for liver cancer HLA-A*11:01 neoantigen target (schematic diagram of screening strategy is shown in Figure 1):

[0106] 1. Through the largest liver cancer multi-omics sequencing cohort in China, the WES and RNA-seq sequencing data of liver cancer patients with HLA-A*11:01 typing were selected for potential antigen prediction;

[0107] 2. The potential antigen source considers the possible sources of tumor neoantigens such as tumor mutations, gene fusions and alternative splicing.

[0108] 3. Mutant prediction algorithm uses Mutect (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); gene fusion prediction algorithm uses STARfusion (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); alternative splicing prediction algorithm uses rMATS (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).

[0109] 4. The core sequence of the HLA-A*11:01 neoantigen target screened is shown in Table 1 as SEQ ID NO: 1-14, and the affinity detection results are shown in Figure 2, and the T cell activation images after adding HLA-A*11:01 antigen peptide in vitro are shown in Figure 3.

[0110] Table 1 HLA-A*1101 neoantigen target sequence

[0111] 5. ELISpot method for detecting antigen peptide-specific T cell immune response

[0112] Firstly, PBMCs from three HLA-typed healthy individuals (P05, P29, P33) were isolated. Then, the 14 antigen peptides (SEQ ID NO: 1-14) 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 14 antigen peptides was evaluated by counting the number of IFN-γ positive spots.

[0113] 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 20 spots in at least one PBMC) were produced by all the 14 antigen peptides.

[0114] The above results show that the 14 antigen peptides (SEQ ID NO: 1-14) screened by the application can all induce specific T cell immune responses.

[0115] 6. Flow detection of the activation level of antigen peptide-specific T cells

[0116] PBMCs from one HLA-typed healthy individual were selected for detection. Firstly, the 14 antigen peptides (SEQ ID NO: 1-14) 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 14 antigen peptides can all increase the expression level of 4-1BB of T cells (at least the 4-1BB expression level of 1 kind of T cell is higher than that of the control sample).

[0117] The above results show that the 14 antigen peptides (SEQ ID NO: 1-14) screened by the application can all increase the activation level of antigen peptide-specific T cells.

[0118] (ii) Construction of mRNA sequences of the tandem neoantigen targets:

[0119] 1. The HLA-A*1101 neoantigen targets (SEQ ID NO: 1-14) screened above are modified to obtain extended amino acid sequences (SEQ ID NO: 15-28) with physiological activity. The SEQ ID NO: 15-28 are connected by a linker, and the amino acid sequence of the linker is: GGSGGGGSGG (SEQ ID NO: 29) or GGSLGGGGSG (SEQ ID NO: 30). 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: 31 (Figure 6). Further, without changing the amino acid sequence, the codon is optimized and modified according to the codon bias of E. coli as an mRNA ORF, and the ORF sequence is shown in SEQ ID NO: 32 (Figure 7). The ORF sequence is cloned into a modified pUC57 vector (the plasmid map of which is shown in Figure 8) to synthesize the plasmid. This step is entrusted to GenScript. In this step, pUC57 is selected as the plasmid vector, and the antigen coding sequence and DNA expression elements of the mRNA vaccine are introduced by conventional molecular biology methods.

[0120] 2. The synthesized plasmid containing the tandem antigen sequence is used to generate mRNA in vitro by T7 transcription. The obtained mRNA is composed of five elements: 5' cap (cap structure), 5' UTR (non-coding region), ORF (protein-coding open reading frame), 3' UTR, and 3' poly A tail (polyadenylic acid tail).

[0121] 3. The generated mRNA is modified in vitro, including 3' end modification and 5' end modification; wherein the poly A tail is modified by cytosine (C); and the 5' cap is modified by Cap1m6AG.

[0122] (Three) Lipid nanoparticles (LNP):

[0123] The composition of LNP includes ionizable cationic lipids, phospholipids, and cholesterol.

