Therapeutic nucleic acids for liver cancer

Abstract

The present invention provides a therapeutic nucleic acid for liver cancer, characterized in that the therapeutic nucleic acid for liver cancer encodes at least two of the liver cancer antigens GPC3(1) to GPC3(17).

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] The first objective of this invention is to provide a therapeutic nucleic acid for liver cancer, wherein the therapeutic nucleic acid for liver cancer encodes at least two of the liver cancer antigens GPC3(1) to GPC3(17).

[0006] In an optional embodiment, the therapeutic nucleic acid for liver cancer encodes liver cancer antigens GPC3 (1) and GPC3 (2), the amino acid sequence of liver cancer antigen GPC3 (1) is shown in SEQ ID NO.28, and the amino acid sequence of liver cancer antigen GPC3 (2) is shown in SEQ ID NO.29.

[0007] In an optional embodiment, the therapeutic nucleic acid for liver cancer encodes at least one co-stimulatory molecule.

[0008] In an optional implementation, the co-stimulatory molecule is selected from at least one member of the B7 family and a member of the tumor necrosis factor family.

[0009] In an optional embodiment, the co-stimulatory molecules are CD80 and CD70.

[0010] In an optional embodiment, 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 an optional embodiment, 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 thereof.

[0011] In an optional embodiment, 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 an optional embodiment, the CD70 further comprises the amino acid sequence shown in SEQ ID NO.19, or a functional fragment thereof, or a transmembrane domain thereof.

[0012] In an optional embodiment, the co-stimulatory molecule encoded by the liver cancer therapeutic nucleic acid further includes 4-1BBL.

[0013] In an optional embodiment, the co-stimulatory molecule amino acid sequence encoded by the hepatocellular carcinoma therapeutic nucleic acid may or may not be linked to the hepatocellular carcinoma-associated antigen amino acid sequence.

[0014] In an optional implementation, the non-connected cases include: (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 sequence is located in the same ORF and is connected by a nucleotide sequence encoding a cleavable linker (such as a 2A self-cleaving peptide).

[0015] In optional embodiments, the connection includes: (1) the C-terminus of the co-stimulatory molecule amino acid sequence is directly connected to the N-terminus of the antigen amino acid sequence; (2) the two are connected by a GS linker peptide.

[0016] In an optional embodiment, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide, and its amino acid sequence is shown as SEQ ID NO.9, 8, 10 or 7 respectively.

[0017] In an optional implementation, 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.

[0018] In an optional implementation, the therapeutic nucleic acid for liver cancer is a DNA molecule or an RNA molecule.

[0019] In an optional embodiment, the liver cancer therapeutic nucleic acid further encodes at least one genetic adjuvant selected from STING, cGAS, Flagellin16a, Flagellin, LTB, and TLR4.

[0020] A second object of the present invention is to provide a protein composition comprising a polypeptide encoded by a therapeutic nucleic acid for liver cancer as described above.

[0021] A third object of the present invention is to provide a vaccine comprising the aforementioned therapeutic nucleic acid for liver cancer, or the aforementioned protein composition.

[0022] A fourth objective of the present invention is to provide a biomaterial comprising any one of an expression cassette, a vector, engineered bacteria, or a cell line, wherein the biomaterial contains or expresses a liver cancer therapeutic nucleic acid as described in any of the preceding claims. Attached Figure Description

[0023] 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.

[0024] Figure 1 The changes in average tumor volume in mice of each experimental group in Example 2 are shown.

[0025] Figure 2 The changes in average tumor volume in mice of each experimental group in Example 3 are shown.

[0026] Figure 3 The changes in average tumor volume in mice of each experimental group in Example 4 are shown.

[0027] Figure 4 The table shows the CD8+ T cell content of IFNγ or TNFα expressed in the spleen cells of mice in each experimental group in Example 5.

[0028] Figure 5 The content of IFNγ or TNFα in the supernatant of spleen cells of mice in each experimental group in Example 5 is shown.

[0029] Figure 6 The study demonstrates the killing effect of spleen cells on tumor cells in each experimental group in Example 5.

[0030] Figure 7 The changes in average tumor volume in mice of each experimental group in Example 6 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] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In a particular embodiment, the 3'UTR sequence is as shown in SEQ ID NO. 4 to 6. In an alternative embodiment, the 3'UTR sequence is as shown in SEQ ID NO. 4.

[0045] 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).

[0046] 1. Structure of the coding region of therapeutic nucleic acid for liver cancer

[0047] 1.1 Basic Structure

[0048] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein includes at least one coding region. The coding region is an open reading frame (ORF) encoding a single peptide or protein, or contains at least two ORFs, each independently encoding a peptide or protein. When the coding region contains multiple ORFs, the encoded peptides and / or proteins may be the same or different from each other.

