mRNA vaccine for treating HPV infection-related diseases

Abstract

A polynucleotide molecule for preventing or treating HPV infection-related diseases, and an mRNA vaccine, pharmaceutical composition or pharmaceutical product containing the polynucleotide.

Description

mRNA vaccines for the treatment of HPV infection-related diseases Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an mRNA vaccine for treating HPV infection-related diseases by inducing HPV antigen-specific immune responses. Background Art

[0002] Diseases caused by high-risk human papillomavirus (HPV) infection account for 5% of all diseases worldwide, with 70% of cervical cancers caused by persistent infection with HPV types 16 and 18. The HPV genome contains up to seven early genes (E1-E7) and two late genes (L1 and L2). The E6 and E7 proteins are expressed in nearly all cervical cancer cells and are essential for maintaining the disease phenotype, making them ideal targets for therapeutic vaccines.

[0003] HPV therapeutic vaccines currently under development primarily include DNA vaccines, subunit vaccines, and recombinant vector vaccines. mRNA vaccines offer advantages such as no integration risk, a short half-life, and improved safety. They can express viral antigens to induce antigen-specific immune responses, killing infected cells, such as tumor cells, and thereby treating related tumors. The purpose of this application is to develop an mRNA vaccine for the treatment of HPV-related diseases.

[0004] SUMMARY OF THE INVENTION

[0005] The present application provides preventive or therapeutic nucleic acids and fusion polypeptides for HPV infection-related diseases, pharmaceutical compositions or pharmaceutical products comprising the therapeutic nucleic acids or fusion polypeptides, and uses of the nucleic acids and fusion polypeptides.

[0006] Specifically, on one hand, the present application provides a polynucleotide molecule comprising at least a coding sequence of an HPV antigen polypeptide, wherein the antigen polypeptide comprises at least the following from the N-terminus to the C-terminus:

[0007] 1) amino acid sequence A, and amino acid sequence B;

[0008] 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B;

[0009] 3) amino acid sequence B, and amino acid sequence A;

[0010] 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A;

[0011] 5) amino acid sequence A, amino acid sequence B and amino acid sequence C;

[0012] 6) amino acid sequence B, amino acid sequence A, and amino acid sequence C;

[0013] 7) amino acid sequence A, amino acid sequence C and amino acid sequence B or

[0014] 8) Amino acid sequence B, amino acid sequence C and amino acid sequence A.

[0015] Wherein, the amino acid sequence A comprises SEQ ID NO: 1, 2, 3, 4 or variants thereof from N-terminus to C-terminus, and the amino acid sequences shown in the SEQ ID NOs are sequentially connected directly or sequentially through connecting peptides;

[0016] The amino acid sequence B comprises SEQ ID NO: 5, 6, 7, 8 or variants thereof from N-terminus to C-terminus, and the amino acid sequences represented by the SEQ ID NOs are sequentially connected directly or sequentially through connecting peptides;

[0017] Amino acid sequence C comprises the HPV E2 antigen sequence.

[0018] In some embodiments, the variants are conservative substitution variants. In some embodiments, the amino acid sequence of each of the variants of SEQ ID NOs: 1-4 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% sequence identity with the amino acid sequence of one of SEQ ID NOs: 1-4, respectively. In some embodiments, the amino acid sequence of each of the variants of SEQ ID NOs: 5-8 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% sequence identity with the amino acid sequence of one of SEQ ID NOs: 5-8, respectively.

[0019] In some embodiments, the amino acid sequence C is an HPV E2 antigen sequence.

[0020] In some embodiments, the HPV E2 antigen sequence is SEQ ID NO: 9 or a variant thereof. In some embodiments, the variant of SEQ ID NO: 9 is a conservative substitution variant. In some embodiments, the variant of SEQ ID NO: 9 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO: 9.

[0021] In some embodiments, the connecting peptide comprises one, two or more amino acid residues. In some embodiments, the connecting peptide is a flexible connecting peptide, a rigid connecting peptide, or a combination thereof. In some embodiments, each segment of the amino acid sequence shown in each SEQ ID NO is connected by a different connecting peptide. In some embodiments, each segment of the amino acid sequence shown in each SEQ ID NO is connected by the same connecting peptide. In some embodiments, the connecting peptide consists of 2-10 amino acid residues. In some embodiments, the amino acid residues are glycine, serine and / or alanine residues. In some embodiments, the connecting peptide is selected from GS linker, (Gly)8, α-helical peptide, (XP)n, etc. In some embodiments, each segment of the amino acid sequence shown in SEQ ID NO is connected by two alanine residues.

[0022] In some embodiments, the HPV antigen polypeptide comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or comprises an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity thereto. In some embodiments, the HPV antigen polypeptide comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or comprises an amino acid sequence having 98%, 98.5%, 99%, or 99.5% or greater sequence identity thereto. In some embodiments, the HPV antigen polypeptide is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0023] In some embodiments, the polynucleotide molecule further comprises a coding sequence for an immunostimulatory factor or its functional domain. In some embodiments, the coding sequence for the immunostimulatory factor or its functional domain is located on the 3' or 5' side of the coding sequence for the HPV antigen polypeptide. In some embodiments, the immunostimulatory factor is selected from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, Pre-IFNα-2β, MIP-α, STING, HSP70, immune checkpoint inhibitors. In some embodiments, the immunostimulatory factor is an antibody or antigen-binding fragment thereof against any one or more of the following checkpoint molecules: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, PD-1, VSIG8. In some embodiments, the immunostimulatory factor is Flt3L. In some embodiments, the polypeptide sequence of the immunostimulatory factor comprises at least the amino acid sequence of SEQ ID NO: 10, or a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 10. In some embodiments, the polypeptide sequence of the immunostimulatory factor is the amino acid sequence of SEQ ID NO: 10, or a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 10. In some embodiments, the coding sequence of the immunostimulatory factor comprises the polynucleotide sequence of SEQ ID NO: 29 or is the polynucleotide sequence of SEQ ID NO: 29. In some embodiments, the coding sequence of the immunostimulatory factor is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 29.

[0024] In some embodiments, the coding sequence of the immunostimulatory factor comprises a synonymous variant of SEQ ID NO: 29 or is a synonymous variant of SEQ ID NO: 29.

[0025] In some embodiments, the polynucleotide molecule further comprises a signal peptide coding sequence. In some embodiments, the signal peptide coding sequence is located 5' of the coding sequence for the HPV antigen polypeptide. In some embodiments, the signal peptide is a secretory signal peptide. In some embodiments, the secretory signal peptide is selected from the signal peptides of secretory proteins of mammals. In some embodiments, the mammal is a human. In some embodiments, the secretory signal peptide is tPA-SP. In some embodiments, the secretory signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 11, or a conservatively substituted variant of SEQ ID NO: 11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 11. In some embodiments, the secretory signal peptide is the amino acid sequence set forth in SEQ ID NO: 11, or a conservatively substituted variant of SEQ ID NO: 11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 11. In some embodiments, the coding sequence of the secretory signal peptide comprises the polynucleotide sequence shown in SEQ ID NO: 28 or is the polynucleotide sequence shown in SEQ ID NO: 28. In some embodiments, the coding sequence of the secretory signal peptide is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 28.

[0026] In some embodiments, the coding sequence of the secretory signal peptide comprises or is a synonymous mutant of SEQ ID NO: 28.

[0027] In some embodiments, the polynucleotide molecule comprises the coding sequence of the signal peptide, the immunostimulatory factor and the HPV antigen polypeptide connected in sequence from the 5' end to the 3' end. In some embodiments, the coding sequence of the signal peptide, the coding sequence of the immunostimulatory factor and the coding sequence of the HPV antigen polypeptide are directly connected or connected through a polynucleotide chain. In some embodiments, the polynucleotide chain comprises 3 or multiples of 3 nucleotides. Or in some embodiments, the polynucleotide molecule consists of the coding sequence of the signal peptide, the coding sequence of the immunostimulatory factor and the coding sequence of the HPV antigen polypeptide connected in sequence from the 5' end to the 3' end. In some embodiments, the coding sequence of the signal peptide, the coding sequence of the immunostimulatory factor and the coding sequence of the HPV antigen polypeptide are located in the same reading frame. In some embodiments, the coding sequence of the reading frame is any one of the polynucleotide sequences shown in SEQ ID NOs: 47-54, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to any one of the polynucleotide sequences shown in SEQ ID NOs: 47-54; in some embodiments, the reading frame encodes a protein as shown in any one of SEQ ID NOs: 17-21, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to any one of the proteins shown in SEQ ID NOs: 17-21.

[0028] In some embodiments, the polynucleotide molecule is DNA, RNA, or a hybrid of DNA and RNA. In some embodiments, the polynucleotide molecule is extracted from a cell. In some embodiments, the polynucleotide molecule is chemically synthesized. In some embodiments, the polynucleotide molecule is not chemically modified in vitro. In some embodiments, the polynucleotide molecule is chemically modified in vitro. In some embodiments, the chemical modification is selected from one or more of the following: m6A, m1A, m5C, m7G, ac4C, 2'-O-methylation, and pseudouracil substitution.

[0029] In some embodiments, the polynucleotide molecule comprises any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 39-54. In some embodiments, the polynucleotide molecule comprises a nucleic acid fragment encoded by any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule is encoded by a nucleic acid encoded by any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule is encoded by a nucleic acid encoded by any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 39-54. In some embodiments, the polynucleotide molecule comprises a sequence complementary to any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of a sequence complementary to any polynucleotide sequence selected from the group consisting of SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of a sequence complementary to any one of the polynucleotide sequences selected from SEQ ID NOs: 39-54.