[0124] Ionizable cationic lipids (ICLs) include DLin-MC3-DMA (MC3), SM-102, and ALC-0315, all of which are monoamine lipids. ALC-0315, MC3, and SM-102 are three ionizable lipids that can be used in humans. They can be assembled in a specific ratio under acidic conditions to form ICLs. Cationic liposomes can bind to negatively charged mRNA through electrostatic interaction and wrapping according to their hydrophobicity, forming mRNA-loaded lipid nanoparticles (LNPs).

[0125] (IV) Preparation of mRNA-lipid nanoparticles (mRNA-LNP) using microfluidic mixing

[0126] 1. Most of the published formulations recommend 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.

[0127] 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 lipid is calculated, so there are 0.5 moles of N in each mole of mixed lipid. The total lipid can be loaded with

[0128] 3. Prepare mRNA-citric acid buffer: 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) solutions each 50 mL using ultrapure water. Take 33.0 mL of citric acid solution and 17.0 mL of sodium citrate solution, mix, then add DEPC (diethypyrocarbonate), let stand for 30 minutes, then remove DEPC under high pressure, sterilize, and then use DEPC water to make up to 100 mL to obtain 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.

[0129] 4. Prepare lipid solution and encapsulate by microfluidic device: this step is entrusted to stemiRNA Therapeutics. The microfluidic device allows the lipid solution and mRNA solution to be mixed thoroughly in the mixer, quickly forming LNP with uniform particle size. 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.

[0130] 5. After preparation, use mRNA-LNP to verify in vitro transfection efficiency, in vivo infection efficiency, and in vivo liver targeting.

[0131] (1) In vitro transfection efficiency verification of mRNA-LNP

[0132] To verify the transfection efficiency, HLA-A*11:01 cell huh7 was transfected with LNP encapsulating eGFP-mRNA, and the results are shown in Figures 9-10: Figure 9, upper left, is the cell morphology observed under 100x light microscope; upper right, is the cell transfection efficiency observed under 100x fluorescence microscope; lower left, is the cell morphology observed under 200x light microscope; lower right, is the cell transfection efficiency observed under 200x fluorescence microscope; it can be seen that the LNP added with eGFP-mRNA can enter the cell and express after 24h, and the green fluorescence is mainly expressed in the cytoplasm. Figure 10 is the flow detection of the proportion of eGFP positive cells, it can be seen that the positive rate of HLA-A*11:01 cell huh7 transfected with LNP added with eGFP-mRNA is more than 90% compared with the negative cells.

[0133] (2) In vivo transfection efficiency verification of mRNA-LNP

[0134] Figure 11 is the live imaging picture of mice after injection of eGFP-mRNA vaccine, and the results show that the mRNA of the application targets and accumulates in the liver after injection in the animal body.

[0135] The above results show that the mRNA of the application has good in vitro and in vivo transfection efficiency, and good targeting.

[0136] 6. Animal in vivo experiment

[0137] Construction of HLA-A*11:01 subcutaneous tumor mouse model: the animal model uses NZGL (NOD-Prkdcem26Cd52 Il2rgem26Cd22 / Gpt-GM-SCF-IL3) mice. Within one week of birth, CD34 + stem cells with appropriate HLA matching (HLA-A*11:01) are selected for injection to reconstitute the mouse immune system. At 8 weeks, peripheral blood is taken for detection, and the detection results are: hCD45>30%, hCD3>25%, and the mouse is qualified. At 10 weeks, the mouse is inoculated with tumor (tumor cell line Huh7 HLA-A*11:01 that meets HLA), and regular detection is performed. After the mouse tumor grows stably, the experiment is performed.

[0138] The HLA-A*11:01 subcutaneous tumor mouse model constructed above is divided into two groups, one group is inoculated with the HLA-A*11:01 mRNA tumor vaccine (mRNA-LNP) constructed by the application, and the other group is inoculated with blank mRNA as a control group (vector group), and the tumor growth of the mouse is detected at 1-28 days after inoculation, and the results of the mouse tumor volume detection are shown in Figure 12. 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.