[0049] In a specific implementation, multiple ORFs are separated by non-coding sequences. These non-coding sequences include internal ribosome entry sites (IRES). In this disclosure, "IRES" refers to a specific nucleotide sequence present in certain mRNA molecules that enables ribosomes to initiate translation directly at the internal site without relying on the 5' cap structure of the mRNA, thereby allowing a single mRNA molecule to translate into multiple different proteins.

[0050] 1.2 Topological structure of mRNA

[0051] In a particular embodiment, the mRNA contains at least one ORF sequence whose structure is selected from any of the following: (1) a single-molecule tandem structure: consisting of a 5' cap, a 5' UTR, an ORF (1), an ORF (2), ..., an ORF (n), a 3' UTR and a 3' poly (A) tail, from the 5' end to the 3' end, where n is a natural number;

[0052] (2) Multimolecular mixed structure: contains two or more independent mRNA molecules, each mRNA molecule independently contains a 5' cap, 5' UTR, ORF, 3' UTR and 3' poly(A) tail, wherein the 5' cap, 5' UTR, 3' UTR and 3' poly(A) tail of each molecule may be the same or different from each other.

[0053] The open reading frame (ORF) described in this invention is a continuous RNA segment, which generally begins with a start codon (such as AUG) and ends with a stop codon (such as TGA, or UAA, UAG, or UGA).

[0054] 1.3 Sequence Optimization

[0055] In a specific implementation, the nucleotide molecule can be improved in vivo by sequence optimization, including but not limited to improving mRNA stability, increasing translation efficiency in target tissues, reducing the number of truncated proteins, improving protein folding, reducing the toxicity of the expression product, reducing cell death, and regulating protein aggregation.

[0056] Sequence optimization also aims to: optimize formulation and delivery characteristics, overcome expression thresholds, increase expression rate and half-life, optimize protein localization, and avoid immune responses and degradation pathways.

[0057] Sequence optimization techniques include: (1) codon optimization based on the codon frequency of the host organism; (2) adjusting the G / C content to increase mRNA stability or reduce secondary structures; (3) eliminating tandem repeat codons or base strings; (4) customizing transcription and translation control regions; and (5) reducing problematic secondary structures within polynucleotides.

[0058] 2. Polypeptides or proteins encoded by therapeutic nucleic acids for liver cancer

[0059] 2.1 Liver cancer-related antigens

[0060] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen.

[0061] 2.1.1 Types of liver cancer-related antigens

[0062] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more liver cancer-associated antigens. The liver cancer-associated antigens are selected from at least one of AFP, GPC3, HBsAg, and HBcAg. The amino acid sequence of GPC3 is shown in Table 1.

[0063] Table 1: Amino acid and nucleotide sequences of some liver cancer-related antigens

[0064]

[0065] Note: The "Amino Acid Sequence Number" and "Nucleotide Sequence Number" in Table 1 are derived from the sequence listing of this invention and can be abbreviated as (SEQ ID NO.). All truncated versions used in the tables above were constructed with reference to the amino acid sequence of the wild-type GPC3 protein (full length 580 amino acid residues, UniProt number P51654). The position numbers indicated in the tables correspond to the amino acid residue numbers of wild-type GPC3.

[0066] 2.1.2 Connection methods between antigens

[0067] In some embodiments, the liver cancer-associated antigen is GPC3, which may be wild-type GPC3 or engineered GPC3. In this disclosure, "engineered" refers to macromolecules with specific functions synthesized by modifying or designing natural macromolecules through genetic engineering or protein engineering techniques.

[0068] In some embodiments, the therapeutic nucleic acid for liver cancer of this disclosure encodes at least one liver cancer-associated antigen, said liver cancer-associated antigen being GPC3(1) and GPC3(2). In a particular embodiment, the nucleotide sequences encoding GPC3(1) and GPC3(2) are arranged in a repetitive manner; for example, the connection between the functional regions in the liver cancer-associated antigen encoded by the therapeutic nucleic acid for liver cancer is: GPC3(1)-GPC3(2)-GPC3(1)-GPC3(2); for another example, the connection between the functional regions in the liver cancer-associated antigen encoded by the therapeutic nucleic acid for liver cancer is: GPC3(1)-GPC3(1)-GPC3(2)-GPC3(2); for yet another example, the connection between the functional regions in the liver cancer-associated antigen encoded by the therapeutic nucleic acid for liver cancer also includes other forms, which are those readily available to those skilled in the art.

[0069] In this disclosure, "the connection pattern between functional regions" describes the sequence of functional regions from the N-terminus to the C-terminus of a polypeptide expressed by engineered nucleotides. Functional regions connected by "-" have both their nucleotide and amino acid sequences interconnected; functional regions connected by "..." have their nucleotide sequences interconnected, but their amino acid sequences are not interconnected.

[0070] In some implementations, the liver cancer-associated antigens are directly linked in sequence.

[0071] In other embodiments, at least two of the liver cancer-associated antigens are linked end-to-end by a REKR sequence.