[0030] In some embodiments, the polynucleotide molecule further comprises a 5'UTR structure. In some embodiments, the polynucleotide molecule comprises a 3'UTR structure. In some embodiments, the polynucleotide molecule further comprises a 5'UTR structure and a 3'UTR structure. In some embodiments, the 5'UTR structure comprises at least the polynucleotide sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85%, or 80% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the 5'UTR structure is a polynucleotide sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85%, or 80% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the 3'UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 23 or SEQ ID NO: 26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85% or 80% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26. In some embodiments, the 3'UTR structure is the polynucleotide sequence shown in SEQ ID NO: 23 or SEQ ID NO: 26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85% or 80% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26.

[0031] In some embodiments, the polynucleotide molecule is an mRNA molecule. In some embodiments, some or all of the uridine in the mRNA molecule is pseudouridine or 1-methyl-pseudouridine. In some embodiments, the mRNA further comprises a 5' cap structure. In some embodiments, the 5' cap structure is type O, type I, and type II. In some embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the mRNA further comprises a polyA tail. In some embodiments, the polyA tail sequence comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 adenylate residues. In some embodiments, the polyA tail comprises at most 500, at most 400, at most 300, at most 200, at most 150, at most 140, at most 130, at most 120, at most 110, at most 100, at most 90, at most 80, at most 70, at most 60 adenylate nucleotides (A), in particular about 120 A. In some embodiments, the polyA tail comprises at least the polynucleotide sequence as shown in SEQ ID NO: 24 or SEQ ID NO: 27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity thereto. In some embodiments, the polyA tail is a polynucleotide sequence as shown in SEQ ID NO:24 or SEQ ID NO:27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:24 or SEQ ID NO:27.

[0032] In addition, the present application also provides a polynucleotide molecule comprising a sequence complementary to the polynucleotide sequence of the above-mentioned polynucleotide molecule.

[0033] The polynucleotide molecules provided herein are, in some embodiments, single-stranded molecules, double-stranded molecules, or circular molecules. In some embodiments, the polynucleotide molecules provided herein comprise single-stranded and double-stranded structures.

[0034] In a preferred embodiment, the polynucleotide molecule is an mRNA molecule, all uridines in the mRNA molecule are 1-methyl-pseudouridine, and the mRNA further comprises a 5' cap structure, wherein the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG,

[0035] The mRNA molecule sequence comprises or is 5'UTR, ORF, 3'UTR, and poly A tail in sequence from the 5' end to the 3' end, and the 5'UTR, ORF, 3'UTR, and poly A tail are operably connected to each other.

[0036] The 5'UTR sequence is SEQ ID NO: 25, the ORF sequence is SEQ ID NO: 54, the 3'UTR sequence is SEQ ID NO: 26, and the poly A tail sequence is SEQ ID NO: 27.

[0037] In some embodiments, the mRNA molecule comprises a polynucleotide sequence as shown in SEQ ID NO: 54, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity thereto.

[0038] In a second aspect, the present application further provides a fusion polypeptide. In some embodiments, the fusion polypeptide is encoded by the polynucleotide molecule described in the first aspect. In some embodiments, the fusion polypeptide comprises at least:

[0039] 1) amino acid sequence A, and amino acid sequence B;

[0040] 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B;

[0041] 3) amino acid sequence B, and amino acid sequence A;

[0042] 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A;

[0043] 5) amino acid sequence A, amino acid sequence B and amino acid sequence C;

[0044] 6) amino acid sequence B, amino acid sequence A, and amino acid sequence C;

[0045] 7) amino acid sequence A, amino acid sequence C and amino acid sequence B or

[0046] 8) Amino acid sequence B, amino acid sequence C and amino acid sequence A.

[0047] Wherein, the amino acid sequence A comprises SEQ ID NO: 1, 2, 3, 4 or variants thereof from N-terminus to C-terminus, and the amino acid sequences shown in the SEQ ID NOs are sequentially connected directly or sequentially through connecting peptides;

[0048] Amino acid sequence B comprises SEQ ID NO: 5, 6, 7, 8 or variants thereof, and the amino acid sequences represented by the SEQ ID NOs are sequentially connected directly or sequentially via connecting peptides;

[0049] Amino acid sequence C comprises the HPV E2 antigen sequence.

[0050] Preferably, the variants are conservative substitution variants.

[0051] In some embodiments, the amino acid sequence C is an HPV E2 antigen sequence.

[0052] In some embodiments, the HPV E2 antigen sequence is SEQ ID NO:9.

[0053] In some embodiments, the connecting peptide comprises one, two, or more amino acid residues. In some embodiments, the connecting peptide is a flexible connecting peptide, a rigid connecting peptide, or a combination thereof. In some embodiments, the amino acid sequences of each SEQ ID NO are connected by different connecting peptides. In some embodiments, the amino acid sequences of each SEQ ID NO are connected by the same connecting peptide. In some embodiments, the amino acid sequences of each SEQ ID NO are connected by two alanine residues.

[0054] In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity thereto. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an amino acid sequence having 98%, 98.5%, 99%, or 99.5% or greater sequence identity thereto. In some embodiments, the fusion polypeptide is the amino acid sequence shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0055] In some embodiments, the fusion polypeptide further comprises a full-length immunostimulatory factor polypeptide or a functional domain thereof. In some embodiments, the immunostimulatory factor protein or its functional domain is located at the C-terminus or N-terminus of the fusion polypeptide. In some embodiments, the immunostimulatory factor is selected from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, Pre-IFNα-2β, MIP-α, STING, MHSP70, immune checkpoint inhibitors. In some embodiments, the immunostimulatory factor is an antibody or antigen-binding fragment thereof against any one or more of the following checkpoint molecules: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, PD-1, VSIG8. In some embodiments, the immunostimulatory factor is Flt3L. In some embodiments, the polypeptide sequence of the immunostimulatory factor comprises at least the amino acid sequence as shown in SEQ ID NO: 10, or a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 10. In some embodiments, the polypeptide sequence of the immunostimulatory factor is the amino acid sequence as shown in SEQ ID NO: 10, or a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 10.

[0056] In some embodiments, the fusion polypeptide further comprises a signal peptide. In some embodiments, the signal peptide is located at the C-terminus or N-terminus of the fusion polypeptide. In some embodiments, the signal peptide is a secretory signal peptide. In some embodiments, the secretory signal peptide is selected from the signal peptides of secretory proteins of mammals. In some embodiments, the mammal is a human. In some embodiments, the secretory signal peptide is tPA-SP. In some embodiments, the secretory signal peptide comprises the amino acid sequence set forth in SEQ ID NO:11, or a conservatively substituted variant of SEQ ID NO:11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:11. In some embodiments, the secretory signal peptide is the amino acid sequence set forth in SEQ ID NO:11, or a conservatively substituted variant of SEQ ID NO:11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:11.

[0057] In some embodiments, the fusion polypeptide comprises the signal peptide, the immunostimulatory factor, and the coding sequence of the fusion polypeptide linked sequentially from the N-terminus to the C-terminus.

[0058] In addition, the present application also provides HPV E2 antigen polypeptides, polynucleotide molecules encoding the HPV E2 antigen polypeptides, and uses thereof. In some embodiments, the HPV E2 antigen polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the HPV E2 antigen polypeptide is shown in SEQ ID NO: 9. In some embodiments, the HPV E2 antigen polypeptide comprises or consists of a conservative substitution variant of SEQ ID NO: 9. The uses of the HPV E2 antigen polypeptide include administering it in combination with other HPV antigen polypeptides to an individual in need, or administering it to an individual in need after fusing it with other HPV antigen polypeptides, so that the individual obtains a stronger immune response against HPV, and the stronger immune response refers to an immune response that is stronger than the immune response obtained by the individual when only the other HPV antigen polypeptides are administered. In some embodiments, the polynucleotide molecule encoding the HPV E2 antigen polypeptide comprises or consists of a polynucleotide sequence as SEQ ID NO: 38. In some embodiments, the polynucleotide molecule encoding the HPV E2 antigen polypeptide comprises or consists of a conservatively substituted variant of the polynucleotide sequence of SEQ ID NO: 38, or is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 38. The use of the polynucleotide molecule encoding the HPV E2 antigen polypeptide comprises administering it in combination with a polynucleotide molecule encoding other HPV antigen polypeptides to an individual in need thereof, or ligating the polynucleotide sequence to a sequence of a polynucleotide molecule encoding other HPV antigen polypeptides to form a new polynucleotide molecule and then administering it to an individual in need thereof to express the HPV E2 antigen polypeptide and the other HPV antigen polypeptide, or a fusion protein of the HPV E2 antigen polypeptide and the other HPV antigen polypeptide. In some embodiments, the individual in need thereof suffers from cervical cancer.

[0059] The third aspect of the present application provides a kind of delivery body, it comprises the polynucleotide molecule of above-mentioned first aspect or the fusion polypeptide of above-mentioned second aspect.In some embodiments, described delivery body is liposome, viral particle or quantum dot.In some embodiments, described delivery body is LNP (lipid nanoparticle).In some embodiments, described LNP comprises the lipid, non-cationic lipid, sterol, ionizable lipid or its any combination that PEG modifies.In some embodiments, described LNP is made up of ionizable lipid, phospholipid, cholesterol, polyethylene glycol (PEG)-lipid and the polynucleotide molecule of above-mentioned first aspect.

