Therapeutic nucleic acids for liver cancer

Abstract

The present application provides a liver cancer therapeutic nucleic acid, which comprises at least two independent nucleotide sequences, wherein one of the independent nucleotide sequences is a first nucleotide sequence encoding at least one genetic adjuvant; and at least one of the remaining one or more independent nucleotide sequences encodes at least one liver cancer-associated antigen.

Description

Technical Field

[0001] This invention relates to the field of gene therapy technology, and in particular to therapeutic nucleic acids for liver cancer. Background Technology

[0002] Since the smallpox vaccination experiment, more and more new vaccines and new ways of using vaccines have been developed. In addition to preventive vaccines, therapeutic vaccines have also been proposed and developed.

[0003] Therapeutic vaccines carry and express specific endogenous or exogenous antigens, primarily inducing or enhancing cell-mediated immunity by stimulating cytotoxic T cells, or inducing humoral immunity by activating B cells to produce specific antibodies. Among therapeutic vaccines, therapeutic cancer vaccines are a hot research area. These vaccines aim to enhance the patient's own immune system response and help the body recognize and attack existing tumor cells. Therapeutic cancer vaccines can work in various ways, such as promoting T-cell immune responses, activating antibody responses, or enhancing the function of immune cells. Currently, therapeutic cancer vaccines targeting various cancers are still in the experimental stage.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] In a first aspect, the present invention provides a therapeutic nucleic acid for liver cancer, characterized in that the therapeutic nucleic acid for liver cancer comprises at least two independent nucleotide sequences, wherein one independent nucleotide sequence is a first nucleotide sequence, the first nucleotide sequence encoding at least one genetic adjuvant; and the remaining one or more independent nucleotide sequences encode at least one liver cancer-related antigen.

[0006] In some embodiments, the genetic adjuvant is selected from at least one of STING, cGAS, Flagellin16a, Flagellin, LTB, and TLR4.

[0007] In some embodiments, the STING is wild-type STING or an artificial STING mutant; the amino acid sequence of the wild-type STING is shown in SEQ ID NO.67; the amino acid sequence of the artificial STING mutant is based on wild-type STING and includes one or more mutations selected from V147L, N154S, V155M, R284M, R284K, R284T, E315Q, R375A, or a combination thereof.

[0008] In some embodiments, the amino acid sequence of cGAS is shown in SEQ ID NO. 69; the amino acid sequence of Flagellin16a is shown in SEQ ID NO. 71; the amino acid sequence of Flagellin is shown in SEQ ID NO. 73; the amino acid sequence of TLR4 is shown in SEQ ID NO. 75; and the amino acid sequence of LTB is shown in Uniprot P32890.

[0009] In some embodiments, the therapeutic nucleic acid for liver cancer encodes at least one liver cancer-associated antigen selected from at least one of AFP, GPC3, HBsAg, HBcAg, NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1, MAGE-C2, CLDN6, hTERT, Survivin, TPTE, PSA, KK-LC-1, KRAS, EGFR, P53, CEA, Tyrosinase, gp100, Melan-A, and HER2.

[0010] In some implementations, the therapeutic nucleic acid for liver cancer encodes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more liver cancer-associated antigens.

[0011] In some embodiments, the therapeutic nucleic acid for liver cancer encodes multiple liver cancer-associated antigens, which are derived from multiple antigen proteins; in some embodiments, the liver cancer-associated antigens are selected from at least one of AFP, GPC3, HBsAg, and HBcAg.

[0012] In some embodiments, the therapeutic nucleic acid for liver cancer encodes at least one co-stimulatory molecule selected from at least one member of the B7 family and the tumor necrosis factor family.

[0013] In some embodiments, the co-stimulatory molecule is CD80; the CD80 comprises the amino acid sequence shown in SEQ ID NO.11 or SEQ ID NO.12 or a functional fragment thereof, or an extracellular domain thereof; the functional fragment may comprise an IgV domain and / or an IgC domain; in some embodiments, the CD80 further comprises the amino acid sequence shown in SEQ ID NO.17, SEQ ID NO.18, or SEQ ID NO.19 or a functional fragment thereof, or a transmembrane domain thereof.

[0014] In some embodiments, the co-stimulatory molecule further includes CD70; the CD70 comprises the amino acid sequence shown in SEQ ID NO.22 or SEQ ID NO.23 or a functional fragment thereof, or an extracellular domain thereof; in some embodiments, the CD70 further comprises the amino acid sequence shown in SEQ ID NO.19 or a functional fragment thereof, or a transmembrane domain thereof.

[0015] In some implementations, the co-stimulatory molecule encoded by the hepatocellular carcinoma therapeutic nucleic acid also includes 4-1BBL.

[0016] In some embodiments, the co-stimulatory molecule amino acid sequence encoded by the hepatocellular carcinoma therapeutic nucleic acid exists in a linked or unlinked form with the hepatocellular carcinoma-associated antigen amino acid sequence.

[0017] In some embodiments, the unconnected form is selected from at least one of the following: (1) the coding sequence is located in two separate nucleotide sequences; (2) the coding sequence is located in the same nucleotide sequence but in different ORFs; (3) the coding sequences are located in the same ORF and are connected by nucleotide sequences encoding cleavable linkers (such as 2A self-cleaving peptides).

[0018] In some embodiments, the connection is selected from at least one of the following: (4) the C-terminus of the co-stimulatory molecule amino acid sequence is directly connected to the N-terminus of the antigen amino acid sequence; (5) the two are connected by a GS linker peptide.

[0019] In some embodiments, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide, and its amino acid sequence is shown in SEQ ID NO. 8, 7, 9 or 10 respectively.

[0020] In some implementations, the GS sequence is selected from (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, where n is an integer from 1 to 20, m is an integer from 1 to 20, o is an integer from 1 to 5, p is an integer from 1 to 40, and q is an integer from 1 to 5.

[0021] In some embodiments, the total mass ratio of the first nucleotide sequence to the remaining one or more independent nucleotide sequences is (10~1):(1~10); in some embodiments, the therapeutic nucleic acid for liver cancer is a DNA molecule or an RNA molecule.

[0022] In a second aspect, the present invention provides a protein composition comprising a polypeptide encoded by a therapeutic nucleic acid for liver cancer as described in any of the preceding claims.

[0023] Thirdly, the present invention provides a vaccine comprising a liver cancer therapeutic nucleic acid as described in any of the preceding claims, or a protein composition as described above.

[0024] Fourthly, the present invention provides a method for preparing the vaccine as described above, the method comprising: mixing a liver cancer therapeutic nucleic acid as described in any one of the preceding methods with a delivery formulation; wherein the delivery formulation is selected from lipid nanoparticles or cationic liposomes, thereby obtaining the vaccine.

[0025] In some embodiments, the preparation method includes: mixing each independent nucleotide sequence with a delivery formulation, and then mixing the delivery formulation encapsulating the first nucleotide sequence with the delivery formulation encapsulating the remaining one or more independent nucleotide sequences at a mass ratio of (10~1):(1~10) to obtain the vaccine.

[0026] Fifthly, the present invention provides a biomaterial comprising any one of an expression cassette, a vector, engineered bacteria, or a cell line, wherein the biomaterial contains or expresses RNA of a liver cancer therapeutic nucleic acid as described in any of the preceding claims. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 The changes in average tumor volume in mice of each experimental group in Example 2 are shown.

[0029] Figure 2 The changes in average tumor volume in mice of each experimental group in Example 3 are shown.

[0030] Figure 3 The changes in average tumor volume in mice of each experimental group in Example 4 are shown. Detailed Implementation

[0031] Although the invention is described in more detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein. It should also be understood that the terminology used herein is for the purpose of describing some specific embodiments only and is not intended to limit the scope of the invention, which is to be limited only by the appended claims.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The elements of the present invention will be described in more detail below. These elements are listed by way of specific embodiments; however, it should be understood that they can be combined in any manner and in any number to form further embodiments.

[0033] The numerous described embodiments and preferred embodiments should not be construed as limiting the scope of the invention to the explicitly described embodiments. This specification should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as disclosed in this specification.

[0034] Unless otherwise specified herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “for example”) provided herein is intended only to better illustrate the disclosure of this invention and does not constitute a limitation on the scope of the additionally claimed disclosure. The language used in this specification should not be construed as indicating any unclaimed element necessary to practice the disclosure of this invention.