[0072] In a further implementation scheme, some liver cancer-related antigens are directly linked end-to-end, while others are linked via REKR sequences.

[0073] 2.1.3 Tag Sequence

[0074] In some embodiments, a Flag tag sequence is attached to the N-terminus or C-terminus of the amino acid sequence of at least one of the liver cancer-associated antigens. The amino acid sequence of the Flag tag sequence is shown in SEQ ID NO. 65.

[0075] 2.2 Enhance antigen presentation molecules

[0076] 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 enhanced antigen-presenting molecule.

[0077] In some embodiments, the enhanced antigen-presenting molecule is selected from at least one of the following: co-stimulatory molecules, MHC molecules, Ubiquitin molecules, cytokines, PADRE, Tetanus toxin P2, chemokines, chemokine receptors, and co-expressed immune checkpoint inhibitors / antibodies.

[0078] In some implementations, the co-expressed immune checkpoint inhibitor / antibody is selected from at least one of anti-PD1, anti-PD-L1, anti-TIGIT, anti-LAG3, and anti-TIM3.

[0079] In some implementations, the amino acids encoded by the RNA encoding the enhanced antigen-presenting molecule increase the immunogenicity of the antigen by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%).

[0080] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein encodes at least one liver cancer-associated antigen and at least one co-stimulatory molecule.

[0081] In this disclosure, "co-stimulatory molecules" refer to cell surface molecules and their ligands involved in co-stimulatory signal transduction, providing the necessary co-stimulatory signals for the complete activation of T cells or B cells, including members of the B7 family and the tumor necrosis factor family. Their main biological functions include enhancing T cell activation and immune responses, and participating in the regulation of tumor immune responses. For example, the binding of the B7 family member CD80 to CD28 on T cells generates a positive signal, thereby enhancing the immune response; while the binding of CTLA-4 to the B7 family member CD80 generates a negative signal, blocking T cell activation induced by CD28 and downregulating the immune response.

[0082] In some embodiments, the co-stimulatory molecule is selected from at least one member of the B7 family and the tumor necrosis factor family.

[0083] In some implementations, the B7 family member 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.

[0084] In some embodiments, the tumor necrosis factor family member is selected from at least one of TNF-β, TNF-α, LT-β, ​​CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, and GITRL.

[0085] 2.2.1 CD80 molecule

[0086] In some implementations, the co-stimulatory molecule is CD80.

[0087] Natural / wild-type CD80 is a transmembrane polypeptide structure, comprising an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is divided into an IgV domain (positions 35-135) and an IgC domain (positions 145-230), both of which play important roles in the binding of CD80 to its 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).

[0088] In some implementations, the disclosed co-stimulatory molecule CD80 may be wild-type or engineered CD80.

[0089] Engineered CD80 is a molecule that is modified from the natural CD80 molecule or designed and constructed using its principles in order to obtain specific functions or improve properties. It includes, but is not limited to: (1) truncated versions with sequence deletions or mutations (such as those containing only extracellular and transmembrane domains); (2) fusion proteins that are fused with other molecules (such as the fusion of the CD80 extracellular domain with the 4-1BB intracellular domain).

[0090] In some 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 thereof. The functional fragment may comprise an IgV domain and / or an IgC domain.

[0091] In some embodiments, the CD80 further comprises the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19, or functional fragments thereof, or transmembrane domains thereof.

[0092] In a specific implementation, the nucleotide sequence encoding CD80 is shown in SEQ ID NO.24 or SEQ ID NO.67.

[0093] 2.2.2 CD70 molecule

[0094] In optional embodiments, the co-stimulatory molecule of the present invention may also 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. The CD70 may be wild-type or engineered CD70.

[0095] In some 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 thereof.

[0096] 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.

[0097] In a specific implementation, the nucleotide sequence encoding CD70 is shown in SEQ ID NO.26 or SEQ ID NO.68.

[0098] 2.2.3 4-1BBL molecule

[0099] In some embodiments, the co-stimulatory molecule includes 4-1BBL. The 4-1BBL may be wild-type or engineered 4-1BBL.

[0100] In some embodiments, 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.

[0101] In some embodiments, the 4-1BBL further comprises the amino acid sequence shown in SEQ ID NO.19 or a functional fragment thereof, or a transmembrane domain thereof.

[0102] In a specific implementation, the nucleotide sequence encoding 4-1BBL is shown in SEQ ID NO.25.

[0103] 2.2.3.4 Co-stimulatory molecular signal peptides and transmembrane regions

[0104] The co-stimulatory molecule CD70 or 4-1BBL involved in this invention may contain an amino acid sequence or fragment thereof that is present or not present in nature.

[0105] In an optional embodiment, the costimulatory molecule CD70 or 4-1BBL described in this invention can be a wild-type costimulatory molecule CD70 or 4-1BBL, whose amino acid sequence is consistent with the amino acid sequence of any natural / wild costimulatory molecule CD70 or 4-1BBL.