[0060] In the lipid nanoparticle composition of the present application, the ionizable lipid may further include, for example, one or more of the following: 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2 ...4-(2-dimethylaminoethyl)-[1,3]-dioxolane Oleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3 ]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl-3-morpholinopropane (DLin-MA), 1,2 -Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazin-o)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2 ...OTAP), N,N-dimethyl-N-carboxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), dioctadecylaminoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybut-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β- oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',1-2'-octadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), MC3, SM-102 or ALC-0315 and their salts or isomers.

[0061] In the present application, the phospholipids may be selected from one or more of the following compounds:

[0062] Dilauroyl phosphatidylcholine (DLPC),

[0063] Dimyristoylphosphatidylcholine (DMPC),

[0064] Dioleoylphosphatidylcholine (DOPC),

[0065] Dipalmitoylphosphatidylcholine (DPPC),

[0066] Distearoylphosphatidylcholine (DSPC),

[0067] Dioleoylphosphatidylcholine (DUPC),

[0068] Palmitoyloleoylphosphatidylcholine (POPC),

[0069] 1,2-Di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC),

[0070] 1-oleoyl-2-cholesteryldimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0071] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0072] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0073] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0074] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0075] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0076] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0077] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0078] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0079] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0080] 1,2-Dioleoyl-sn-glycero-3-phospho-(1-glycerol) sodium salt (DOPG) or sphingomyelin.

[0081] In a specific embodiment, the phospholipid is DSPC.

[0082] In a specific embodiment, the phospholipid is DOPE.

[0083] In a specific embodiment, the phospholipid is a combination of DSPC and DOPE.

[0084] In the present application, the structured lipid can be selected from the group consisting of, but not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0085] In the present application, the PEG lipid is alternatively referred to as a PEGylated lipid. The PEG lipid is a lipid modified with polyethylene glycol. The PEG lipid can be selected from the non-limiting group of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

[0086] In a specific embodiment, the PEG lipid is DMG-PEG2000.

[0087] In some embodiments of the present application, in the lipid nanoparticle composition, the content of the ionizable lipid is 35 mol%-65 mol%, for example, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, %, 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47. 5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mol%, 4 8.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol %, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54.5 mol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 6 0 mol%, 60.5 mol%, 61 mol%, 61.5 mol%, 62 mol%, 62.5 mol%, 63 mol%, 63.5 mol%, 64 mol%, 64.5 mol%, 65 mol%.

[0088] In some embodiments of the present application, in the lipid nanoparticle composition, the content of the sum of the phospholipids and the structural lipids may be 35 mol%-65 mol%, for example, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%. , 42mol%, 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol %, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mo l%, 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8 mol%, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54 .5mol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 60mol%, 60.5mol%, 61mol%, 61.5mol%, 62mol%, 62.5mol%, 63mol%, 63.5mol%, 64mol%, 64.5mol%, 65mol%.

[0089] In some embodiments of the present application, in the lipid nanoparticle composition, the phospholipid content can be 5 mol%-15 mol%, for example, it can be 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, 15 mol%; for example, it can be 10 mol%-15 mol%.

[0090] In some embodiments of the present application, in the lipid nanoparticle composition, the structural lipid content can be 30 mol%-50 mol%, for example, it can be 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, 42.5 mol%, 43 mol%, 43.5 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7m ol%, 48.8mol%, 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol%, 49.9mol%, 50mol%; for example, it can be 35mol%-45mol%.

[0091] In some embodiments of the present application, in the lipid nanoparticle composition, the content of the PEG lipid is 0.5 mol%-5 mol%, for example, it can be 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%. ol%, 2.5mol%, 2.6mol%, 2.7mol%, 2.8mol%, 2.9mol%, 3mol%, 3.1mol%, 3.2mol%, 3.3mol%, 3.4mol%, 3.5mol%, 3.6mol%, 3.7mo 1%, 3.8mol%, 3.9mol%, 4mol%, 4.1mol%, 4.2mol%, 4.3mol%, 4.4mol%, 4.5mol%, 4.6mol%, 4.7mol%, 4.8mol%, 4.9mol%, 5mol%.

[0092] In a specific embodiment, the lipid nanoparticle composition comprises ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 35 mol%-65 mol%, the content of the sum of phospholipids and cholesterol is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%.

[0093] In a specific embodiment, the lipid nanoparticle composition comprises ionizable lipid, phospholipid, cholesterol, and PEG lipid, wherein the content of ionizable lipid is 40 mol%-50 mol%, the content of phospholipid is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipid is 1.5 mol%-2.5 mol%.

[0094] In a specific embodiment, the lipid nanoparticle composition comprises ionizable lipid, phospholipid, cholesterol, and PEG lipid, wherein the content of ionizable lipid is 48 mol%-50 mol%, the content of phospholipid is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipid is 1.5 mol%-2.5 mol%.

[0095] In some embodiments of the present application, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is 35-65:35-65:0.5-5.

[0096] In the lipid nanoparticle composition of the present application, the molar ratio of the sum of the ionizable lipid, the phospholipid and the structural lipid, and the PEG lipid is (35-65): (35-65): (0.5-5), and 35-65 can take any value between 35-65, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 0.5-5 can take any value between 0.5-5, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.

[0097] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is (40-50): (35-65): (1-3).

[0098] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the sum of the phospholipid and the structural lipid, and the PEG lipid is (50-65): (35-65): (1-3).

[0099] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-65):(5-15):(30-50):(1-3).

[0100] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-65):(10-15):(35-45):(1.5-2.5).

[0101] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(30-50):(1-3). Wherein, for the molar ratio (40-50): (5-15): (30-50): (1-3), 40-50 can take any value between 40-50, for example, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 5-15 can take any value between 5-15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can take any value between 30-50, for example, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46 , 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1-3, for example, it can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0102] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (40-50):(5-15):(35-45):(1.5-2.5). Wherein, for the molar ratio (40-50): (5-15): (35-45): (1.5-2.5), 40-50 can take any value between 40-50, for example, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 5-15 can take any value between 5-15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can take any value between 35-45, for example, it can be 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can take any value between 1.5-2.5, for example, it can be 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0103] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(5-15):(30-50):(1-3). In the molar ratio (50-65):(5-15):(30-50):(1-3), 50-65 can take any value between 50-65, for example, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60. 5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can take any value between 5-15, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can take any value between 5-15, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; Take any value between 30-50, for example, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45 , 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1-3, for example, it can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0104] In a specific embodiment, in the lipid nanoparticle composition, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (50-65):(5-15):(35-45):(1.5-2.5). Wherein, for the molar ratio (50-65): (5-15): (35-45): (1.5-2.5), 50-65 can take any value between 50-65, for example, it can be 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can take any value between 5-15, for example, it can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can take any value between 35-45, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can take any value between 1.5-2.5, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0105] In a specific embodiment, the therapeutic and / or prophylactic agent is mRNA, and the N / P ratio of the ionizable lipid to the mRNA in the lipid nanoparticles is 3.5-5.5, for example, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5.

[0106] The fourth aspect of the present application provides a cell comprising the polynucleotide molecule of the first aspect or the fusion polypeptide of the second aspect. In some embodiments, the cell is a bacterial, fungal or mammalian cell.

[0107] The fifth aspect of the present application provides a pharmaceutical composition or pharmaceutical product or kit, which comprises the polynucleotide molecule of the first aspect, the fusion polypeptide of the second aspect, the delivery body of the third aspect, and / or the cell of the fourth aspect. In some embodiments, the pharmaceutical composition or pharmaceutical product is an mRNA vaccine, and it comprises the mRNA in the polynucleotide molecule of the first aspect. In some embodiments, the mRNA is selected from any one of the polynucleotides shown in SEQ ID NOs: 28-54 or is encoded by a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NOs: 28-54. In some embodiments, the mRNA is selected from any one of the polynucleotides shown in SEQ ID NOs: 39-54 or is encoded by a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NOs: 39-54.

[0108] In some embodiments, the mRNA further comprises a 5'UTR structure. In some embodiments, the mRNA comprises a 3'UTR structure. In some embodiments, the mRNA further comprises a 5'UTR structure and a 3'UTR structure. In some embodiments, the 5'UTR structure comprises at least the polynucleotide sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the 5'UTR structure is a polynucleotide sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the 3'UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 23 or SEQ ID NO: 26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26. In some embodiments, the 3'UTR structure is the polynucleotide sequence shown in SEQ ID NO: 23 or SEQ ID NO: 26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26.

[0109] In some embodiments, the mRNA molecule of the present invention is a mature mRNA molecule, which comprises, from the 5' to the 3' end, a 5' cap, a 5' UTR, the coding sequence of the secretory signal peptide, the coding sequence of the immunostimulatory factor, the coding sequence of the HPV antigen polypeptide, a 3' UTR, and a poly A tail, wherein the 5' UTR, the coding sequence of the secretory signal peptide, the coding sequence of the immunostimulatory factor, the coding sequence of the HPV antigen polypeptide, the 3' UTR, and the poly A tail are operably linked to each other. In some embodiments, the 5' cap is a m7G(5')ppp(5')(2'-OMeA)pG structure.

[0110] In some embodiments, the 5' end of the 5' UTR further comprises an AGG, AUG, or other nucleotide triplet; or the 5' UTR and one or two bases on its 5' end together form an AGG, AUG, or other nucleotide triplet, to facilitate use with different capping systems. The initiation sites required for different capping systems, such as Clean Cap AG and Clean Cap AU, are known in the art and can be routinely selected by those skilled in the art.

[0111] In some embodiments, the mRNA molecule of the present invention comprises a Kozak sequence. In some specific embodiments, the Kozak sequence comprises GCCACC located on the 5' side of the coding sequence of the secretory signal peptide.

[0112] In some embodiments, some or all of the uridines in the mRNA are chemically modified uridines. In some embodiments, some or all of the uridines in the mRNA are pseudouridines or 1-methyl-pseudouridines.