[0035] When referring to an amino acid sequence (peptide or protein), a “fragment” refers to a portion of the amino acid sequence, specifically a sequence shortened at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame (OPF) lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated OPF lacking the 5' end, provided the truncated OPF contains a start codon for initiating translation. The amino acid sequence fragment comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. The amino acid sequence fragment preferably comprises at least 6, particularly at least 8, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0036] According to the disclosure of this invention, a portion or fragment of a peptide or protein preferably possesses at least one functional property of the peptide or protein from which it originates. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective binding to nucleic acids. For example, a pharmacologically active fragment of a peptide or protein possesses at least one pharmacological activity of the peptide or protein from which the fragment originates. A portion or fragment of a peptide or protein preferably comprises a sequence of at least six, particularly at least eight, at least ten, at least twelve, at least fifteen, at least twenty, at least thirty, or at least fifty consecutive amino acids. A portion or fragment of a peptide or protein preferably comprises a sequence of up to eight, particularly up to ten, up to twelve, up to fifteen, up to twenty, up to thirty, or up to fifty-five consecutive amino acids.

[0037] Those skilled in the art will understand that both uracil and thymine can be represented by 't', rather than uracil by 'u' and thymine by 't'; in the case of ribonucleic acid, it should be understood that unless otherwise specified, 't' is used to represent uracil.

[0038] 1. Therapeutic nucleic acids for liver cancer

[0039] In one aspect, this article provides therapeutic nucleic acid molecules for the control and treatment of liver cancer. In some embodiments, the liver cancer therapeutic nucleic acid encodes a peptide or polypeptide, which, when administered to a subject in need, is expressed by cells in the subject to produce the encoded peptide or polypeptide.

[0040] In some embodiments, the therapeutic nucleic acid for liver cancer is a DNA molecule. In other embodiments, the therapeutic nucleic acid for liver cancer is an RNA molecule. In specific embodiments, the therapeutic nucleic acid for liver cancer is an mRNA molecule or circular RNA.

[0041] In a specific embodiment, based on the provided mRNA sequence, those skilled in the art can obtain the corresponding circular RNA sequence and, in accordance with the content of publications such as CN202180048567.4, prepare a complete circular RNA sequence encoding the same amino acid sequence. In an optional embodiment, based on the provided mRNA sequence, those skilled in the art can obtain the corresponding DNA sequence (e.g., uracil to thymine). Similarly, based on the provided DNA sequence, those skilled in the art can obtain the corresponding RNA sequence (e.g., thymine to uracil). In an optional embodiment, based on the provided RNA or DNA sequence, those skilled in the art can obtain the corresponding amino acid sequence.

[0042] In a particular embodiment, the mRNA described in this invention consists of a sequence comprising a 5' cap, a 5' UTR, an ORF, a 3' UTR, and a 3' poly(A) tail, sequentially from the 5' end to the 3' end.

[0043] In a particular implementation, the 5' end cap is selected from ARCA, mCAP, dmCAP, m7G(5')ppp(5')(2'OMeA)pG, tmCAP, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG; in an optional implementation, the 5' end cap is m7Gppp(5')(2'-OMeA)pG.

[0044] In a particular embodiment, the length of the 5'UTR is preferably 10 to 200 nucleotides. In a particular embodiment, the length of the 5'UTR is 15 to 100 nucleotides. In a particular embodiment, the 5'UTR nucleotide sequence is as shown in SEQ ID NO. 1 to 3. In a particular embodiment, the 5'UTR nucleotide sequence is as shown in SEQ ID NO. 1.

[0045] In a particular embodiment, the 3'UTR sequence is as shown in SEQ ID NO.4-6. In an alternative embodiment, the 3'UTR sequence is as shown in SEQ ID NO.4.

[0046] In a particular embodiment, one or more uridines in the mRNA are replaced with a modified nucleoside. In a particular embodiment, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U). In an optional embodiment, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0047] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein comprises at least one coding region. In some embodiments, the coding region is an open reading frame (ORF) encoding a single peptide or protein. In some embodiments, the coding region comprises at least two ORFs, each encoding a peptide or protein. In embodiments where the coding region comprises more than one ORF, the encoded peptides and / or proteins may be the same as or different from each other. In some embodiments, multiple ORFs in the coding region are separated by non-coding sequences. In a specific embodiment, the non-coding sequence separating two ORFs comprises an internal ribosome entry site (IRES).

[0048] The internal ribosome entry site (IRES) described in this invention refers to a special nucleotide sequence present in certain mRNA molecules. The internal ribosome entry site (IRES) enables ribosomes to initiate the translation process directly at the internal site without relying on the 5' cap structure of the mRNA, thereby allowing one mRNA molecule to be translated into multiple different proteins, playing an important role in the gene expression regulation of eukaryotes and some viruses.

[0049] In a specific embodiment, the mRNA described in this invention comprises at least one ORF sequence. For example, the mRNA comprises two ORF sequences, which are sequentially arranged between the 5'UTR and the 3'UTR; specifically, the mRNA consists of a sequence comprising a 5' cap, a 5'UTR, an ORF(1), an ORF(2), a 3'UTR, and a 3' poly(A) tail from the 5' end to the 3' end. For example, the mRNA comprises n ORF sequences (n is a natural number), which are sequentially arranged between the 5'UTR and the 3'UTR; specifically, the mRNA consists of a sequence comprising a 5' cap, a 5'UTR, an ORF(1), an ORF(2), ..., an ORF(n-1), an ORF(n), a 3'UTR, and a 3' poly(A) tail from the 5' end to the 3' end. For example, the mRNA contains two ORF sequences, which are located in two independent nucleotide sequences. Specifically, the mRNA includes two independent (unconnected) nucleotide sequences, denoted as mRNA1 and mRNA2. mRNA1 consists of a sequence from the 5' end to the 3' end, which includes a 5' cap, 5' UTR, ORF(1), 3' UTR, and a 3' poly(A) tail. mRNA2 consists of a sequence from the 5' end to the 3' end, which includes a 5' cap, 5' UTR, ORF(2), 3' UTR, and a 3' poly(A) tail. Among the several independent nucleotide sequences, the 5' cap, 5' UTR, 3' UTR, and 3' poly(A) tail can be the same or different from each other.

[0050] In specific implementations, nucleotide molecules can be sequence-optimized to enhance mRNA sequences and improve properties related to expression efficacy after in vivo administration. These improvements include: increasing mRNA stability; enhancing translational efficiency in target tissues; reducing the number of truncated proteins expressed; improving protein folding or preventing misfolding; reducing the toxicity of the expression product; reducing cell death induced by the expression product; and increasing and / or decreasing protein aggregation, resulting in mRNAs with improved properties. Sequence optimization also aims to: optimize the formulation and delivery characteristics of nucleotide-based therapeutics while maintaining structural and functional integrity; overcome expression thresholds; increase expression rates; improve half-life and / or protein concentration; optimize protein localization; and avoid adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization techniques include: (1) codon optimization based on codon frequencies in specific organs and / or host organisms to ensure proper folding and expression; (2) adjusting G / C content to increase mRNA stability or reduce secondary structures; (3) minimizing tandem repeat codons or base runs that may impair gene construction or expression; (4) customizing transcription and translation control regions; and (5) reducing or eliminating problematic secondary structures within polynucleotides.

[0051] In some embodiments, the liver cancer therapeutic nucleic acid of this disclosure comprises at least two independent nucleotide sequences, wherein one independent nucleotide sequence is a first nucleotide sequence encoding at least one genetic adjuvant; and at least one of the remaining one or more independent nucleotide sequences encodes at least one liver cancer-associated antigen.

[0052] Encoding at least one liver cancer-associated antigen and at least one genetic adjuvant, said genetic adjuvant being selected from STING, cGAS, Flagellin16a, Flagellin, LTB, and TLR4.

[0053] In some implementations, the STING is wild-type STING or an artificial STING mutant.

[0054] In some embodiments, the amino acid sequence of the wild-type STING is shown in SEQ ID NO.67.

[0055] The amino acid sequence of the artificial STING mutant is based on wild-type STING and includes one or more mutations selected from V147L, N154S, V155M, R284M, R284K, R284T, E315Q, R375A, or combinations thereof.

[0056] In some embodiments, the amino acid sequence of STING undergoes a V155M mutation, and the nucleotide sequence encoding the V155M mutated STING is shown in SEQ ID NO. 68.

[0057] In some embodiments, the amino acid sequence of cGAS is shown in SEQ ID NO. 69. In some embodiments, the nucleotide sequence of cGAS is shown in SEQ ID NO. 70.

[0058] In some embodiments, the amino acid sequence of Flagellin16a is shown in SEQ ID NO.71. In some embodiments, the nucleotide sequence of Flagellin16a is shown in SEQ ID NO.72.

[0059] In some embodiments, the amino acid sequence of Flagellin is shown in SEQ ID NO. 73. In some embodiments, the nucleotide sequence of Flagellin is shown in SEQ ID NO. 74.