[0106] In an optional embodiment, the co-stimulatory molecule CD70 or 4-1BBL described in this invention can be an engineered co-stimulatory molecule CD70 or 4-1BBL.

[0107] "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.

[0108] In some embodiments, the amino acid sequence expressed by the coding sequence of said 4-1BBL or CD70, from N-terminus to C-terminus, is as follows: CD8a signal peptide (SEQ ID NO. 21), extracellular domain (SEQ ID NO. 13 / 14 or 22 / 23), CD8a Hinge region (SEQ ID NO. 19), and CD8a TM region (SEQ ID NO. 20). The nucleotide sequence encoding the CD8a signal peptide is shown in SEQ ID NO. 27, and the nucleotide sequences encoding the CD8a Hinge region and TM region are shown in SEQ ID NO. 66.

[0109] 2.3 The connection mechanism between co-stimulatory molecules and antigens

[0110] 2.3.1 Classification of Connection Methods

[0111] In one embodiment, the co-stimulatory molecule amino acid sequence encoded by the hepatocellular carcinoma therapeutic nucleic acid may or may not be linked to the hepatocellular carcinoma-associated antigen amino acid sequence.

[0112] In one embodiment, the non-connected cases include: (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).

[0113] In one implementation, the connection includes: (1) the C-terminus of the co-stimulatory molecule amino acid sequence is directly connected to the N-terminus of the antigen amino acid sequence; and (2) the two are connected by a GS linker peptide.

[0114] The 2A peptides described in this disclosure are peptide fragments of 18-22 amino acid residues in length that can induce self-cleavage of recombinant proteins containing 2A peptides. In some embodiments, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide, or F2A peptide, and their amino acid sequences are shown in SEQ ID NO. 9, 8, 10, and 7, respectively.

[0115] In some embodiments, the GS sequence described in this disclosure 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.

[0116] 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. 40. SEQ ID NO. 39 or SEQ ID NO. 40 are longer nucleotide sequences that include the above sequences and also encode other functional regions.

[0117] 2.3.2 Active considerations for connection methods

[0118] In one embodiment, the disclosed therapeutic nucleic acid for liver cancer comprises: (i) an ORF encoding a co-stimulatory molecule; (ii) an ORF encoding a liver cancer-associated antigen; and (iii) an ORF encoding a 2A self-cleaving peptide; and / or (iv) an ORF encoding a GS-linked polypeptide.

[0119] The connection sequence and connection method between the ORFs of types (i) to (iv) are intended to avoid reducing the activity of the encoded co-stimulatory molecules or hepatocellular carcinoma-associated antigens. For example, if using GS linking peptides to link ORFs encoding transmembrane co-stimulatory molecules results in a slowdown in transmembrane transduction or failure to transmembrane transduction, thereby affecting activity, this connection method will not be adopted.

[0120] 2.3.3 Specific Implementation Plan (Combination Form)

[0121] In some embodiments, the therapeutic nucleic acid for liver cancer encodes liver cancer-associated antigens GPC3(1) and GPC3(2), as well as the co-stimulatory molecule CD80. In a particular embodiment, the connection pattern of the functional regions of the therapeutic nucleic acid for liver cancer is selected from at least one of CD80-GPC3(1)…P2A…GPC3(2) or CD80-GPC3(1)-GPC3(2).

[0122] In some embodiments, the therapeutic nucleic acid for liver cancer encodes liver cancer-associated antigens GPC3(1) and GPC3(2), as well as co-stimulatory molecules CD80 and CD70. In a particular embodiment, the connection pattern of the functional regions of the therapeutic nucleic acid for liver cancer is selected from at least one of CD80-GPC3(1)…P2A…GPC3(2)…T2A…CD70 or CD80-GPC3(1)-GPC3(2)…P2A…CD70.

[0123] In some embodiments, the therapeutic nucleic acid for liver cancer encodes liver cancer-associated antigens GPC3(1) and GPC3(2), as well as co-stimulatory molecules CD80, 4-1BBL, and CD70. In a particular embodiment, the functional regions of the therapeutic nucleic acid for liver cancer are connected in the form of CD80-GPC3(1)…P2A…4-1BBL-GPC3(2)…T2A…CD70.

[0124] 2.4 Genetic adjuvants

[0125] 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 genetic adjuvant, said genetic adjuvant being selected from STING, cGAS, Flagellin16a, Flagellin, LTB, and TLR4.

[0126] In some implementations, the amino acid sequence of STING is shown in SEQ ID NO.71.

[0127] 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.72.

[0128] In some embodiments, the amino acid sequence of STING contains one or more mutations selected from V147L, N154S, V155M, R284M, R284K, R284T, E315Q, R375A and combinations thereof.