[0113] In some embodiments, some or all of the uridine nucleotides in the mRNA are substituted with pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.

[0114] In some embodiments, the mRNA further comprises a 5' cap structure. In some embodiments, the 5' cap structure is type O, type I, and type II. In some embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the mRNA further comprises a polyA tail. In some embodiments, the polyA tail sequence comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 adenylate residues. In some embodiments, the polyA tail sequence comprises at most 500, at most 400, at most 300, at most 200, at most 150, at most 140, at most 130, at most 120, at most 110, at most 100, at most 90, at most 80, at most 70, at most 60 adenylate residues (A), particularly about 120 A's. In some embodiments, the polyA tail comprises at least the polynucleotide sequence shown in SEQ ID NO: 24 or SEQ ID NO: 27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 27. In some embodiments, the polyA tail is the polynucleotide sequence shown in SEQ ID NO: 24 or SEQ ID NO: 27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 27.

[0115] In some embodiments, the pharmaceutical composition or pharmaceutical product or kit further comprises an immunostimulatory factor and / or adjuvant. In some embodiments, the immunostimulatory factor is selected from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, Pre-IFNα-2β, MIP-α, STING, HSP70, immune checkpoint inhibitors, or their encoding polynucleotides. In some embodiments, the STING is STING V155M In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the encoding polynucleotide is mRNA.

[0116] In a sixth aspect of the present application, a tumor therapeutic vaccine for treating HPV infection-related diseases is provided, which includes mRNA encoding HPV antigens, and the mRNA is encapsulated in a lipid nanoparticle composition (LNP).

[0117] In a specific embodiment, the encoded HPV antigen sequence is SEQ ID NO:21.

[0118] In a specific embodiment, all uridines in the mRNA molecule are 1-methyl-pseudouridine, and the mRNA further comprises a 5' cap structure, wherein the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG,

[0119] The mRNA molecule sequence comprises or is 5'UTR, ORF, 3'UTR, and poly A tail in sequence from the 5' end to the 3' end, wherein the 5'UTR, ORF, 3'UTR, and poly A tail are operably connected to each other.

[0120] In a specific embodiment, the mRNA ORF sequence encoding the HPV antigen is SEQ ID NO: 54.

[0121] In a specific embodiment, the 5'UTR sequence is SEQ ID NO: 25,

[0122] In a specific embodiment, the 3'UTR sequence is SEQ ID NO: 26,

[0123] In a specific embodiment, the poly A tail sequence is SEQ ID NO: 27.

[0124] In a specific embodiment, the lipid nanoparticle composition comprises ionizable lipid, phospholipid, cholesterol, and PEG lipid, wherein the content of ionizable lipid is 40 mol%-50 mol%, the content of phospholipid is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipid is 1.5 mol%-2.5 mol%.

[0125] In a specific embodiment, the lipid nanoparticle composition comprises ionizable lipid, phospholipid, cholesterol, and PEG lipid, wherein the content of ionizable lipid is 48 mol%-50 mol%, the content of phospholipid is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipid is 1.5 mol%-2.5 mol%.

[0126] In a specific embodiment, the PEG lipid is DMG-PEG2000.

[0127] In a specific embodiment, the phospholipid is selected from DSPC or DOPE, preferably DSPC.

[0128] In the seventh aspect of the present application, a vaccine preparation is provided, which includes the above-mentioned tumor therapeutic vaccine for treating HPV infection-related diseases and a pharmaceutically acceptable carrier.

[0129] In a specific embodiment, the pharmaceutically acceptable carrier includes a cryoprotectant, a pH buffer, a pH adjuster, and water for injection.

[0130] In a specific embodiment, the cryoprotectant is sucrose.

[0131] In a specific embodiment, the pH buffer is glacial acetic acid and / or tromethamine.

[0132] In a specific embodiment, the pH adjuster is sodium hydroxide and / or hydrochloric acid.

[0133] In a specific embodiment, the pH of the formulation is 7.2-8.

[0134] The eighth aspect of the present application provides a method for treating or preventing diseases related to HPV infection, comprising administering to an individual the polynucleotide molecule of the first aspect, the fusion polypeptide of the second aspect, the delivery body of the third aspect, the cell of the fourth aspect, the pharmaceutical composition or pharmaceutical product of the fifth aspect, the therapeutic vaccine for HPV infection-related diseases of the sixth aspect, or the vaccine preparation of the seventh aspect.

[0135] Also provided is the use of the polynucleotide molecule of the first aspect, the fusion polypeptide of the second aspect, the delivery body of the third aspect, the cell of the fourth aspect, the pharmaceutical composition or pharmaceutical product of the fifth aspect, the therapeutic vaccine for HPV infection-related diseases of the sixth aspect, or the vaccine preparation of the seventh aspect in the preparation of drugs for treating or preventing HPV infection-related diseases.

[0136] In some embodiments, the HPV infection-related disease is cervical cancer.

[0137] In some embodiments, the HPV infection-related disease is cervical precancerous lesions.

[0138] In some embodiments, the HPV infection-related disease is cervical high-grade intraepithelial lesion.

[0139] In some embodiments, the HPV infection-related disease is high-grade squamous intraepithelial lesion (HSIL).

[0140] In some embodiments, the HPV infection-related disease is cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN2 / 3).

[0141] In some embodiments, the HPV infection-related disease is low-grade LSIL intraepithelial neoplasia (CIN 1).

[0142] In some embodiments, the HPV infection-related disease is cervical adenocarcinoma in situ (AIS).

[0143] In some embodiments, the HPV infection-related disease is head and neck squamous cell carcinoma (HNSCC).

[0144] In some embodiments, the HPV infection-related disease is cervical cancer (CC).

[0145] In some embodiments, the HPV infection-related disease is oropharyngeal squamous cell carcinoma.

[0146] In some embodiments, the HPV infection-related disease is one of anal cancer, vaginal cancer, vulvar cancer, and penile cancer.

[0147] In the above embodiment, the HPV infection-related disease is a disease associated with human papillomavirus (HPV) type 16 and / or type 18.

[0148] Preferably, the HPV infection-related disease is human papillomavirus (HPV) type 16 and / or type 18-related grade 2 or grade 3 cervical intraepithelial neoplasia (CIN2 / 3).

[0149] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related cervical cancer.

[0150] Preferably, the HPV infection-related disease is human papillomavirus (HPV) type 16 and / or type 18-related human papillomavirus (HPV) 16 and / or HPV18-related head and neck squamous cell carcinoma.

[0151] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related anal cancer.

[0152] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related vaginal cancer.

[0153] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related vulvar cancer.

[0154] Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV 18-related penile cancer.

[0155] In some embodiments, the administration is intratumoral or perilumphal injection or intramuscular injection. In some embodiments, the method further comprises administering to the individual an immunostimulatory factor, chemotherapy, radiotherapy, and / or targeted therapy. In some embodiments, the targeted therapy is an antibody or its functional domain directed against a cervical cancer-specific tumor target.

[0156] In some embodiments, the method further comprises administering an immune checkpoint inhibitor to the individual. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the PD-1 or PD-L1 inhibitor is one or more of pembrolizumab, nivolumab, tislelizumab, toripalimab, sintilimab, camrelizumab, penampalimab, sepalimab, envolimab, sugemalimab, slulizumab, putalimab, and adebelimumab. In some preferred embodiments, the PD-1 inhibitor is pembrolizumab.

[0157] It should be understood that the aspects and embodiments of the present application described herein include aspects and embodiments that "comprise," "consist of," and "consist essentially of." The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple variations of the technical solution of the present application may be made, including combining the various technical features in any other suitable manner. These simple variations and combinations should also be considered as disclosed in the present application and fall within the scope of protection of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 shows the cellular immune response induced by different doses of HPV-M mRNA vaccine in normal mice.

[0159] Figure 2 Anti-tumor effect of HPV-M mRNA vaccine in TC-1 tumor model mice after intramuscular or intratumoral injection.

[0160] Figure 3 Anti-tumor effect of HPV-M mRNA vaccine after injection into the muscle or lymph nodes near the tumor in TC-1 tumor model mice.

[0161] Figure 4 ELISpot was used to detect the levels of HPV16 and 18 type E6 and E7-specific IFNγ and IL-2 induced by two injections of five different mRNA vaccines (5 μg).

[0162] Figure 5 Flow cytometry detection of HPV16 and 18 E6 and E7-specific T cell responses induced by two injections of five different mRNA vaccines (5 μg).

[0163] Figure 6 ELISpot was used to detect the levels of HPV16 and 18 type E6 and E7-specific IFNγ and IL-2 induced by three injections of four different mRNA vaccines (12.5 μg).

[0164] Figure 7 Flow cytometry was used to detect the HPV16 and 18 E6 and E7-specific T cell responses induced by three injections of four different mRNA vaccines (12.5 μg).

[0165] Figure 8 Anti-tumor effects of three injections of four different mRNA vaccines (12.5 μg) in TC-1 tumor model mice.

[0166] Figure 9 Anti-tumor effects of three injections of four different mRNA vaccines (12.5 μg) in TC-1 tumor model mice.

[0167] Figure 10 Evaluation of the anti-tumor effect of HPV-5 mRNA vaccine combined with PD-L1 antibody.

[0168] Detailed Description of the Invention

[0169] This application provides novel polynucleotide sequences that can be used to prepare preventive or therapeutic nucleic acids and fusion polypeptides for HPV infection-related diseases. This application also provides pharmaceutical compositions or pharmaceutical products, such as mRNA vaccines, containing these therapeutic nucleic acids or fusion polypeptides, and their use in treating diseases.