[0060] In some embodiments, the amino acid sequence of TLR4 is shown in SEQ ID NO. 75. In some embodiments, the amino acid sequence of LTB is shown in Uniprot P32890.

[0061] In some embodiments, the total mass ratio of the first nucleotide sequence to the remaining one or more independent nucleotide sequences is (10~1):(1~10).

[0062] In some embodiments, the total mass ratio of the first nucleotide sequence to the remaining one or more independent nucleotide sequences is (10~1):(1~10). In some embodiments, the total mass ratio of the first nucleotide sequence to the remaining one or more independent nucleotide sequences is one of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0063] According to the disclosure of this invention, "antigen" encompasses any substance or molecular structure that can bind to antibodies or T-cell receptors. The presence of an antigen in the body can elicit an immune response. Therefore, "antigen" encompasses any substance targeted by an immune response or immune mechanism. This also includes cases where the antigen is processed into an antigenic peptide, and the immune response or immune mechanism targets one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, "antigen" refers to any substance that specifically reacts with antibodies or T lymphocytes (T cells), preferably peptides or proteins. According to the disclosure of this invention, the term "antigen" includes any molecule containing at least one epitope (e.g., a T-cell epitope). Preferably, in the context of the disclosure of this invention, an antigen is a molecule that optionally induces an immune response after processing, which preferably specifically targets the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, or an epitope derived from such an antigen.

[0064] The tumor-associated antigens described in this invention refer to antigen molecules that are highly expressed in tumor cells, including differentiation-associated antigens (DAA), overexpressed antigens, and cancer-testis antigens. For example, DDAA includes alpha-fetoprotein (AFP), which is highly expressed in liver cancer; carcinoembryonic antigen (CEA), which is highly expressed in colorectal cancer; and melanocyte antigen 1 (MART-1 / Melan-A), which is highly expressed in melanoma. As further examples, overexpressed antigens, such as human epidermal growth factor receptor (EGFR), are highly expressed in non-small cell lung cancer, breast cancer, and pancreatic cancer; human epidermal growth factor receptor-2 (HER2) is highly expressed in breast cancer, gastric cancer, colorectal cancer, and bladder cancer; and mucin 1 (MUC1) is highly expressed in epidermal-associated cancers such as breast cancer and ovarian cancer. For example, cancer testis antigens include human esophageal squamous cell carcinoma antigen 1 (NY-ESO-1), melanoma-associated antigen 4 (MAGE-A4), and preferentially expressed antigen in melanoma (PRAME), which are highly expressed in melanoma.

[0065] Tumor-associated antigens are "common" antigens, widely expressed in a large patient population and across a variety of cancer types.

[0066] Therefore, compositions / drugs / vaccines / mixtures containing tumor-associated antigens can be used as off-the-shelf therapies for antigen-positive patient populations (typically a subgroup of patients with a specific tumor type), and are therefore versatile.

[0067] In this invention, off-the-shelf therapy refers to standardized treatment products that are pre-prepared and ready to be used to treat patients at any time. In contrast to personalized therapy, it eliminates the need for individual collection and preparation of cells or drugs for each patient. Off-the-shelf therapy is similar to "on-demand" goods in clinical practice, which can be quickly obtained from inventory and applied to patients, significantly shortening the time interval between deciding on treatment and starting treatment.

[0068] In some embodiments, the liver cancer-associated antigen is selected from at least one of AFP, GPC3, HBsAg, HBcAg, NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1, MAGE-C2, CLDN6, hTERT, Survivin, TPTE, PSA, KK-LC-1, KRAS, EGFR, P53, CEA, Tyrosinase, gp100, Melan-A, and HER2.

[0069] In this invention, the liver cancer-related antigen can be a full-length protein / peptide of the aforementioned antigens (at least one of AFP, GPC3, HBsAg, HBcAg, NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1, MAGE-C2, CLDN6, hTERT, Survivin, TPTE, PSA, KK-LC-1, KRAS, EGFR, P53, CEA, Tyrosinase, gp100, Melan-A, HER2), or a truncated version of the aforementioned antigens, or a fusion protein of the aforementioned antigens, or a mutant protein of the aforementioned antigens, or any other immunogenic fragment (including but not limited to polypeptides, peptides, proteins, RNA, DNA, etc.) that can specifically react with antibodies or T lymphocytes (T cells). For example, the liver cancer-associated antigen is P53. According to those skilled in the art, the wild-type protein amino acid sequence of P53 is Uniprot P04637. The amino acid sequence of the liver cancer-associated antigen P53 should have at least one of the following amino acid residue mutations: R175, R213, G245, R248, R273, or R282. It should be understood that, taking "R175" as an example, "R" means that position "175" of the P53 wild-type protein amino acid sequence (UniprotP04637) is an arginine (Arg) residue.

[0070] The amino acid sequences of the liver cancer-related antigens NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1, MAGE-C2, CLDN6, hTERT, Survivin, TPTE, PSA, KK-LC-1, CEA, Tyrosinase, gp100, Melan-A, and HER2 are shown in Table 1.

[0071] Table 1: Amino acid sequences of some liver cancer-related antigens

[0072]

[0073] In this invention, "Uniprot" is the abbreviation for Universal Protein. The characters in the "Uniprot" column of Table 1 are the protein sequence numbers in the protein database (https: / / www.uniprot.org / ), corresponding to unique protein sequences in that database.

[0074] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 liver cancer-associated antigens. In a particular embodiment, the therapeutic nucleic acid for liver cancer of this disclosure encodes multiple liver cancer-associated antigens, which are derived from different immunogenic fragments of the same antigen protein (at least one of AFP, GPC3, HBsAg, HBcAg, NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1, MAGE-C2, CLDN6, hTERT, Survivin, TPTE, PSA, KK-LC-1, KRAS, EGFR, P53, CEA, Tyrosinase, gp100, Melan-A, HER2). In a particular embodiment, the therapeutic nucleic acid for liver cancer of this disclosure encodes multiple liver cancer-associated antigens, which are derived from multiple antigen proteins.

[0075] In some embodiments, the liver cancer-associated antigen is selected from at least one of AFP, GPC3, HBsAg, and HBcAg. The amino acid sequences (or immunogenic fragment amino acid sequences) of the liver cancer-associated antigen AFP, the liver cancer-associated antigen GPC3, the liver cancer-associated antigen HBsAg, and the liver cancer-associated antigen HBcAg are shown in Table 2.

[0076] Table 2: Amino acid and nucleotide sequences of some liver cancer-related antigens

[0077]

[0078] Note: The “Serial Number” in Table 2 comes from the sequence list of this invention and can be abbreviated as (SEQ ID NO.).

[0079] In some implementations, the liver cancer-associated antigen is GPC3. This can be understood as either wild-type GPC3 or engineered GPC3.

[0080] In this invention, "engineering" refers to the modification or design and synthesis of natural macromolecules with specific functions and properties through technologies such as genetic engineering and protein engineering.

[0081] In some embodiments, one of the liver cancer-associated antigens may be a repeating unit. In a particular embodiment, the liver cancer-associated antigen is GPC3(1), wherein GPC3(1) is a repeating unit. For example, a liver cancer therapeutic nucleic acid encodes a two-unit GPC3(1). As another example, a liver cancer therapeutic nucleic acid encodes a three-unit GPC3(1).

[0082] In some implementations, the liver cancer-associated antigens are GPC3(1) and GPC3(2).

[0083] In some implementations, the liver cancer-associated antigens are GPC3(3), GPC3(4), GPC3(5), and GPC3(6). In a specific embodiment, the nucleotide sequences encoding GPC3(3), GPC3(4), GPC3(5), and GPC3(6) are arranged in a repeating pattern; for example, the connection between functional regions in the liver cancer-associated antigen encoded by the liver cancer therapeutic nucleic acid is: GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6); for another example, the connection between functional regions in the liver cancer-associated antigen encoded by the liver cancer therapeutic nucleic acid is: GPC3(3)-GPC3(3)-GPC3(4)-GPC3(4)-GPC3(5)-GPC3(5)-GPC3(6)-GPC3(6); for yet another example, the connection between functional regions in the liver cancer-associated antigen encoded by the liver cancer therapeutic nucleic acid also includes other forms, which are those that can be obtained without objection by those skilled in the art.

[0084] In this invention, "the connection form between each functional region" describes the sequence of each functional region from the N-terminus to the C-terminus of the polypeptide expressed by engineered nucleotides; for functional regions connected by "-", not only are the nucleotide sequences encoding them connected, but the amino acid sequences are also connected; for functional regions connected by "...", the nucleotide sequences encoding them are connected, but the amino acid sequences are not connected.