[0129] In some embodiments, the amino acid sequence of cGAS is shown in SEQ ID NO. 73. In some embodiments, the nucleotide sequence of cGAS is shown in SEQ ID NO. 74.

[0130] In some embodiments, the amino acid sequence of Flagellin16a is shown in SEQ ID NO.75. In some embodiments, the nucleotide sequence of Flagellin16a is shown in SEQ ID NO.76.

[0131] In some embodiments, the amino acid sequence of Flagellin is shown in SEQ ID NO. 77. In some embodiments, the nucleotide sequence of Flagellin is shown in SEQ ID NO. 78.

[0132] In some embodiments, the amino acid sequence of TLR4 is shown in SEQ ID NO. 79. In some embodiments, the amino acid sequence of LTB is shown in Uniprot P32890.

[0133] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein encodes at least one liver cancer-associated antigen, at least one genetic adjuvant, and at least one antigen-presenting enhancement molecule.

[0134] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein encodes at least one liver cancer-associated antigen, at least one genetic adjuvant, and at least one co-stimulatory molecule.

[0135] In some embodiments, the therapeutic nucleic acid for liver cancer disclosed herein encodes at least one liver cancer-associated antigen, at least one genetic adjuvant, at least one antigen-presenting enhancement molecule, and at least one co-stimulatory molecule.

[0136] 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.

[0137] In some embodiments, the genetic adjuvant is selected from at least one of STING, cGAS, Flagellin16a, Flagellin, LTB, and TLR4.

[0138] 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).

[0139] 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.

[0140] 3. Regarding protein compositions

[0141] The present invention provides a protein composition comprising a polypeptide encoded by a therapeutic nucleic acid for liver cancer as described above.

[0142] The present invention provides a protein composition comprising a polypeptide encoded by RNA as described above, which encodes an antigen-presenting molecule that enhances antigen presentation.

[0143] The present invention provides a protein composition comprising a polypeptide encoded by RNA as described above, which encodes a T-cell co-stimulatory molecule.

[0144] 4. Regarding vaccines

[0145] 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).

[0146] The present invention provides a vaccine, characterized in that the vaccine comprises the liver cancer therapeutic nucleic acid as described above.

[0147] In one embodiment, the vaccine further includes a delivery formulation of RNA encapsulating the therapeutic nucleic acid for liver cancer.

[0148] In an optional embodiment, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0149] In an optional embodiment, the present invention provides a vaccine preparation method according to any of the foregoing embodiments, characterized in that it includes: mixing the liver cancer therapeutic nucleic acid and a delivery formulation to form a vaccine; the delivery formulation includes lipid nanoparticles or cationic liposomes.

[0150] 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, wherein one independent nucleotide sequence is a first nucleotide sequence encoding at least one genetic adjuvant; at least one of the remaining one or more independent nucleotide sequences encodes at least one liver cancer-associated antigen; 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. 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.

[0151] The present invention provides a vaccine comprising the protein composition as described above.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] Example 1: Process for preparing LNPs from RNA

[0162] A lipid nanoparticle comprising a nucleic acid encoding a therapeutic liver cancer, wherein the lipid nanoparticle comprises, by molar percentage, 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol and 1% PEG-DMG.

[0163] 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.

[0164] (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.

[0165] (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 nucleic acids encoding therapeutic nucleic acids for liver cancer.

[0166] Example 2

[0167] 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 3Mice were randomly divided into four groups based on tumor volume, with a tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. The four groups were designated as eLNP group (injected with empty LNP), LRIO-202 group, LRIO-210 group, and LRIO-211 group, with six mice in each group. Mice were administered the drug via intramuscular injection on Days 0, 7, and 14 at a dose of 20 μg / mouse / dose. Tumor volume was measured 2-3 times per week after administration.

[0168] Among them, the LRIO-202 group, LRIO-210 group and LRIO-211 group were injected with lipid nanoparticles containing liver cancer therapeutic nucleic acid encoding GPC3. The lipid nanoparticles were prepared according to the method described in Example 1.

[0169] The liver cancer therapeutic nucleic acid contained in the LRIO-202 group injected contains T-cell co-stimulatory polypeptides encoded by CD80 and CD70, which encode liver cancer antigens GPC3(1) (amino acid sequence as shown in SEQ ID NO. 28) and GPC3(2) (amino acid sequence as shown in SEQ ID NO. 29). The connection form of the functional regions of the liver cancer therapeutic nucleic acid contained in the LRIO-202 group injected is CD80-GPC3(1)…P2A…GPC3(2)…T2A…CD70. The RNA sequence contained in the LRIO-202 group injected is shown in SEQ ID NO. 40, and the encoded amino acid sequence is shown in SEQ ID NO. 69.