[0170] the term

[0171] As used herein, "coding sequence" may refer to a ribonucleotide sequence in a mature mRNA that can be translated into a protein, or may refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence that serves as a template for transcribing the ribonucleotide (RNA) sequence. Furthermore, the "coding sequence" of the present application may further include polynucleotide sequences encoding functional nucleic acids, such as miRNA, shRNA, dsRNA, and the like.

[0172] As used herein, the term "HPV E2 antigen sequence" is used to refer to an immunogenic amino acid sequence derived from the HPV E2 protein. In some embodiments, the HPV E2 antigen sequence is derived from the E2 protein sequence of any wild-type or artificially mutated HPV subtype, or is a fusion protein of multiple wild-type or artificially mutated HPV subtype E2 protein sequences. In some embodiments, the HPV E2 antigen sequence is derived from a conserved peptide sequence of the HPV E2 protein, or is a combination of two or more conserved peptide sequences. In some embodiments, the HPV E2 antigen sequence is a fusion protein of one or more conserved peptide sequences of the HPV E2 protein and one or more specific wild-type and / or artificially mutated HPV subtype E2 protein sequences. The conserved peptide sequence can be a conserved peptide sequence within a genotype, that is, a conserved amino acid sequence in different mutants of a certain HPV subtype E2 protein; it can also be a conserved peptide sequence between genotypes, that is, a conserved amino acid sequence in multiple HPV subtype E2 proteins. Methods for obtaining conserved peptide sequences (or methods for conservative assessment) are known in the art. Exemplary, for example, the available full-length sequences of E2 proteins from different HPV genotypes can be collected from protein databases such as NCBI and used as raw data input. All available full-length sequences are used to ensure that the selected conserved peptide sequences will equivalently represent the entire environmental population. For example, before conservative assessment, all genotypes are aligned and the sequences within each genotype are weighted to ensure equivalent representation of genotype diversity and therefore ensure that the HPV E2 antigen sequence candidates represent the entire environmental population. Conservation within the genotype is then assessed using, for example, a sliding window of 15 amino acids (intra-genotype conservation), thereby determining the conservation value of each window based on the amino acid prevalence within the combined window and the weighting of the value of each sequence to identify fragments conserved within each genotype, as well as the intra-genotype conserved peptide sequences created for each window. 'Intra-genotype conserved peptide sequences' means amino acid sequences representing a weighted set of genotype sequences, rather than the most common amino acids at each position. To be classified as conservative, the window must have a conservation value within the first quartile of all window conservation values ​​for the protein. Subsequently, the conserved genotype window at the same position in all genotypes was identified, regardless of the percent identity of the standardized consensus sequence within the genotype shared between the genotypes (conserved between genotypes). The phylogeny of the resulting region was then created and the group sequences in the combined tree were used to generate conserved peptide sequences between the genotypes with a high level of shared consensus identity.

[0173] As used herein, "linker" refers to an amino acid residue in a fusion protein that connects two polypeptide fragments to each other, or a peptide chain containing two or more amino acid residues. In some embodiments, the linker is a flexible linker that allows the two amino acid fragments to have a certain degree of mobility. The addition of Ser and Thr can form hydrogen bonds between the linker and water molecules, giving the linker stability in aqueous solution, thereby reducing the interaction between the linker and the two proteins before and after. Common flexible linkers are composed of Gly and Ser residues ("GS" linker). In addition to the GS flexible linker, there are some other flexible linkers, such as (Gly)8, which are all known in the art. In some embodiments, the linker is a rigid linker that can be used to completely isolate the two connected proteins and maintain their independent functions. Commonly used rigid linkers include α-helical peptides, (XP)n, etc., where P represents proline, X can be any amino acid, preferably Ala, Lys, Glu, and n represents the number of times XP is repeated. Those skilled in the art can independently adjust and select different linkers according to the specific application scenario and the 3D structure requirements of the fusion protein.

[0174] In this application, the term "5' end" is used to describe the relative positional relationship between two sequences within the same polynucleotide sequence. The "5' end" refers to the end of the polynucleotide sequence containing a free 5'-hydroxyl group. For example, "further comprising a coding sequence for an immunostimulatory factor or its functional domain on the 5' end of the coding sequence for an HPV antigen polypeptide" means that the "coding sequence for the immunostimulatory factor or its functional domain" is closer to the 5' end of the polynucleotide sequence in which the two sequences are co-located, relative to the "coding sequence for an HPV antigen polypeptide."

[0175] The term "signal peptide" refers to a short peptide chain that directs the localization or transfer of newly synthesized proteins. Signal peptides that direct the transfer of newly synthesized proteins to the secretory pathway are also called "secretory signal peptides." In most cases, the signal peptide is located at the N-terminus of the amino acid sequence. In mRNA, the coding sequence for the signal peptide is usually located after the start codon and is an RNA region that encodes a hydrophobic amino acid sequence. After the signal peptide guides the protein to complete its localization, it is usually removed by the action of a signal peptidase. The term "tPA-SP" stands for tissue plasminogen activator signal peptide, a secretory signal peptide.

[0176] As used herein, the "hybrid of a DNA sequence and an RNA sequence" is a polynucleotide sequence, wherein the nucleotides constituting the polynucleotide sequence are partly DNA and partly RNA.

[0177] The term "5' cap" is located at the 5' end of the mRNA and contains methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate linkage with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are called type O, type I and type II, respectively. Type O refers to the unmethylated ribose of the terminal nucleotide, type I refers to the methylation of the ribose of one terminal nucleotide, and type II refers to the methylation of the ribose of both terminal nucleotides. In this article, "CleanCap AG" is used to refer to the m7G(5')ppp(5')(2'-OMeA)pG cap.

[0178] As used herein, the term "Poly A tail" or "Poly A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3'-end of an RNA molecule. Poly-A tails or Poly-A sequences are known to those skilled in the art and can be selected according to actual needs. In mRNA, in the presence of a 3'-UTR, the Poly-A sequence is connected to the 3' end of the 3'-UTR. An uninterrupted poly-A tail is characterized by continuous adenylate residues. The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 adenylate residues (A), in particular about 120 A's. Typically, the vast majority of nucleotides in the poly A tail are adenosine, where the vast majority refers to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A, such as U (uridylic acid), G (guanylic acid), or C (cytidylic acid).

[0179] As used herein, percentages of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity, refer to a degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, the percentage of positions with identical bases or amino acid residues is determined as a ratio of the total number of positions after two sequences have been aligned to have identical residues at as many positions as possible, such as by introducing gaps. Percentages of "identity" can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the Internet at the following address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0180] As used herein, the "complementarity" of nucleic acids refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, 10 out of 10 are about 50%, 60%, 70%, 80%, 90% and 100% complementary, respectively). "Completely complementary" refers to that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. For single bases or nucleotides, according to the Watson-Crick base pairing principle, when A pairs with T or U, or C pairs with G or I, they are considered complementary or matching, and vice versa; all other base pairings are considered non-complementary. In this application, the "complementary polynucleotide sequence" of a polynucleotide sequence refers to a polynucleotide sequence that is completely complementary to the polynucleotide sequence.

[0181] As used herein, "delivery body" refers to a structure that is packaged or wrapped to assist larger biomolecules such as polynucleotides and polypeptides to enter the cell and to form a structure with a higher affinity for the cell membrane and easier to transport across the membrane from the extracellular to the intracellular. Delivery bodies and their preparation methods are all known in the art, including but not limited to liposomes (such as lipid nanoparticles (LNP)), viruses (such as AAV, lentivirus), and quantum dots. The preparation method of LNP is known in the art, such as those disclosed in CN114901360A and CN113941011A. In some embodiments, the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, an ionizable lipid, or any combination thereof.

[0182] As used herein, the "immune stimulatory factor" in this application specifically refers to a protein, polypeptide or nucleic acid molecule that can be produced in the mammalian body itself and can enhance the immune system's response to antigens, including but not limited to: cytokines that enhance immune cell processing and / or antigen presentation capabilities, such as dendritic cell growth factors (such as Flt3L, etc.); molecules that break immunosuppression, including but not limited to immune checkpoint inhibitors; proinflammatory cytokines (such as granulocyte-macrophage colony-stimulating factor, IFNα-2a, IFNα-2β, Pre-IFNα-2β, IL-2), etc.

[0183] As used herein, the term "adjuvant" refers to an exogenous substance added to a pharmaceutical composition or formulation to enhance the response of an individual's immune system to an antigen, including but not limited to chemical adjuvants and bacterial antigens.

[0184] As used herein, "Flt3L" refers to FMS-like tyrosine kinase 3 ligand. In some embodiments of the present application, Flt3L is human Flt3L, such as the Flt3L recorded in the NCBI database with gene ID: 2323.

[0185] The term "immune checkpoint" refers to molecules in the immune system that can turn a signal (co-stimulatory molecules) on or off. Many cancers protect themselves from the immune system by suppressing T cell signaling. As used herein, the term "immune checkpoint inhibitor" can help block this protective mechanism of cancer by acting on immune checkpoints. For example, an immune checkpoint inhibitor can be an antibody or antigen-binding fragment thereof against any one or more of the following checkpoint molecules: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, VSIG8. Among them, "PD-1" (programmed T cell death receptor) is a transmembrane protein found on the surface of T cells. When it binds to PD-L1 (programmed T cell death ligand 1) on tumor cells, it leads to the inhibition of T cell activity and the reduction of T cell-mediated cytotoxicity. Therefore, PD-1 and PD-L1 are immune downregulators or "off switches" of immune checkpoint signaling.