[0085] In some implementations, the liver cancer-associated antigen is GPC3 (7).

[0086] In some implementations, the liver cancer-associated antigen is GPC3(8).

[0087] In some implementations, the liver cancer-associated antigen is GPC3 (9).

[0088] In some implementations, the liver cancer-associated antigen is GPC3 (10).

[0089] In some implementations, the liver cancer-associated antigen is GPC3 (11) to (17).

[0090] In some implementations, the liver cancer-associated antigen is AFP. This can be understood as either wild-type AFP or engineered AFP.

[0091] In some implementations, the liver cancer-associated antigen is HBsAg. This can be understood as either wild-type HBsAg or engineered HBsAg.

[0092] In some implementations, the liver cancer-associated antigen is HBcAg. This can be understood as either wild-type HBcAg or engineered HBcAg.

[0093] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen. In a particular embodiment, the liver cancer-associated antigen is directly linked in sequence.

[0094] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen. In a particular embodiment, at least two of the liver cancer-associated antigens are linked end-to-end by a REKR sequence. The amino acid sequence of the REKR sequence is shown in SEQ ID NO. 65.

[0095] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen. In a particular embodiment, the liver cancer-associated antigen may be partially directly linked end-to-end, and at least two of the liver cancer-associated antigens may be linked end-to-end via a REKR sequence.

[0096] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen. In a particular embodiment, a Flag tag sequence is attached to the N-terminus or C-terminus of the amino acid sequence of at least one of said liver cancer-associated antigens. The amino acid sequence of said Flag tag sequence is shown in SEQ ID NO. 66.

[0097] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein also encodes at least one co-stimulatory molecule.

[0098] "Co-stimulatory molecules" are cell surface molecules and their ligands involved in co-stimulatory signal transduction, providing the necessary co-stimulatory signals for the complete activation of T (or B) cells. Co-stimulatory molecules include members of the B7 family and the tumor necrosis factor family, among others. The main biological functions of co-stimulatory molecules include enhancing T cell activation and immune responses, and they also participate in the regulation of tumor immune responses. For example, the binding of the B7 family member CD80 to CD28 on T cells produces a positive signal, thereby enhancing the immune response; while the binding of CTLA-4 to CD80 produces a negative signal, blocking T cell activation induced by CD28 and downregulating the immune response. Other examples of B7 family members include CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2. For example, members of the tumor necrosis factor family include TNF-β, TNF-α, LT-β, ​​CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, GITRL, etc.

[0099] In some embodiments, the co-stimulatory molecule is selected from at least one member of the tumor necrosis factor family and the B7 family. For example, the co-stimulatory molecule is selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2; another example is that the co-stimulatory molecule includes at least CD80.

[0100] The natural / wild-nature costimulatory molecule CD80 is a transmembrane polypeptide structure comprising an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain of natural / wild-nature CD80 is the binding site for receptors CD28 or CTLA-4, participating in signal transduction during immune regulation. The transmembrane domain of natural / wild-nature CD80, composed of hydrophobic amino acids, is embedded in the cell membrane, ensuring stable localization of the natural / wild-nature CD80 protein and supporting protein localization and stability. The intracellular domain of natural / wild-nature CD80 contains several signal transduction-related amino acid sequences, potentially interacting with intracellular signal transduction pathways and regulating T cell activation.

[0101] The extracellular domain of natural / wild CD80 is further subdivided into two domains, named the IgV domain and the IgC domain, which are believed to play important roles in the binding of CD80 to receptors CD28 or CTLA-4 (Both Extracellular Immunoglobin-like Domains of CD80 Contain Residues Critical for Binding T Cell Surface Receptors CTLA-4 and CD28, Peach, Robert J. et al. Journal of Biological Chemistry, Volume 270, Issue 36, 21181-21187). The IgV domain is located at positions 35-135 of the CD80 extracellular domain, and the IgC domain is located at positions 145-230 of the CD80 extracellular domain.

[0102] In some embodiments, the costimulatory molecule CD80 can be a wild-type costimulatory molecule CD80, whose amino acid sequence is identical to that of any natural / wild-type costimulatory molecule CD80. In optional embodiments, the costimulatory molecule CD80 can be an engineered costimulatory molecule CD80.

[0103] "Engineering co-stimulatory molecule CD80" refers to a CD80 molecule that is modified from a natural / wild-type CD80 molecule or designed and constructed using its principles to obtain specific functions or improved properties. In optional embodiments, the engineered co-stimulatory molecule CD80 of this invention is obtained by performing sequence deletion or sequence mutation on a natural / wild-type CD80 molecule. In optional embodiments, the engineered co-stimulatory molecule CD80 is a truncated version of the natural / wild-type CD80 molecule; for example, the engineered co-stimulatory molecule CD80 contains only the extracellular domain and transmembrane domain of the natural / wild-type CD80; or, for another example, the engineered co-stimulatory molecule CD80 contains only the extracellular domain and transmembrane domain of the natural / wild-type CD80.

[0104] In an optional embodiment, the engineered co-stimulatory molecule CD80 of the present invention is a CD80 fusion protein constructed by fusing the extracellular domain of CD80 with other molecules. In an optional embodiment, the CD80 extracellular domain of the CD80 fusion protein can be the full-length or truncated version of the extracellular domain of natural / wild-type CD80 (e.g., the CD80 extracellular domain of the CD80 fusion protein contains only the IgV and IgC domains); in an optional embodiment, the CD80 extracellular domain of the CD80 fusion protein is designed based on the extracellular domain of natural / wild-type CD80 through site mutation. In an optional embodiment, the CD80 fusion protein is obtained by fusing the extracellular domain of CD80 with the intracellular domain of 4-1BB.

[0105] In optional embodiments, the CD80 comprises the amino acid sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12, or a functional fragment thereof, or an extracellular domain composed of therefrom. For example, the CD80 comprises an extracellular domain of the amino acid sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12. For example, the CD80 comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO. 12, the functional fragment comprising an IgV domain and an IgC domain. For example, the CD80 comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO. 12 and a functional fragment thereof, the functional fragment comprising an IgV domain and an IgC domain. For example, the CD80 comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO. 12 and a functional fragment thereof, the functional fragment comprising an IgV domain. For example, CD80 contains an extracellular domain composed of functional fragments of the amino acid sequences shown in SEQ ID NO.12 and SEQ ID NO.162, wherein the functional fragments contain an IgC domain.

[0106] In an optional embodiment, the CD80 further comprises an amino acid sequence or a functional fragment thereof as shown in SEQ ID NO.17, SEQ ID NO.18, or SEQ ID NO.19, or a transmembrane domain thereof.

[0107] In optional embodiments, the co-stimulatory molecule may be selected from at least one member of the tumor necrosis factor family and the B7 family. In optional embodiments, the co-stimulatory molecule may further include CD70. In optional embodiments, the CD70 comprises the amino acid sequence shown in SEQ ID NO. 22 or SEQ ID NO. 23, or a functional fragment thereof, or an extracellular domain composed of therefrom. For example, the CD70 of the present invention comprises an extracellular domain of the amino acid sequence shown in SEQ ID NO. 22 or SEQ ID NO. 23. As another example, the CD70 of the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO. 22. As another example, the CD70 of the present invention comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO. 22 and a functional fragment of the amino acid sequence shown in SEQ ID NO. 22. As another example, the CD70 of the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO. 23.

[0108] In an optional embodiment, the CD70 of the present invention further comprises an amino acid sequence as shown in SEQ ID NO.19 or a functional fragment thereof, or a transmembrane domain thereof.

[0109] In an optional embodiment, the co-stimulatory molecule may further include 4-1BBL. In an optional embodiment, the 4-1BBL comprises, or is composed of, the amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 14, or a functional fragment thereof. For example, the 4-1BBL of the present invention comprises an extracellular domain of the amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 14. As another example, the 4-1BBL of the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO. 13. As another example, the 4-1BBL of the present invention comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO. 13 and a functional fragment of the amino acid sequence shown in SEQ ID NO. 13. As yet another example, the 4-1BBL of the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO. 14.

[0110] "Engineering co-stimulatory molecules CD70 or 4-1BBL" are CD70 or 4-1BBL molecules that are modified from natural / wild CD70 or 4-1BBL molecules or designed and constructed using their principles to obtain specific functions or improved properties. For specific forms of engineered co-stimulatory molecules CD70 or 4-1BBL, please refer to engineered co-stimulatory molecules CD80.

[0111] In an optional embodiment, the 4-1BBL of the present invention further comprises an amino acid sequence or a functional fragment thereof as shown in SEQ ID NO.19, or a transmembrane domain thereof.