[0170] The liver cancer therapeutic nucleic acid contained in the LRIO-210 group injected contains T-cell co-stimulatory polypeptides encoded by CD80 and CD70, which encode the liver cancer antigen GPC3 (9) (amino acid sequence as shown in SEQ ID NO. 36, nucleotide sequence as shown in SEQ ID NO. 56). The functional regions of the liver cancer therapeutic nucleic acid contained in the LRIO-210 group injected are linked in the form of CD80-GPC3 (9)…T2A…CD70. The CD80 RNA sequence contained in the LRIO-210 group injected is shown in SEQ ID NO. 24, and the CD80 is composed of sequentially linked amino acid sequences SEQ ID NO. 11 and SEQ ID NO. 17.

[0171] The liver cancer therapeutic nucleic acid contained in the LRIO-211 group contains T-cell co-stimulatory polypeptides encoded by CD80 and CD70, which encode liver cancer antigens GPC3(11)-GPC3(17) (amino acid sequences are shown in SEQ ID NO.38 and SEQ ID NO.42-47, respectively, and nucleotide sequences are shown in SEQ ID NO.58-64, respectively). The connection of the functional regions of the liver cancer therapeutic nucleic acid contained in the LRIO-211 group is CD80-GPC3(11)-GPC3(12)-GPC3(13)-GPC3(14)-GPC3(15)-GPC3(16)-GPC3(17) …T2A…CD70. The CD80 RNA sequence contained in the LRIO-211 group is shown in SEQ ID NO.24, and the CD80 is composed of sequentially linked amino acid sequences SEQ ID NO.11 and SEQ ID NO.17.

[0172] In addition to the open reading frame sequence described 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. The "GPC3(1)" and "GPC3(2)" described in this invention are truncated amino acid sequences of GPC3, respectively selected from the full-length GPC3 protein, and there is no sequence overlap between them.

[0173] The results of the average tumor volume change in each experimental group of mice during the experiment in this embodiment are shown in [the table below]. Figure 1 The results showed that, compared with the eLNP group, all other experimental groups could inhibit tumor growth, with the LRIO-202 group showing the best inhibitory effect on tumor growth.

[0174] Example 3

[0175] 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 3Mice were randomly divided into four groups based on tumor volume, with a tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. The four groups were designated as NC group (injected with empty LNP), LRIO-060 group, LRIO-202 group, and LRIO-201 group, with 6 mice in each group. Mice were administered the drug via intramuscular injection on Days 0, 7, and 14 at a dose of 20 μg / mouse / dose. Tumor volume was measured 2-3 times per week after administration.

[0176] Among them, the LRIO-060 group, LRIO-202 group and LRIO-201 group were injected with lipid nanoparticles containing liver cancer therapeutic nucleic acid encoding GPC3. The lipid nanoparticles were prepared according to the method described in Example 1.

[0177] The LRIO-060 group injected samples contained T-cell co-stimulatory peptides encoded by therapeutic nucleic acids for liver cancer, including CD80, CD70, and 4-1BBL. The antigen was GPC3 protein (GPC3, also known as DGSX, GTR2-2, MXR7, and OCI-5, is a member of the heparan sulfate (HS) proteoglycan family encoded by the GPC3 gene. The GPC3 gene is located on chromosome Xq26, and the protein it encodes contains a 580-amino acid core protein and two C-terminal HS chains. The GPC3 core protein is anchored to the cell membrane surface via glycosylphosphatidylinositol (GPI) and can be cleaved into an approximately 40 kDa N-terminal soluble protein (sGPC3) and a 30 kDa... C-terminal membrane protein. ), in which the connection between functional regions of the co-stimulatory molecular polypeptide encoded by the hepatocellular carcinoma therapeutic nucleic acid contained in the LRIO-060 group injection sample is as follows: CD80-GPC3(1)…P2A…4-1BBL-GPC3(2)…T2A…CD70.

[0178] The samples injected into the LRIO-202 group contained T-cell co-stimulatory molecular peptides encoded by therapeutic nucleic acids for liver cancer, including CD80 and CD70, with GPC3 protein as the antigen. The connection between the functional regions of the co-stimulatory molecular peptides encoded by therapeutic nucleic acids for liver cancer in the samples injected into the LRIO-202 group was as follows: CD80-GPC3(1)…P2A…GPC3(2)…T2A…CD70.

[0179] The liver cancer therapeutic nucleic acid contained in the LRIO-201 group injection sample does not encode T cell co-stimulatory molecule polypeptide, but only encodes antigen GPC3 protein. The connection form between the functional regions of the polypeptide encoded by the liver cancer therapeutic nucleic acid contained in the LRIO-201 group injection sample is: GPC3(1)…P2A…GPC3(2).

[0180] In this embodiment, the co-stimulatory molecules CD80, CD70, and 4-1BBL are all mouse-derived molecules; CD80 includes an amino acid sequence as shown in SEQ ID NO.11 and SEQ ID NO.17; the amino acid sequence of CD70 is shown in SEQ ID NO.15; and the amino acid sequence of 4-1BBL is shown in SEQ ID NO.21, SEQ ID NO.13, SEQ ID NO.19, and SEQ ID NO.20.