[0186] In this application, "immune checkpoint inhibitors" also include agonists of costimulatory molecules. For example, agonists of CD28, CD122, CD137, etc. CD28 is constitutively expressed on almost all human CD4+T cells and about half of CD8+T cells, prompting T cell expansion. CD122 can increase the proliferation of CD8+ effector T cells. 4-1BB (also known as CD137) is involved in T cell proliferation and can protect T cells, especially CD8+T cells from activation-induced cell death by mediating signal transduction.

[0187] As used herein, "HPV infection-related disease" refers to any disease caused primarily or in part by infection with human papillomavirus. Most HPV infections do not cause symptoms and resolve on their own. However, in some cases, they persist and this may lead to the appearance of common warts or precancerous lesions. In this application, "HPV infection-related disease" includes but is not limited to cervical cancer caused or partially caused by HPV. Methods for determining whether a disease is caused or partially caused by HPV are known in the art, for example, by a history of previous HPV infection, detecting HPV antigens and / or antibodies in the patient's lesion tissue, blood, body fluids, or other related tissues or tissue fluids, and other methods for determination. In this application, HPV may cover any subtype of human papillomavirus, including but not limited to HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV58 and HPV59, etc.

[0188] As used herein, "mRNA" (messenger RNA) is any RNA of a naturally occurring, non-naturally occurring, or modified amino acid polymer that encodes at least one protein and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. It will be appreciated by those skilled in the art that, unless otherwise indicated, the polynucleotide sequences described herein may use "T" to refer to thymine when representing a DNA sequence, but when the polynucleotide sequence represents RNA (e.g., mRNA), the "T" will be replaced by "U" (uracil). Thus, any DNA disclosed and identified by a particular sequence number (SEQ ID NO) herein also discloses an RNA (e.g., mRNA) sequence that is complementary or corresponding to the DNA, wherein each "T" of the DNA sequence is replaced by a "U."

[0189] As used herein, an "open reading frame (ORF)" is a continuous DNA or RNA segment that begins with a start codon, such as a methionine codon (ATG or AUG), and ends with a stop codon, such as TAA, TAG, or TGA, or UAA, UAG, or UGA. An ORF typically encodes a protein.

[0190] As used herein, the terms "fusion polypeptide" and "fusion protein" are used interchangeably and should be understood to mean a polypeptide comprising a combination of sequences derived from different gene products (e.g., homologous proteins of different HPV subtypes, different proteins of the same HPV subtype, and non-homologous proteins of different HPV subtypes) or a combination of sequences from the same gene product (e.g., a single HPV protein), wherein these sequences are derived from different / separate regions of the wild-type gene product. For example, a fusion polypeptide can comprise a combination of sequences that are typically separated by other sequence segments in the wild type, as well as a fusion of the remaining peptide segments after removing one or more sequences.

[0191] As used herein, "at least includes" means including or being. For example,

[0192] "From N-terminus to C-terminus, it contains at least:

[0193] 1) amino acid sequence A, and amino acid sequence B;

[0194] 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B;

[0195] 3) amino acid sequence B, and amino acid sequence A; or

[0196] 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A"

[0197] means:

[0198] a):

[0199] From N-terminus to C-terminus, it contains:

[0200] “1) amino acid sequence A, and amino acid sequence B;

[0201] 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B;

[0202] 3) amino acid sequence B, and amino acid sequence A; or

[0203] 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A", wherein at least two adjacent amino acid sequences (e.g., sequences A and B, A and C, C and B) further comprise one or more amino acids; or

[0204] b): From N-terminus to C-terminus, it contains:

[0205] “1) amino acid sequence A, and amino acid sequence B;

[0206] 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B;

[0207] 3) amino acid sequence B, and amino acid sequence A; or

[0208] 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A", wherein there are no more amino acids between any two adjacent amino acid sequences (for example, sequences A and B, A and C, C and B).

[0209] As used herein, when "directly connected" is used to describe the relationship between two amino acid sequences, it means that no other amino acids are contained between the amino acid sequences at both ends; in some embodiments, "directly connected" means that the amino acid sequences at both ends are connected by a chemical bond; in some embodiments, "directly connected" means that the amino acid sequences at both ends are connected by a peptide bond (amide bond).

[0210] Polynucleotide sequence

[0211] This application provides, on one hand, polynucleotide sequences that can be used to prevent or treat diseases associated with HPV infection. Through extensive comparisons and experiments, the inventors ultimately determined that the polynucleotide sequences of this application can induce a significant HPV-specific immune response in healthy mice and exhibit significant anti-tumor activity in HPV-positive tumor model mice.

[0212] The "variant" refers to a sequence or molecule that retains the same or substantially the same biological activity as the original sequence. The variant may be from the same or different species (e.g., homologous proteins from different mutants of the same HPV subtype), or may be a synthetic sequence based on a natural molecule or an existing molecule. In this application, "variant" may be used to refer to a variant of a protein, polypeptide, or amino acid sequence, and may also be used to refer to a variant of a nucleic acid molecule or a polynucleotide sequence.

[0213] Those skilled in the art can readily identify variants of SEQ ID NOs: 1 to 9. For example, through sequence alignment, the positional segments of each of the aforementioned amino acid sequences within the amino acid sequences of the corresponding HPV subtype proteins can be determined. The sequences of all mutant strains of HPV subtypes that exhibit mutations within these positions and segments are considered variants of the corresponding amino acid sequences. Therefore, the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof comprises the amino acid sequence of E6 protein corresponding to amino acid positions 1 to 85 of the E6 protein reference sequence (NCBI accession number QHA94929 or AAL96630.1) in the HPV-16 subtype mutant strain; the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof comprises the amino acid sequence of E7 protein corresponding to amino acid positions 1 to 65 of the E7 protein reference sequence (NCBI accession number ATI99837 or NP_041326.1) in the HPV-16 subtype mutant strain; the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof comprises the amino acid sequence of E6 protein corresponding to amino acid positions 71 to 158 of the E6 protein reference sequence in the HPV-16 subtype mutant strain; the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof comprises the amino acid sequence of E7 protein corresponding to amino acid positions 51 to 98 of the E7 protein reference sequence in the HPV-16 subtype mutant strain; The amino acid sequence shown in NO:5 or its variant comprises the amino acid sequence of E6 protein corresponding to the 1st to 85th amino acid positions of the E6 protein reference sequence (NCBI accession number ABP99784) in the HPV-18 subtype mutant strain, the amino acid sequence shown in SEQ ID NO:6 or its variant comprises the E7 protein reference sequence corresponding to the 1st to 65th amino acid positions of the E7 protein reference sequence (NCBI accession number UZQ21949 or ABP99785.1) in the HPV-18 subtype mutant strain, the amino acid sequence shown in SEQ ID NO:7 or its variant comprises the E6 protein amino acid sequence corresponding to the 71st to 158th amino acid positions of the E6 protein reference sequence in the HPV-18 subtype mutant strain, and the amino acid sequence shown in SEQ ID NO:8 or its variant comprises the E7 protein amino acid sequence corresponding to the 51st to 105th amino acid positions of the E7 protein reference sequence in the HPV-18 subtype mutant strain. The amino acid sequence shown in SEQ ID NO: 9 is formed by the fusion of multiple segments of HPV16, 18 and 31 E2 proteins. The inventors of the present application have confirmed that the HPV E2 antigen sequence has the effect of enhancing the immunogenicity of HPV proteins.

[0214] In some embodiments, a "variant" of the amino acid sequence has at least one amino acid difference relative to the amino acid sequence, for example, at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution can be a conservative amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitutions" can be polar to polar amino acids, such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); non-polar to non-polar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe); acidic to acidic amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Gln); u); basic to basic amino acids, such as arginine (R, Arg), histidine (H, His), lysine (K, Lys); charged amino acids to charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys) and arginine (R, Arg); hydrophobic to hydrophobic amino acids, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp) and methionine (M, Met). In some other embodiments, the variant may also comprise non-conservative substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity relative to the amino acid sequence. Compared to the amino acid sequence, the "variant" of the amino acid sequence may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or a range consisting of any two of the aforementioned values. As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one or more amino acid residues that undergo amino acid substitution without changing the overall conformation and function of the protein or enzyme, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm."Conservative substitution variants" also include polypeptides or enzymes having more than 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably more than 75%, preferably more than 85%, and even more than 90%, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

[0215] Therefore, the HPV antigen polypeptides and the fusion polypeptides described in the present application should encompass the above-mentioned variants.

[0216] Those skilled in the art will be aware that variants of protein-encoding nucleic acid molecules or polynucleotide sequences include "synonymous mutants", which refer to nucleic acid molecules or polynucleotide sequences obtained by replacing one or more codons in the nucleic acid molecule or polynucleotide sequence with other codons encoding the same amino acid as the codons.

[0217] The term "at least comprises" means that the polynucleotide sequence may consist of the coding sequence of the above-mentioned HPV antigen polypeptide, or may further comprise other polynucleotide sequences on the basis of comprising the coding sequence of the above-mentioned HPV antigen polypeptide.

[0218] For example, sequences that regulate the expression of the above-mentioned HPV antigen polypeptides, sequences that make the polynucleotide more stable, and other polynucleotide sequences that can promote the above-mentioned HPV antigen polypeptides to stimulate immune responses in the subject.

[0219] In some embodiments, the other polynucleotide sequence can use the coding sequence of any immunostimulatory factor. Preferred immunostimulatory factors include, for example, Flt3L.

[0220] The polynucleotide sequence may be a DNA sequence, an RNA sequence, or a hybrid of a DNA sequence and an RNA sequence.