[0112] In one embodiment, in the co-stimulatory molecule polypeptide / amino acid encoded by the hepatocellular carcinoma therapeutic nucleic acid, the co-stimulatory molecule amino acid sequence may or may not be linked to the hepatocellular carcinoma-associated antigen amino acid sequence.

[0113] For example, the amino acid sequence of the co-stimulatory molecule is not linked to the amino acid sequence of the liver cancer-associated antigen. In an optional embodiment, the nucleotide sequence encoding the co-stimulatory molecule and the nucleotide sequence encoding the liver cancer-associated antigen are located in two separate, unconnected nucleotide sequences. In an optional embodiment, the nucleotide sequence encoding the co-stimulatory molecule and the nucleotide sequence encoding the liver cancer-associated antigen are located in the same nucleotide sequence, but in different open reading frames (ORFs). In an optional embodiment, the nucleotide sequence encoding the co-stimulatory molecule and the nucleotide sequence encoding the liver cancer-associated antigen are linked by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located in the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides, specifically, the cleavable linker is selected from T2A peptide, P2A peptide, E2A peptide, or F2A peptide.

[0114] For example, the co-stimulatory molecule amino acid sequence is linked to the liver cancer-associated antigen amino acid sequence. In an optional embodiment, the C-terminus of the co-stimulatory molecule amino acid sequence is directly linked to the N-terminus of the liver cancer-associated antigen amino acid sequence. In an optional embodiment, the C-terminus of the co-stimulatory molecule amino acid sequence and the N-terminus of the liver cancer-associated antigen amino acid sequence are linked by a GS linking peptide.

[0115] In one embodiment, the nucleotide sequence encoding CD80 and the nucleotide sequence encoding the hepatocellular carcinoma-associated antigen (HCC-Associated Antigen) are located in two separate, unconnected nucleotide sequences, and the CD80 amino acid sequence is not connected to the HCC-Associated Antigen amino acid sequence. In another embodiment, the nucleotide sequence encoding CD80 and the nucleotide sequence encoding the HCC-Associated Antigen are located in the same nucleotide sequence but in different open reading frames (ORFs), and the CD80 amino acid sequence is not connected to the HCC-Associated Antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding CD80 and the nucleotide sequence encoding the HCC-Associated Antigen are connected by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located in the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides. In one embodiment, the C-terminus of the CD80 amino acid sequence is directly connected to the N-terminus of the HCC-Associated Antigen amino acid sequence. In one embodiment, the C-terminus of the CD80 amino acid sequence is connected to the N-terminus of the HCC-Associated Antigen amino acid sequence by a GS linker polypeptide.

[0116] In one embodiment, the nucleotide sequence encoding CD70 and the nucleotide sequence encoding the hepatocellular carcinoma-associated antigen (HCC-Associated Antigen) are located in two separate, unconnected nucleotide sequences, and the CD70 amino acid sequence is not connected to the HCC-Associated Antigen amino acid sequence. In another embodiment, the nucleotide sequence encoding CD70 and the nucleotide sequence encoding the HCC-Associated Antigen are located in the same nucleotide sequence but in different open reading frames (ORFs), and the CD70 amino acid sequence is not connected to the HCC-Associated Antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding CD70 and the nucleotide sequence encoding the HCC-Associated Antigen are connected by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located in the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides. In one embodiment, the C-terminus of the CD70 amino acid sequence is directly connected to the N-terminus of the HCC-Associated Antigen amino acid sequence. In one embodiment, the C-terminus of the CD70 amino acid sequence is connected to the N-terminus of the HCC-Associated Antigen amino acid sequence by a GS linker polypeptide.

[0117] In one embodiment, the nucleotide sequence encoding the 4-1BBL and the nucleotide sequence encoding the hepatocellular carcinoma-associated antigen (HCC-Associated Antigen) are located in two separate, unconnected nucleotide sequences, and the 4-1BBL amino acid sequence is not connected to the HCC-Associated Antigen amino acid sequence. In another embodiment, the nucleotide sequence encoding the 4-1BBL and the nucleotide sequence encoding the HCC-Associated Antigen are located in the same nucleotide sequence but in different open reading frames (ORFs), and the 4-1BBL amino acid sequence is not connected to the HCC-Associated Antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding the 4-1BBL and the nucleotide sequence encoding the HCC-Associated Antigen are connected by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located in the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides. In one embodiment, the C-terminus of the 4-1BBL amino acid sequence is directly connected to the N-terminus of the HCC-Associated Antigen amino acid sequence. In one embodiment, the C-terminus of the 4-1BBL amino acid sequence is connected to the N-terminus of the HCC-Associated Antigen amino acid sequence by a GS linker polypeptide.

[0118] In an optional embodiment, the 4-1BBL amino acid sequence or the CD70 amino acid sequence is linked to the liver cancer-associated antigen amino acid sequence. In an optional embodiment, the amino acid sequence expressed / encoded by the nucleotide sequence encoding the 4-1BBL, from N-terminus to C-terminus, is as shown in the CD8a signal peptide, SEQ ID NO. 13 or SEQ ID NO. 14, the Hinge region of CD8a, and the CD8a TM region. The amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO. 21, the amino acid sequence of the CD8a Hinge region is shown in SEQ ID NO. 19, and the amino acid sequence of the CD8a TM region is shown in SEQ ID NO. 20. In an optional embodiment, the amino acid sequence expressed / encoded by the nucleotide sequence of CD70, from N-terminus to C-terminus, is as shown in the CD8a signal peptide, SEQ ID NO. 22 or SEQ ID NO. 23, the Hinge region of CD8a, and the CD8a TM region. The amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO.21, the amino acid sequence of the CD8a Hinge region is shown in SEQ ID NO.19, and the amino acid sequence of the CD8a TM region is shown in SEQ ID NO.20.

[0119] In one embodiment, the therapeutic nucleic acid for liver cancer of the present invention comprises at least one of the following: (i) an open reading frame nucleotide sequence encoding a co-stimulatory polypeptide / protein; (ii) an open reading frame nucleotide sequence encoding an amino acid sequence of a liver cancer-associated antigen; and (iii) an open reading frame nucleotide sequence encoding a 2A self-cleaving peptide, or (iv) an open reading frame nucleotide sequence encoding a GS-linked polypeptide; if the open reading frame nucleotide sequences of types (i) to (iv) are linked, the order and manner of linking are such that, as would be understood by those skilled in the art, the purpose is not to reduce the activity of the co-stimulatory polypeptide / protein or the liver cancer-associated antigen encoded by the open reading frame nucleotide sequence. For example, if linking an open reading frame nucleotide sequence encoding a co-stimulatory polypeptide / protein with an open reading frame nucleotide sequence encoding a GS-linked polypeptide results in at least one transmembrane-functional co-stimulatory polypeptide / protein having a slower or impossible transmembrane crossing speed in the cell, thereby affecting its activity, such a linking method will not be adopted.

[0120] 2A peptides (2A self-cleaving peptides) are a class of peptide fragments with 18-22 amino acid residues in length that can induce self-cleavage of recombinant proteins containing 2A peptides within cells. In optional embodiments, the 2A self-cleaving peptide is selected from T2A peptides, P2A peptides, E2A peptides, or F2A peptides. In optional embodiments, the amino acid sequence of F2A peptide is shown in SEQ ID NO.7; the amino acid sequence of P2A peptide is shown in SEQ ID NO.8; the amino acid sequence of T2A peptide is shown in SEQ ID NO.9; and the amino acid sequence of E2A peptide is shown in SEQ ID NO.10.

[0121] In an optional implementation, the GS sequence includes (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q (where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; and q is an integer from 1 to 5).

[0122] In this invention, unless otherwise specified, the nucleotide sequences of the GS sequence and the 2A peptide may be the nucleotide sequences encoding the GS sequence and the 2A peptide as specified in SEQ ID NO. 39 or SEQ ID NO. 79. SEQ ID NO. 39 or SEQ ID NO. 79 are longer nucleotide sequences that include the aforementioned sequences and also encode other functional regions.

[0123] In one embodiment, the nucleotide sequence encoding CD80 is shown in SEQ ID NO.24 or SEQ ID NO.77.

[0124] In one embodiment, the nucleotide sequence encoding 4-1BBL is shown in SEQ ID NO.25.

[0125] In one embodiment, the nucleotide sequence encoding CD70 is shown in SEQ ID NO.26 or SEQ ID NO.78.

[0126] In one embodiment, the nucleotide sequence encoding the CD8a signal peptide is shown in SEQ ID NO.27.

[0127] In one embodiment, the nucleotide sequences encoding the Hinge region and CD8a™ region of CD8a are shown in SEQ ID NO. 76.