[0181] In this embodiment, the amino acid sequence of antigen GPC3(1) protein is shown in SEQ ID NO.28, and the amino acid sequence of antigen GPC3(2) protein is shown in SEQ ID NO.29.

[0182] In this embodiment, the liver cancer therapeutic nucleic acid contained in the sample injected into the LRIO-060 group is RNA, and the RNA sequence is shown in SEQ ID NO. 39. The liver cancer therapeutic nucleic acid contained in the sample injected into the LRIO-202 group is RNA, and the RNA sequence is shown in SEQ ID NO. 40. The liver cancer therapeutic nucleic acid contained in the sample injected into the LRIO-201 group is RNA, and the RNA sequence is shown in SEQ ID NO. 41.

[0183] The therapeutic nucleic acid for liver cancer in this example is RNA. In addition to the open reading frame sequence described above, the RNA 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. The "GPC3(1)" and "GPC3(2)" described in this invention are truncated amino acid sequences of GPC3, respectively selected from the full-length GPC3 protein, and there is no sequence overlap between them.

[0184] The results of the average tumor volume change in each experimental group of mice during the experiment in this embodiment are shown in the figure. Figure 2 The results showed that, compared with the NC group, all other experimental groups could inhibit tumor growth. Among them, the tumor-inhibiting effects of the LRIO-060 group and the LRIO-202 group were comparable and superior to the LRIO-201 group.

[0185] Example 4

[0186] 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 mm3 Mice were randomly divided into three groups based on tumor volume, with a tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. The three groups were designated as G1 (injection of empty LNP), G2, and G3, with 6 mice in each group. The drugs were administered intramuscularly on Days 0, 7, and 14 at a dose of 20 μg / mouse / dose. Tumor volume was measured 2-3 times per week after administration.

[0187] In both groups G2 and G3, lipid nanoparticles containing hepatocellular carcinoma therapeutic nucleic acid encoding GPC3 were injected. The lipid nanoparticles were prepared according to the method described in Example 1.

[0188] The sample injected into group (LRIO-203) contained CD80, a T-cell co-stimulatory molecule polypeptide encoded by a therapeutic nucleic acid for liver cancer, and the antigen was GPC3 protein (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, is a member of the heparan sulfate (HS) proteoglycan family encoded by the GPC3 gene). The connection between the functional regions of the polypeptide encoded by the therapeutic nucleic acid for liver cancer in the sample injected into group G2 was: CD80-GPC3(1)…P2A…GPC3(2).

[0189] The liver cancer therapeutic nucleic acid contained in the sample injected in the G3 (LRIO-201) group does not encode the T cell co-stimulatory molecule polypeptide, but only encodes the antigen GPC3 protein. The connection form between the functional regions of the polypeptide encoded by the liver cancer therapeutic nucleic acid contained in the sample injected in the G3 group is: GPC3(1)…P2A…GPC3(2).

[0190] In this embodiment, the co-stimulatory molecule CD80 is a mouse-derived molecule; wherein CD80 includes an amino acid sequence such as SEQ ID NO.11 and SEQ ID NO.17.

[0191] In this embodiment, the amino acid sequence of antigen GPC3(1) protein is shown in SEQ ID NO.28, and the amino acid sequence of antigen GPC3(2) protein is shown in SEQ ID NO.29.

[0192] In this embodiment, the liver cancer therapeutic nucleic acid sequence contained in the sample injected in group G2 is shown in SEQ ID NO.70, and the liver cancer therapeutic nucleic acid sequence contained in the sample injected in group G3 is shown in SEQ ID NO.41.

[0193] The therapeutic nucleic acid for liver cancer in this example is RNA. In addition to the open reading frame sequence described above, the RNA 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. The "GPC3(1)" and "GPC3(2)" described in this invention are truncated amino acid sequences of GPC3, respectively selected from the full-length GPC3 protein, and there is no sequence overlap between them.

[0194] The results of the average tumor volume change in each experimental group of mice during the experiment in this embodiment are shown in the figure. Figure 3 The results showed that, compared with the G1 group, all other experimental groups were able to inhibit tumor growth.

[0195] Example 5

[0196] C57BL / 6 mice (female, 5-6 weeks old, average weight 15-20g, purchased from Zhuhai Beston Biotechnology Co., Ltd.) were selected and randomly divided into two groups, with the day of grouping defined as D0. The two groups were designated as Empty LNP group and LRIO-060 group, with 4 mice in each group. The mice were administered the drug via intramuscular injection on Days 0, 7, and 14, at a dose of 20 μg / mouse / dose.