[0221] mRNA vaccines

[0222] This application also provides an mRNA vaccine for the prevention or treatment of HPV-related diseases, comprising an RNA sequence from the aforementioned polynucleotide sequence. The mRNA vaccine can induce a specific immune response by introducing a polynucleotide sequence encoding an HPV antigen polypeptide into a subject, directly translating it to form the corresponding antigen protein, thereby achieving a preventive immune response. Furthermore, the vaccine can also target tumor cells containing HPV antigens to kill them.

[0223] Methods for preparing mRNA vaccines are known in the art. In particular, the mRNA in the mRNA vaccine, while containing the above-mentioned polynucleotide sequence, further contains coding sequences of multiple necessary functional components to express, regulate, or enhance the expression level of the above-mentioned HPV antigen polypeptides. The functional components include, but are not limited to, a 5' cap, a 5' UTR, a 3' UTR, a poly tail, and the like. The functional components are known in the art, and those skilled in the art can select and combine them according to actual needs. Both the 5' UTR and the 3' UTR are usually transcribed from genomic DNA and are elements possessed by pre-mature mRNA (or mRNA precursor or pre-mRNA). The characteristic structural features of mature mRNA (such as the 5'-cap and the 3'-poly (A) tail) are usually added to the transcribed (pre-mature) mRNA during mRNA processing. Therefore, in some embodiments, the mRNA is an mRNA precursor. In some embodiments, the mRNA is a mature mRNA.

[0224] In some embodiments, the mRNA vaccine comprises the above-described RNA polynucleotide sequence having an open reading frame encoding at least one antigenic polypeptide having at least one modification, at least one 5' cap, and is formulated within a lipid nanoparticle. 5' capping of the polynucleotide can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to produce a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA), or m7G(5')ppp(5')(2'-OMeA)pG (CleanCapAG) according to the manufacturer's protocol. 5' capping of modified RNA can be accomplished post-transcriptionally using vaccinia virus capping enzyme to produce a type O cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Type I cap structures can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to produce m7G(5')ppp(5')(2'-OMeA)pG, which can also be generated by the CleanCap method. Type II cap structures can be generated from type I cap structures by 2'-O-methylation of the 5'-penultimate nucleotide using 2'-O methyl-transferase. Type III cap structures can be generated from type II cap structures by 2'-O-methylation of the 5'-penultimate nucleotide using 2'-O methyl-transferase.

[0225] A 3'-poly(A) tail is typically added to the 3' end of a transcribed mRNA. In some embodiments, it may comprise up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail may be essential for the stability of a particular mRNA.

[0226] In some embodiments, the mRNA further comprises a stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. In some embodiments, the mRNA comprises a coding region, at least one histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop. Although the two have alternative mechanisms in nature, their synergistic effect can increase protein expression to levels exceeding that observed with either element alone. The synergistic effect of the combination of poly(A) and at least one histone stem-loop is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem-loop is typically derived from a histone gene and comprises two adjacent partially or completely reverse-complementary sequences separated by a spacer (composed of a short sequence) that form a loop through intramolecular base pairing. Unpaired loop regions are generally unable to base pair with either of the stem-loop elements. The stability of the stem-loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) can be generated. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length. In some embodiments, the mRNA does not comprise a histone downstream element (HDE). A "histone downstream element" (HDE) is included in a polynucleotide segment of approximately 15 to 20 nucleotides rich in purine at the 3' of the naturally occurring stem-loop, which represents a binding site for the U7 snRNA involved in processing histone pre-mRNA into mature histone mRNA.

[0227] In some embodiments, one or more AU-rich sequences of the mRNA can be removed. These sequences are sometimes referred to as AURES, which are destabilizing sequences found in the 3' UTR. AURES can be removed from the mRNA. Alternatively, AURES can be retained in the mRNA.

[0228] In some embodiments, the mRNA is configured in a lipid nanoparticle (LNP). In some embodiments, lipids are mixed with the mRNA to form lipid nanoparticles. In some embodiments, RNA is formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles are first formed into empty lipid nanoparticles and are combined or wrapped with the mRNA of the vaccine just before administration (e.g., within a few minutes to an hour).

[0229] The lipid nanoparticles generally comprise ionizable lipids, non-cationic lipids, sterols and PEG lipid components and target nucleic acids, such as the above-mentioned mRNA. Lipid nanoparticles of the present disclosure can be produced using components, compositions and methods as generally known in the art, see, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2017 / 037551, PCT / US2017 / 037552, PCT / US2017 / 037551, PCT / US2017 / 0 S2016 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, and PCT / US2016 / 069491, all of which are incorporated herein by reference in their entirety.

[0230] In some embodiments, the mRNA vaccine may further comprise one or more adjuvants. Adjuvants are known in the art and can be selected based on the specific antigen and disease condition. Exemplary adjuvants include: aluminum salt adjuvants (such as aluminum hydroxide or aluminum phosphate solution), nucleic acid adjuvants (such as CpG-ODN), lipid adjuvants (such as LPS), mixed adjuvants (such as MF59, Freund's adjuvant), and aggregate structure adjuvants (such as RAM1, RAM2, RAM3).

[0231] Pharmaceutical composition or pharmaceutical product

[0232] The pharmaceutical composition or pharmaceutical product provided herein comprises the above-mentioned polynucleotide sequence, a nucleic acid molecule comprising the polynucleotide sequence, a delivery vehicle or cell, a fusion polypeptide encoded by the above-mentioned polynucleotide sequence, or an mRNA vaccine, wherein the nucleic acid molecule, delivery vehicle, cell, fusion polypeptide, and mRNA in the mRNA vaccine have a purity that meets clinical needs.

[0233] Optionally, the pharmaceutical composition or pharmaceutical product of the present application may further include one or more other active compounds according to the needs of the specific indications for treatment. Preferably, the compound has a complementary activity, auxiliary, or promoting effect on the aforementioned nucleic acid molecules, delivery bodies, cells, and fusion polypeptides, such as enhancing the ability of the nucleic acid molecules, delivery bodies, cells, and fusion polypeptides to cause an immune response, or enhancing the immune response of the immune system to the nucleic acid molecules, delivery bodies, cells, and fusion polypeptides, and does not adversely affect each other. Such compounds can be present in the pharmaceutical composition or pharmaceutical product in an amount effective for the intended purpose. For example, in some embodiments, the other active compounds may include one or more immunostimulatory factors, such as Flt3L, granulocyte-macrophage colony stimulating factor, IFNα-2a, IFNα-2β, Pre-IFNα-2β, IL-2, and immune checkpoint inhibitors such as PD-1 or PD-L1 antibodies.

[0234] The above-mentioned active ingredients, such as nucleic acid molecules, delivery vehicles, cells, fusion polypeptides, etc., can be encapsulated or contained in delivery vehicles or colloidal drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, or nanocapsules. When the pharmaceutical composition or pharmaceutical product contains two or more active ingredients, the active ingredients can be mixed with each other or separated from each other, such as being present in the same delivery vehicle, colloidal particle, or microcapsule, or being present in different delivery vehicles, colloidal particles, or microcapsules.

[0235] Optionally, the pharmaceutical composition or pharmaceutical product further comprises one or more pharmaceutically acceptable carriers, excipients or stabilizers (Remington: Remington: The Science and Practice of Pharmacy 20th edition (2000)), in the form of an aqueous solution, lyophilized or other dry preparation. The pharmaceutically acceptable carriers, excipients or stabilizers are non-toxic to the subject at the dosage and concentration used, and include buffers (such as phosphate, citrate, histidine and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives, low molecular weight (less than about 10 amino acid residues) polypeptides, proteins (such as serum albumin, gelatin or immunoglobulins); hydrophilic polymers, such as polyvinylpyrrolidone. Amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (such as EDTA), polysaccharides (such as sucrose, mannose, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes; nonionic surfactants (such as TWEEN TM 、PLURONICS TMor polyethylene glycol). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulins of the present invention, which are in the form of heteromorphic materials, such as films or microcapsules.

[0236] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art. Therefore, those of ordinary skill in the art will recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments. Example

[0237] Example 1: Sequence construction and preparation of HPV vaccine

[0238] 1.1 Synthesis of HPV vaccine sequences and construction of recombinant vectors

[0239] In this embodiment, the antigen sequences of the HPV-related tumor mRNA vaccine are the E6 and E7 proteins of HPV types 16 and 18, and their coding fragments are concatenated to obtain a polynucleotide sequence. From the 5' end, the coding nucleotide sequence comprising: a T7 promoter with XbaI at the 5' end, a 5' UTR, tPA-SP, Flt3L, HPV E2 (if present), E6 / E7 protein or variant thereof, a 3' UTR, and / or a poly A tail is double-digested with XbaI and NotI, and ligated with a pUC57-GW-Kan (Jinweizhi) vector backbone fragment digested with XbaI and NotI to construct a recombinant plasmid. In Examples 1-6, the nucleic acid sequence component 1 used has the ORF coding sequences of SEQ ID NOs: 47-48, SEQ ID NOs: 50-52, and SEQ ID NO: 54; in Examples 7-8, the nucleic acid sequence component 2 used has the ORF coding sequences of SEQ ID NOs: 49, SEQ ID NOs: 53, and SEQ ID NOs: 54.

[0240] 1.2 mRNA preparation

[0241] 1.2.1 Plasmid linearization

[0242] The recombinant plasmid constructed in step 1 has a Sap I restriction site after the last A in the polyA tail sequence. Linearize the plasmid containing the target gene with the restriction endonuclease Sap I using the reaction system shown in Table 1. Incubate the enzyme digestion at 37°C for 3 hours.