[0128] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen and at least one co-stimulatory molecule, wherein the liver cancer-associated antigen is GPC3(1) and GPC3(2), and the co-stimulatory molecule is CD80. In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(1)…P2A…GPC3(2). In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(1)-GPC3(2).

[0129] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen and at least one co-stimulatory molecule, wherein the liver cancer-associated antigen is GPC3(1) and GPC3(2), and the co-stimulatory molecule is CD80 and CD70. In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(1)…P2A…GPC3(2)…T2A…CD70. In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(1)-GPC3(2)…P2A…CD70.

[0130] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen and at least one co-stimulatory molecule, wherein the liver cancer-associated antigen is GPC3(1) and GPC3(2), and the co-stimulatory molecule is CD80, 4-1BBL, and CD70. In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(1)…P2A…4-1BBL-GPC3(2)…T2A…CD70.

[0131] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen and at least one co-stimulatory molecule, wherein the liver cancer-associated antigen is GPC3(3), GPC3(4), GPC3(5), and GPC3(6), and the co-stimulatory molecule is CD80. In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6). In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6).

[0132] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen and at least one co-stimulatory molecule, wherein the liver cancer-associated antigen is GPC3(3), GPC3(4), GPC3(5), and GPC3(6), and the co-stimulatory molecule is CD80 and CD70. In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)…P2A…CD70. In a particular embodiment, the connection between the functional regions in the therapeutic nucleic acid for liver cancer is CD80-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)…P2A…CD70.

[0133] 2. Regarding protein compositions

[0134] The present invention provides a protein composition comprising a polypeptide encoded by a therapeutic nucleic acid for liver cancer as described above.

[0135] 3. Regarding vaccines

[0136] Tumor therapeutic nucleic acid vaccines can be mainly divided into three categories: universal tumor vaccines, personalized tumor vaccines, and tumor-associated vaccines caused by viral infections. Personalized tumor vaccines are immunotherapies designed specifically for the tumor characteristics of a particular patient. Their core principle is to utilize the patient's own tumor-specific antigens (TSA) or neoantigens to activate the body's immune system, thereby precisely identifying and eliminating cancer cells. Universal tumor vaccines mainly refer to vaccines encoding tumor-associated antigens (TAAs).

[0137] The present invention provides a vaccine, characterized in that the vaccine comprises the liver cancer therapeutic nucleic acid as described above.

[0138] In one embodiment, the vaccine further includes a delivery formulation that encapsulates the hepatocellular carcinoma therapeutic nucleic acid, RNA encoding an enhanced antigen-presenting molecule, or RNA encoding a T-cell co-stimulatory molecule.

[0139] In an optional embodiment, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0140] In an optional embodiment, the present invention provides a vaccine preparation method according to any of the foregoing embodiments, characterized in that it comprises: mixing the liver cancer therapeutic nucleic acid (or RNA encoding an enhanced antigen-presenting molecule, or RNA encoding a T-cell co-stimulatory molecule) and a delivery formulation to form a vaccine; the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0141] In an optional embodiment, the liver cancer therapeutic nucleic acid (or RNA encoding an enhanced antigen-presenting molecule, or RNA encoding a T-cell co-stimulatory molecule) comprises at least two independent nucleotide sequences, and the vaccine preparation method comprises: mixing each independent nucleotide sequence with a delivery formulation, and then mixing the delivery formulation encapsulating the first nucleotide sequence with the delivery formulation encapsulating the remaining independent nucleotide sequences at a mass ratio of (10~1):(1~10) to form a vaccine.

[0142] In some embodiments, the mass ratio of the delivery formulation encapsulating the first nucleotide sequence to the delivery formulation encapsulating the remaining independent nucleotide sequences is one of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0143] The present invention provides a vaccine comprising the protein composition as described above.

[0144] In one embodiment, the vaccine is a protein vaccine, comprising the protein composition and antigen as described above. In an optional embodiment, the vaccine includes an adjuvant selected from aluminum adjuvants, MF59 adjuvants, AS01 adjuvants, CpG adjuvants, etc. In an optional embodiment, the vaccine may further include a stabilizer and / or a buffer solution.

[0145] The stabilizers in protein vaccines are selected from sugars (such as sucrose, lactose, etc.) and amino acids (such as glycine, etc.), which can prevent the antigen protein from agglomerating and denaturing during processes such as freeze-drying.

[0146] In the field of protein vaccines, phosphate-buffered saline (PBS) is a commonly used buffering system. It can maintain the pH stability of the vaccine, ensuring a suitable acid-base environment during storage and transportation, and preventing denaturation of antigen proteins and other components.

[0147] In an optional embodiment, the present invention provides a biomaterial comprising any one of an expression cassette, a vector, engineered bacteria, or a cell line, wherein the biomaterial contains or expresses RNA of a liver cancer therapeutic nucleic acid (or RNA encoding an enhanced antigen-presenting molecule, or RNA encoding a T-cell co-stimulatory molecule) as described in any of the preceding embodiments.

[0148] In an optional embodiment, the present invention provides a method comprising administering to a subject an effective amount of at least one of the following: a vaccine as described in any one of the preceding claims, a therapeutic nucleic acid for liver cancer as described in any one of the preceding claims, RNA encoding an enhanced antigen-presenting molecule as described in any one of the preceding claims, RNA encoding a T-cell co-stimulatory molecule as described in any one of the preceding claims, or a biological material as described in any one of the preceding claims, to induce a cellular immune response or a humoral immune response in the subject.

[0149] In an optional implementation, the method includes administering to a subject at least two effective doses of any of the preceding vaccines, any of the preceding liver cancer therapeutic nucleic acids (or RNA encoding enhanced antigen-presenting molecules, or RNA encoding T-cell co-stimulatory molecules), or any of the preceding biological materials.

[0150] In an optional implementation, the time interval between the first and second applications of the at least two applications is not less than 14 days.

[0151] In an optional implementation, the subject is immunocompromised; in an optional implementation, the subject is no older than 5 years or no younger than 65 years.

[0152] In optional embodiments, the effective amount of the present invention is at least one of the following: a vaccine, a liver cancer therapeutic nucleic acid (or RNA encoding an enhanced antigen-presenting molecule, or RNA encoding a T-cell co-stimulatory molecule) as low as 40 μg, 30 μg, 25 μg, 20 μg, 15 μg, 10 μg, 5 μg, 3 μg, or 1 μg.

[0153] Example 1: Process for preparing LNPs from RNA

[0154] A lipid nanoparticle comprising an antigen RNA encoding a lipid nanoparticle, wherein the lipid nanoparticle comprises, by molar percentage, 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol and 1% PEG-DMG.

[0155] The preparation method is as follows: (a) The therapeutic nucleic acid for liver cancer is RNA. The RNA is dissolved in citrate buffer at pH 4 and the concentration is adjusted to 0.1 mg / ml to obtain an aqueous phase.

[0156] (b) Dissolve Dlin-MC3-DMA, DOPG, cholesterol and PEG-DMG in anhydrous ethanol according to the formulation amount, and adjust the concentration of lipid components in the organic phase to 6 mg / mL to obtain the organic phase.

[0157] (c) The aqueous phase from step (a) and the organic phase from step (b) were mixed at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min. The mixture was immediately diluted 100 times with PBS solution at pH 7.4, and ethanol was removed from the solution using tangential flow filtration (TFF). The mixture was then concentrated until the concentration of mRNA in the system was 55 μg / ml to obtain lipid nanoparticles containing therapeutic nucleic acids for liver cancer.

[0158] Example 2

[0159] C57BL / 6 mice (female, 5-6 weeks old, average weight 15-20g, purchased from Zhuhai Beston Biotechnology Co., Ltd.) were selected for in vivo antitumor efficacy evaluation. The experimental mice were subcutaneously inoculated with MC38 / GPC3 (MC38 cells overexpressing the human GPC3 gene) tumor cells (5 × 10⁻⁶). 5 / each), when the average tumor volume reaches 100 mm 3 Mice were randomly divided into 6 groups based on tumor volume, with a tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. The six groups were designated as NC group (injected with empty LNP), LRIO-060 group, LRIO-039 group, LRIO-055 group, LRIO-0391 group, and LRIO-0551 group, with 6 mice in each group.

[0160] The dosage for LRIO-060 group was 20 μg / animal / dose, the dosage for LRIO-0391 and LRIO-0551 groups was 25 μg / animal / dose, and the dosage for LRIO-039 and LRIO-055 groups was 5 μg / animal / dose. Tumor volume was measured 2-3 times per week after administration.