[0197] On Day 28, mouse spleen cells were stimulated with GPC3 antigen peptide (purchased from Jiangsu Genscript Biotech Co., Ltd.) for 2 days. The levels of CD8+ T cells expressing IFNγ or TNFα were measured, as were the levels of IFNγ or TNFα in the cell supernatant. Immunized mouse spleen cells and MC38 / GPC3 tumor cells were mixed at ratios of 25:1, 50:1, or 100:1 and co-cultured for 24 hours. The killing effect of spleen cells on tumor cells was then assessed.

[0198] 1. Method for detecting the content of CD8+ T cells expressing IFNγ or TNFα. Mouse spleen cells were stimulated with GPC3 antigen peptide for 32 hours, and then cultured for another 16 hours in Brefeldin A Solution (purchased from BD Biosciences). Spleen cells were collected, surface stained with fluorescently labeled anti-CD3, CD4, and CD8 specific monoclonal antibodies (purchased from BioLegend), and then permeabilized with Fixation / Permeabilization solution (purchased from BD Biosciences). Intracellular staining was then performed with fluorescently labeled anti-TNF-α and anti-IFN-γ antibodies (purchased from BioLegend). Finally, the content of T cells expressing IFNγ or TNFα was detected and analyzed by flow cytometry.

[0199] 2. Method for detecting the content of IFNγ or TNFα in cell supernatant. Mouse spleen cells were stimulated with GPC3 antigen peptide for 48 hours, and the supernatant was collected to detect the content of cytokines IFNγ and TNFα (kit purchased from Dakota).

[0200] 3. Method for detecting the killing effect of spleen cells on tumor cells. Mouse spleen cells were stimulated with GPC3 antigen peptide for 48 hours, and then collected. MC38 / GPC3 tumor cells were inoculated into 96-well plates. After 16-24 hours of adhesion, the stimulated spleen cells were collected and mixed with tumor cells at a ratio of 25:1, 50:1, or 100:1 (E:T). After culturing for 24 hours, the supernatant was collected to detect the LDH (lactate dehydrogenase) content (detection kit purchased from Thermo Fisher Scientific), and the killing effect of spleen cells on tumor cells was calculated.

[0201] The experimental results of CD8+ T cell content in mouse spleen cells expressing IFNγ or TNFα are as follows: Figure 4 As shown, the results indicated that LRIO-060 significantly induced the expression of IFNγ and TNFα in CD8+ T cells compared to Empty LNP (P<0.05). The experimental results of IFNγ or TNFα content in mouse spleen cell supernatant are shown below. Figure 5 As shown in the figure, the results indicated that LRIO-060 significantly induced the release of cytokines IFNγ and TNFα compared to Empty LNP (P<0.05). The results of the spleen cell killing experiment against tumor cells are as follows... Figure 6 As shown, the results indicate that, compared with Empty LNP, GPC3 antigen-specific T cells induced by LRIO-060 can significantly kill MC38 / GPC3 tumor cells (P<0.05).

[0202] Example 6

[0203] 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 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.

[0204] 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.

[0205] The sample used in the LRIO-060 group is the same as that in Example 2. 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.

[0206] In this embodiment, the amino acid sequence of STING is based on the amino acid sequence shown in SEQ ID NO.71, with a V155M mutation. The amino acid sequence of cGAS is shown in SEQ ID NO.73.

[0207] In this embodiment, the sample injected into the LRIO-039 group contains the STING RNA sequence as shown in SEQ ID NO. 72, and the sample injected into the LRIO-055 group contains the cGAS RNA sequence as shown in SEQ ID NO. 74.

[0208] 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.

[0209] 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.

[0210] The results of this embodiment are shown below. Figure 7 The results showed that, compared with the empty LNP 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.

[0211] 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 encodes at least two of the liver cancer antigens GPC3(1) to GPC3(17).

2. The therapeutic nucleic acid for liver cancer as described in claim 1, characterized in that, The therapeutic nucleic acid for liver cancer encodes liver cancer antigens GPC3 (1) and GPC3 (2), the amino acid sequence of liver cancer antigen GPC3 (1) is shown in SEQ ID NO.28, and the amino acid sequence of liver cancer antigen GPC3 (2) is shown in SEQ ID NO.

29.

3. 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.

4. The therapeutic nucleic acid for liver cancer as described in claim 3, characterized in that, The co-stimulatory molecules are CD80 and CD70; 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 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 3, 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, Therapeutic nucleic acids for liver cancer are either DNA or RNA molecules.

7. The therapeutic nucleic acid for liver cancer as described in claim 1, characterized in that, The therapeutic nucleic acid for liver cancer also encodes at least one genetic adjuvant selected from STING, cGAS, Flagellin16a, Flagellin, LTB, and TLR4.

8. A protein composition comprising a polypeptide encoded by a therapeutic nucleic acid for liver cancer as described in claims 1-7.

9. A vaccine, characterized in that, The vaccine comprises the liver cancer therapeutic nucleic acid as described in claims 1-7, or the protein composition as described in claim 8.

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 the therapeutic nucleic acid for liver cancer as described in any one of claims 1-7.

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