[0139] Figure 13 is a slice and HE staining of the spleen, brain, lung, kidney, heart and liver of a mouse after the mouse was killed at the observation endpoint after using the A11 type mRNA vaccine of the present application, and a large number of lymphocyte infiltrations in the spleen of the mouse were observed, while no obvious immune infiltration increase was observed in other important physiological organs. This result proves that the A11 type mRNA vaccine has good tumor targeting and no obvious damage to other organs, and has good safety

[0140] Therefore, patients clinically diagnosed as having hepatocellular carcinoma or patients whose postoperative pathology confirms hepatocellular carcinoma after surgical resection, and patients whose peripheral blood or tumor tissue is detected as HLA-A*11:01, or HLA-A11:02, HLA-A11:03, HLA-A11:04, HLA-A11:05, HLA-A11:06, HLA-A11:07, HLA-A11:08, HLA-A11:09, HLA-A11:10, HLA-A11:11, HLA-A11:12, HLA-A11:100, or other HLA types, can be treated using the mRNA vaccine of the present application; the mRNA vaccine of the present application is a lyophilized powder, which is dissolved and administered by injection when used, and the injection methods include but are not limited to subcutaneous injection, intramuscular injection, intravenous injection and intraperitoneal injection. The mRNA vaccine of the present application can be used alone or in combination with PD-1 and / or PD-L1 monoclonal antibodies, or in combination with other anti-tumor drugs.

[0141] The above is only a preferred embodiment of the present application, and is not a limitation on the form and substance 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 also 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: 1-14, or a combination of one or more of polypeptides with amino acid sequences as shown in SEQ ID NO: 15-28.

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: 1-14, or the nucleic acid encodes a combination of polypeptides with amino acid sequences as shown in SEQ ID NO: 15-28.

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: 1-14, or the combination of polypeptides is in series by linkers from polypeptides with amino acid sequences as shown in SEQ ID NO: 15-28. Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 29 or SEQ ID NO:

30.

4. The nucleic acid of claim 2 or 3, wherein The nucleic acid encodes a polypeptide in series from SEQ ID NO: 15-28; Preferably, the nucleic acid encodes a polypeptide as shown in SEQ ID NO: 31 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: 31, and retains the function of the polypeptide as shown in SEQ ID NO:

31.

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: 32 or with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

32.

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: 32 or with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

32.

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*11 type liver cancer; More preferably, the liver cancer is HLA-A*11:01 type liver cancer.

19. The use of claim 18, 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 as shown in SEQ ID NO: 15-SEQ ID NO: 28 connected by a linker into a vector for plasmid synthesis; Step 2: in vitro transcription of the synthesized plasmid containing the tandem antigen sequence to synthesize mRNA; Step 3: in vitro modification of the synthesized mRNA, including 3' end modification and 5' end modification; Step 4: preparation of mRNA-lipid nanoparticles by microfluidic technology; Preferably, the amino acid sequence of the linker in step 1 is as shown in SEQ ID NO: 29 or SEQ ID NO:

30.

21. A method for preventing and / or treating liver cancer, comprising administering to a subject in need thereof 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; Preferably, the liver cancer is HLA-A*11 type liver cancer; More preferably, the liver cancer is HLA-A*11 :01 type liver cancer.

22. The method of claim 21, wherein, The method further comprises administering to the subject in need thereof a second therapeutic agent; Preferably, the second therapeutic agent is selected from immune checkpoint inhibitors 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 further comprises HLA-A11 :02, HLA-A11 :03, HLA-A11 :04, HLA-A11 :05, HLA-A11 :06, HLA-A11 :07, HLA-A11 :08, HLA-A11 :09, HLA-A11 :10, HLA-A11 :11, HLA-A11 :12, HLA-A11 :13, HLA-A11 :14 HLA-A11 :15, HLA-A11 :16, HLA-A11 :17, HLA-A11 :100, HLA-A11 :101, or HLA-A11 :109 type, but not limited to the above-mentioned typing.

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*11:01 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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