[0161] The samples injected into the LRIO-060 group contained T-cell co-stimulatory polypeptides encoded by hepatocellular carcinoma therapeutic nucleic acids, including CD80, CD70, and 4-1BBL. The antigen was GPC3 protein, and the connection between the functional regions of the co-stimulatory polypeptide encoded by its RNA was: CD80-GPC3(1)…P2A…4-1BBL-GPC3(2)…T2A…CD70. The amino acid sequence encoded by the RNA in the samples injected into the LRIO-060 group is shown in SEQ ID NO.40, and the RNA sequence in the samples injected into the LRIO-060 group is shown in SEQ ID NO.39.

[0162] The LRIO-039 group was injected with lipid nanoparticles containing STING RNA, and the LRIO-055 group was injected with lipid nanoparticles containing cGAS RNA. The lipid nanoparticles were prepared according to the method described in Example 1.

[0163] In this embodiment, STING is an artificial STING mutant, which is based on wild-type STING (amino acid sequence as shown in SEQ ID NO. 67) and undergoes a V155M mutation. The amino acid sequence of cGAS is shown in SEQ ID NO. 69.

[0164] In this embodiment, the RNA sequence contained in the sample injected into the LRIO-039 group is shown in SEQ ID NO.68, and the RNA sequence contained in the sample injected into the LRIO-055 group is shown in SEQ ID NO.70.

[0165] The LRIO-0391 group of injected samples contained LRIO-060 and LRIO-039 group injected samples at a mass ratio of 4:1; the LRIO-055 group of injected samples contained LRIO-060 and LRIO-055 group injected samples at a mass ratio of 4:1.

[0166] The CD80 RNA sequence contained in the sample injected in the experimental group of this embodiment is shown in SEQ ID NO.24. The CD80 sequence is composed of the sequentially linked amino acid sequences SEQ ID NO.11 and SEQ ID NO.17.

[0167] The nucleotide sequence of CD70 contained in the sample injected in the experimental group of this embodiment is shown in SEQ ID NO.26, and the amino acid sequence of CD70 is shown in SEQ ID NO.15. The amino acid sequence expressed / encoded by the nucleotide sequence of CD70 from the N-terminus to the C-terminus is as shown in CD8a signal peptide, the amino acid sequence shown in SEQ ID NO.22, the Hinge region of CD8a, and the CD8aTM region.

[0168] The nucleotide sequence of 4-1BBL contained in the sample injected in the experimental group of this embodiment is shown in SEQ ID NO.25. The amino acid sequence expressed / encoded by the nucleotide sequence of 4-1BBL from the N-terminus to the C-terminus is as shown in CD8a signal peptide, amino acid sequence shown in SEQ ID NO.13, Hinge region of CD8a and CD8a TM region.

[0169] In addition to the open reading frame sequence mentioned above, the RNA in this example also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in SEQ ID NO.1), a 3' UTR (as shown in SEQ ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the RNA is replaced with 5' pseudouracil.

[0170] The results of this embodiment are shown below. Figure 1 The results showed that, compared with the NC group, the LRIO-060 group could inhibit tumor growth, and the addition of LRIO-039 or LRIO-055 could enhance the anti-tumor effect of LRIO-060. Among them, the combination of LRIO-060 and LRIO-055 showed the best effect in controlling tumor growth.

[0171] Example 3

[0172] C57BL / 6 mice (female, 5-6 weeks old, average weight 15-20g, purchased from Zhuhai Beston Biotechnology Co., Ltd.) were selected for in vivo antitumor efficacy evaluation. The experimental mice were subcutaneously inoculated with MC38 / GPC3 tumor cells (5×10⁻⁶). 5 / each), when the average tumor volume reaches 100 mm 3 Mice were randomly divided into 5 groups based on tumor volume, with a tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. The 5 groups were named eLNP, LRIO-134 (16 μg) + LRIO-055 (4 μg), LRIO-134 (18 μg) + LRIO-055 (2 μg), LRIO-134 (10 μg) + LRIO-055 (10 μg), and LRIO-134 (20 μg), with 6 mice in each group.

[0173] The dosage for each experimental group was 20 μg / animal / dose. Specifically, the LRIO-134 (16 μg) + LRIO-055 (4 μg) group received 16 μg of LRIO-134 and 4 μg of LRIO-055 per animal per dose; the LRIO-134 (18 μg) + LRIO-055 (2 μg) group received 18 μg of LRIO-134 and 2 μg of LRIO-055 per animal per dose; the LRIO-134 (10 μg) + LRIO-055 (10 μg) group received 10 μg of LRIO-134 and 10 μg of LRIO-055 per animal per dose; and the LRIO-134 (20 μg) group received 20 μg of LRIO-134 per animal per dose. Tumor volume was measured 2-3 times per week after administration.

[0174] The liver cancer therapeutic nucleic acid encoded by the LRIO-134 group injection sample contains T-cell co-stimulatory polypeptides including CD80 and CD70, which encode liver cancer antigens GPC3(3), GPC3(4), GPC3(5), and GPC3(6) (amino acid sequences are shown in SEQ ID NO. 30-33, and nucleotide sequences are shown in SEQ ID NO. 50-53, respectively). The amino acid sequence of the polypeptide encoded by the liver cancer therapeutic nucleic acid in the LRIO-134 group injection sample is shown in SEQ ID NO. 41, and the nucleotide sequence is shown in SEQ ID NO. 79. The connection form of each functional region of the liver cancer therapeutic nucleic acid in the LRIO-134 group injection sample is CD80-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)…T2A…CD70.

[0175] The CD80 RNA sequence contained in the sample injected in the experimental group of this embodiment is shown in SEQ ID NO.24. The CD80 sequence is composed of the sequentially linked amino acid sequences SEQ ID NO.11 and SEQ ID NO.17.

[0176] The nucleotide sequence of CD70 contained in the sample injected in the experimental group of this embodiment is shown in SEQ ID NO.26, and the amino acid sequence of CD70 is shown in SEQ ID NO.15. The amino acid sequence expressed / encoded by the nucleotide sequence of CD70 from the N-terminus to the C-terminus is as shown in CD8a signal peptide, the amino acid sequence shown in SEQ ID NO.22, the Hinge region of CD8a, and the CD8aTM region.

[0177] The LRIO-055 sample contains lipid nanoparticles encoding cGAS RNA, which were prepared according to the method described in Example 1.

[0178] In this embodiment, the cGAS amino acid sequence encoded by the LRIO-055 sample is shown in SEQ ID NO.69, and the cGAS RNA sequence contained in the LRIO-055 sample is shown in SEQ ID NO.70.

[0179] In addition to the open reading frame sequence mentioned above, the RNA in this example also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in SEQ ID NO.1), a 3' UTR (as shown in SEQ ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the RNA is replaced with 5' pseudouracil.

[0180] The results of this embodiment are shown below. Figure 2 The results showed that, compared with the eLNP group, at the same total dose, the different ratios of LRIO-134 and LRIO-055 mixed samples tested could significantly inhibit tumor growth (P<0.05).

[0181] Example 4

[0182] C57BL / 6 mice (female, 5-6 weeks old, average weight 15-20g, purchased from Zhuhai Beston Biotechnology Co., Ltd.) were selected for in vivo antitumor efficacy evaluation. The experimental mice were subcutaneously inoculated with MC38 / GPC3 tumor cells (5×10⁻⁶). 5 / each), when the average tumor volume reaches 100 mm 3 Mice were randomly divided into 6 groups based on tumor volume, with a tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. The six groups were designated as eLNP group, LRIO-134 group, LRIO-134+LRIO-m011 group, LRIO-134+LRIO-055 group, LRIO-134+LRIO-161 group, and LRIO-134+LRIO-167 group, with 6 mice in each group.

[0183] The dosage for the LRIO-134 group was 20 μg of LRIO-134 per dose; the dosage for the LRIO-134+LRIO-m011 group was 20 μg of LRIO-134 and 5 μg of LRIO-m011 per dose; the dosage for the LRIO-134+LRIO-055 group was 20 μg of LRIO-134 and 5 μg of LRIO-055 per dose; the dosage for the LRIO-134+LRIO-161 group was 20 μg of LRIO-134 and 5 μg of LRIO-161 per dose; and the dosage for the LRIO-134+LRIO-167 group was 20 μg of LRIO-134 and 5 μg of LRIO-167 per dose. Tumor volume was measured 2-3 times per week after administration.

[0184] The T-cell co-stimulatory polypeptide encoded by the hepatocellular carcinoma therapeutic nucleic acid contained in the LRIO-134 sample includes CD80 and CD70, which encode hepatocellular carcinoma antigens GPC3(3), GPC3(4), GPC3(5), and GPC3(6) (the amino acid sequences are shown in SEQ ID NO. 30-33, and the nucleotide sequences are shown in SEQ ID NO. 50-53, respectively). The amino acid sequence of the polypeptide encoded by the hepatocellular carcinoma therapeutic nucleic acid contained in the LRIO-134 sample is shown in SEQ ID NO. 41. The connection form of each functional region of the hepatocellular carcinoma therapeutic nucleic acid contained in the LRIO-134 sample is CD80-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6)-GPC3(3)-GPC3(4)-GPC3(5)-GPC3(6) …T2A…CD70.

[0185] The CD80 RNA sequence contained in the sample injected in the experimental group of this embodiment is shown in SEQ ID NO.24. The CD80 sequence is composed of the sequentially linked amino acid sequences SEQ ID NO.11 and SEQ ID NO.17.

[0186] The nucleotide sequence of CD70 contained in the sample injected in the experimental group of this embodiment is shown in SEQ ID NO.26, and the amino acid sequence of CD70 is shown in SEQ ID NO.15. The amino acid sequence expressed / encoded by the nucleotide sequence of CD70 from the N-terminus to the C-terminus is as shown in CD8a signal peptide, the amino acid sequence shown in SEQ ID NO.22, the Hinge region of CD8a, and the CD8aTM region.

[0187] The LRIO-055 sample contains lipid nanoparticles encoding cGAS RNA. The lipid nanoparticles were prepared according to the method described in Example 1. The amino acid sequence of cGAS is shown in SEQ ID NO. 69, and the RNA sequence encoding cGAS is shown in SEQ ID NO. 70.

[0188] The LRIO-m011 sample contains lipid nanoparticles encoding membrane-bound IL2 RNA. The lipid nanoparticles were prepared according to the method described in Example 1. The RNA sequence encoding membrane-bound IL2 is shown in SEQ ID NO.80.

[0189] The LRIO-161 sample contains lipid nanoparticles encoding the cytokine FLT3LG RNA. The lipid nanoparticles were prepared according to the method described in Example 1, and the RNA sequence encoding FLT3LG is shown in SEQ ID NO.81.

[0190] The LRIO-167 sample contains lipid nanoparticles encoding cGAS, membrane-bound IL2, and cytokine FLT3LG RNA. The lipid nanoparticles were prepared according to the method described in Example 1, and their RNA sequences are shown in SEQ ID NO.82.

[0191] In addition to the open reading frame sequence mentioned above, the RNA in this example also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in SEQ ID NO.1), a 3' UTR (as shown in SEQ ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the RNA is replaced with 5' pseudouracil.

[0192] The results of this embodiment are shown below. Figure 3 The results showed that, compared with the eLNP group, all other experimental groups could significantly inhibit tumor growth; compared with LRIO-134 alone, the combination of LRIO-055, LRIO-161 or LRIO-167 could enhance the anti-tumor activity of LRIO-134, with the combination of LRIO-055 showing the best enhancement effect.

[0193] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure.

Claims

1. A therapeutic nucleic acid for liver cancer, characterized in that, The therapeutic nucleic acid for liver cancer comprises at least two independent nucleotide sequences, wherein one independent nucleotide sequence is a first nucleotide sequence that encodes at least one genetic adjuvant; and at least one of the remaining one or more independent nucleotide sequences encodes at least one liver cancer-associated antigen. The genetic adjuvant is selected from at least one of STING, cGAS, Flagellin16a, Flagellin, LTB, and TLR4; The STING is wild-type STING or an artificial STING mutant; the amino acid sequence of the wild-type STING is shown in SEQ ID NO. 67; the amino acid sequence of the artificial STING mutant is based on wild-type STING and includes one or more mutations selected from V147L, N154S, V155M, R284M, R284K, R284T, E315Q, R375A, or a combination thereof. The amino acid sequence of cGAS is shown in SEQ ID NO. 69; the amino acid sequence of Flagellin16a is shown in SEQ ID NO. 71; the amino acid sequence of Flagellin is shown in SEQ ID NO. 73; the amino acid sequence of TLR4 is shown in SEQ ID NO. 75; and the amino acid sequence of LTB is shown in Uniprot P32890.

2. The therapeutic nucleic acid for liver cancer as described in claim 1, characterized in that, The therapeutic nucleic acid for liver cancer encodes at least one liver cancer-associated antigen, which is selected from at least one of AFP, GPC3, HBsAg, HBcAg, NY-ESO-1, PRAME, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1, MAGE-C2, CLDN6, hTERT, Survivin, TPTE, PSA, KK-LC-1, KRAS, EGFR, P53, CEA, Tyrosinase, gp100, Melan-A, and HER2. Preferably, the therapeutic nucleic acid for liver cancer encodes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more liver cancer-related antigens.

3. The therapeutic nucleic acid for liver cancer as described in claim 2, characterized in that, The therapeutic nucleic acid for liver cancer encodes multiple liver cancer-associated antigens, which are derived from multiple antigen proteins; preferably, the liver cancer-associated antigens are selected from at least one of AFP, GPC3, HBsAg, and HBcAg.

4. The therapeutic nucleic acid for liver cancer as described in claim 1, characterized in that, The therapeutic nucleic acid for liver cancer encodes at least one co-stimulatory molecule, which is selected from at least one member of the B7 family and the tumor necrosis factor family. Preferably, the co-stimulatory molecule is CD80; Preferably, the CD80 comprises the amino acid sequence shown in SEQ ID NO.11 or SEQ ID NO.12 or a functional fragment thereof, or an extracellular domain composed thereof; the functional fragment may comprise an IgV domain and / or an IgC domain; preferably, the CD80 further comprises the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.18, or SEQ ID NO.19 or a functional fragment thereof, or a transmembrane domain composed thereof; Preferably, the co-stimulatory molecule further includes CD70; Preferably, the CD70 comprises the amino acid sequence shown in SEQ ID NO.22 or SEQ ID NO.23 or a functional fragment thereof, or an extracellular domain thereof; preferably, the CD70 further comprises the amino acid sequence shown in SEQ ID NO.19 or a functional fragment thereof, or a transmembrane domain thereof. Preferably, the co-stimulatory molecule encoded by the therapeutic nucleic acid for liver cancer further includes 4-1BBL.

5. The therapeutic nucleic acid for liver cancer as described in claim 4, characterized in that, The co-stimulatory molecule amino acid sequence encoded by the therapeutic nucleic acid for liver cancer exists in a linked or unlinked form with the amino acid sequence of liver cancer-associated antigen. Preferably, the unconnected form is selected from at least one of the following: (1) the coding sequence is located in two independent nucleotide sequences; (2) the coding sequence is located in the same nucleotide sequence but in different ORFs; (3) the coding sequences are located in the same ORF and are connected by nucleotide sequences encoding cleavable linkers (such as 2A self-cleaving peptides). Preferably, the connection is selected from at least one of the following: (4) the C-terminus of the co-stimulatory molecule amino acid sequence is directly connected to the N-terminus of the antigen amino acid sequence; (5) the two are connected by a GS linking peptide; Preferably, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide, and its amino acid sequence is shown in SEQ ID NO.9, 8, 10 and 7 respectively; Preferably, the GS sequence is selected from (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, where n is an integer from 1 to 20, m is an integer from 1 to 20, o is an integer from 1 to 5, p is an integer from 1 to 40, and q is an integer from 1 to 5.

6. The therapeutic nucleic acid for liver cancer as described in claim 1, characterized in that, The total mass ratio of the first nucleotide sequence to the remaining one or more independent nucleotide sequences is (10~1):(1~10); preferably, the therapeutic nucleic acid for liver cancer is a DNA molecule or an RNA molecule.

7. A protein composition, characterized in that, The protein composition comprises a polypeptide encoded by a therapeutic nucleic acid for liver cancer as described in any one of claims 1 to 6.

8. A vaccine, characterized in that, The vaccine comprises a therapeutic nucleic acid for liver cancer as described in any one of claims 1 to 6, or a protein composition as described in claim 7.

9. A method for preparing the vaccine as described in claim 8, characterized in that, The preparation method includes: mixing the liver cancer therapeutic nucleic acid as described in any one of claims 1 to 6 with a delivery formulation; wherein the delivery formulation is selected from lipid nanoparticles or cationic liposomes, thereby obtaining the vaccine; Preferably, the preparation method includes: mixing each independent nucleotide sequence with a delivery formulation, and then mixing the delivery formulation containing the first nucleotide sequence with the delivery formulation containing the remaining independent nucleotide sequences at a mass ratio of (10~1):(1~10) to form a vaccine.

10. A biomaterial, characterized in that, The biomaterial includes any one of expression cassettes, vectors, engineered bacteria, or cell lines, and the biomaterial contains or expresses RNA of the liver cancer therapeutic nucleic acid as described in any one of claims 1 to 6.

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