[0243] Table 1. Plasmid linearization enzyme digestion system

[0244] 2 μL of the digested product was subjected to 1% agarose gel electrophoresis to check the linearization of the plasmid. The linearized plasmid was purified using a PCR product recovery kit (Comvison).

[0245] (2) In vitro transcription and purification

[0246] The linearized recombinant plasmid obtained in step (1) was used as a template for in vitro transcription using a high-yield T7 RNA transcription kit. The high-yield T7 RNA transcription kit is named High Yield T7 RNA Synthesis Kit and is available from Shanghai Zhaowei Technology Development Co., Ltd. with a catalog number of ON-040. 5× Reaction Buffer, 100mM ATP Solution, 100mM CTP Solution, 100mM GTP Solution, Enzyme Mix, DNase I, Ammonium Acetate Stop Solution, and Lithium Chloride (LiCl) Precipitation Solution are all components of the high-yield T7 RNA transcription kit. 100mM ΨUTP Solution (pseudouridine triphosphate), also known as N1-Me-pUTP, is available from Shanghai Zhaowei Technology Development Co., Ltd. with a catalog number of R5-027. Each component was added according to the following system (Table 2) (using a 20μL reaction system as an example), mixed, and reacted at 37°C for 3h.

[0247] Table 2. In vitro transcription system

[0248] Among them, CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG, product number is ON-134, Shanghai Zhaowei.

[0249] After the transcription reaction is completed, add 1 μL of DNase I and react at 37°C for 15 minutes. Add 15 μL of Ammonium Acetate Stop Solution and mix well. Then add 1 / 3 volume of 7.5M Lithium Chloride (LiCl) Precipitation Solution (to a final concentration of 2.5M) and incubate at -20°C for 30 minutes. Centrifuge at 12,000g for 15 minutes to allow the RNA to precipitate at the bottom. Discard the supernatant. Add 1 mL of 70% ethanol to wash the RNA. Centrifuge at 12,000g for 5 minutes and discard the supernatant. After drying, add 50 μL of RNase-free water to dissolve the precipitate and quantify the mRNA using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

[0250] 1.3 Lipid Nanoparticle (LNP) Encapsulation

[0251] The mRNA stock solution obtained in step 1.2 was dispersed in 20 mM acetic acid solution (pH 5.0) to obtain an RNA solution with an mRNA concentration of 200 μg / mL. A lipid mixture was prepared by mixing the mRNA and lipid mixture in a molar ratio of ionizable lipid: cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5. The flow rates of the aqueous and oil phases were controlled to mix the mRNA and lipid mixture using a T-type mixing method. The syringe pump was activated to allow the mRNA solution and lipid mixture to mix to form LNPs. The mRNA solution was then diluted 10-fold with diluent, concentrated by centrifugation through an ultrafiltration tube, and then exchanged three times. The resulting solution was adjusted to a pH of 7.0-8.0 by adding a Tris aqueous solution to obtain an LNP-encapsulated mRNA solution. LNPs are lipid nanoparticles.

[0252] The mRNA concentration and particle size of LNPs were determined using a Ribogreen RNA quantification kit (Invitrogen, R11490) and a Darwin ZetaSizer particle size analyzer, respectively. LNPs without any substance encapsulated were also used as a control.

[0253] Example 2 Cellular Immunological Evaluation of HPV-M mRNA Vaccine

[0254] Twenty-five SPF-grade female C57BL / 6 mice, 6-8 weeks old, were randomly divided into five groups of five mice each. Mice were immunized with the mRNA vaccine three times every two weeks according to the groupings shown in Table 4. Seven days after immunization, mice were sacrificed, spleens were removed, placed on a 70 μm nylon filter, and the cells were thoroughly ground in 2 ml of complete RPMI-1640 medium to prepare a cell suspension. The cells were then counted.

[0255] Table 3. Grouping of mice in vaccine cellular immune response detection

[0256] 2.1 ELISpot detection of HPV16 and 18 E6, E7-specific IFNγ and IL-2 levels

[0257] 1.5×10 per well 5Cells were seeded into an ELISpot plate and an overlapping peptide library of E6 and E7 proteins of HPV types 16 and 18 (synthesized by Sabritech) was added. The peptide contained a 15-amino acid sequence, of which 8 amino acid residues overlapped with each consecutive peptide. Positive stimulants, phorbol methyl parathionate (PMA) and ionomycin, were added to the positive control wells, while no stimulants were added to the negative control wells. The plate was then placed in a 5% CO2 incubator and incubated at 37°C for 20 hours. According to the ELISpot kit instructions (Dakoway, 2210001 and Mabtech, 2210001), the cells in the plate were incubated with antibodies and color development. After air drying, the plate was read using a Mabtech IRIS ELISpot / FluoroSpot plate reader equipped with Mabtech Apex software (version 1.1.45.114) to detect the spot-forming units (SFU) in the plate.

[0258] 2.2 Flow cytometry detection of HPV16 and 18 E6 and E7-specific T cell responses

[0259] In order to comprehensively evaluate the cellular immune response induced by the HPV vaccine, intracellular cytokine staining was performed simultaneously. The single cells obtained above were broken into red blood cells and filtered. 1x10 cells were inoculated into each well of a 96-well U-bottom cell culture plate. 6 Cells were loaded, and an overlapping peptide library of HPV16 and 18 E6 and E7 proteins (synthesized by Cytosine) or PMA and ionomycin were used as stimuli. BFA and monensin (Biolegend, 420601 and 420701) were used as blocking agents. After incubation overnight at 37°C in a 5% CO2 incubator, extracellular staining (CD3-cy5.5, CD4-APC or FITC, and CD8-APC or FITC) was performed. Cells were then permeabilized and fixed, and stained for intracellular cytokines such as IFN-γ-PE or TNF-α-PE. The resulting cells were analyzed using a Cytoflex flow cytometer (Beckman Coulter). IFN-γ and TNF-α levels in CD4+ and CD8+ cells were obtained by gating.

[0260] One-way ANOVA was used to analyze the statistical significance of the experimental and control groups (***p<0.001 vs. LNP group; ###p<0.001 vs. HPV-M-2.5μg; ##p<0.01 vs. HPV-M-2.5μg; &&p<0.01 vs. HPV-M-12.5μg; &p<0.05 vs. HPV-M-12.5μg). As shown in Figure 1, both Elispot and flow cytometry results showed that the cellular immune response to HPV-M was dose-dependent, reaching a plateau after 25μg.

[0261] Example 3 Pharmacodynamic Evaluation of HPV-M mRNA Vaccine

[0262] 3.1 Antitumor effects of HPV-M vaccine after intramuscular or intratumoral injection

[0263] This study used the TC-1 mouse tumor model to evaluate the pharmacodynamics of the HPV-M vaccine. 6-8 week old SPF female C57BL / 6 mice were subcutaneously inoculated with TC-1 cells in the logarithmic growth phase. 3 At 4 hr, mice were randomly divided into groups based on tumor volume, with 10 mice per group. Mice were immunized with the mRNA vaccine three times weekly according to the groupings shown in Table 4. Tumor size was measured twice weekly, and mouse survival was recorded. Tumor volume was calculated using the following formula: Implanted tumor volume = major diameter × minor diameter × minor diameter / 2, i.e., V = ab² / 2. One-way ANOVA and log-rank analysis were used to analyze tumor volume and survival rate among the groups, respectively.

[0264] Table 4 Grouping of mice for HPV-M vaccine efficacy evaluation

[0265] The results are shown in Figure 2. In the TC-1 mouse tumor model, whether injected intramuscularly or intratumorally, the average tumor volume of mice treated with HPV-M in each dose group was significantly reduced, and the survival of the mice was significantly prolonged compared with the mice treated with LNP as the control group.

[0266] 3.2 Antitumor effect of HPV-M vaccine after injection into muscle or perilymph nodes near tumor

[0267] According to the above-mentioned anti-tumor effect evaluation method, mice were immunized with mRNA vaccine according to the grouping shown in Table 5.

[0268] Table 5 Mouse grouping for HPV-M vaccine efficacy evaluation

[0269] The results are shown in Figure 3. In the TC-1 mouse tumor model, regardless of intramuscular injection or perilymphatic injection, all doses of HPV-M significantly inhibited tumor growth and significantly prolonged mouse survival compared with the control group of LNP-treated mice.

[0270] Example 4 Evaluation of cellular immunity after vaccination of mice with different HPV mRNA vaccines

[0271] Thirty-five SPF-grade female C57BL / 6 mice, aged 6-8 weeks, were randomly divided into seven groups of five mice each. Mice were immunized with the mRNA vaccine twice every two weeks according to the groupings shown in Table 4. Seven days after immunization, mice were sacrificed, spleens were removed, placed on a 70 μm nylon cell strainer, and the cells were thoroughly ground in 2 ml of complete RPMI-1640 medium to prepare a cell suspension. Cells were then counted.

[0272] Table 6. Grouping of mice in vaccine cellular immune response detection

[0273] 4.1 ELISpot detection of HPV16 and 18 E6, E7-specific IFNγ and IL-2 levels

[0274] The same ELISpot method as in Example 2 was used to detect the levels of antigen-specific IFNγ and IL-2 induced by the above five HPV vaccines.

[0275] As shown in Figure 4, after two injections of 5 μg low dose, the SFU / 10 6 The mean number of IFN-γ and IL-2 spots on splenocytes was greater than that in the negative control group. Compared with the HPV-M vaccine group, the HPV-1, HPV-2, HPV-3, and HPV-4 vaccine groups significantly increased IFN-γ and IL-2 levels in mouse splenocytes after stimulation with specific peptides, effectively inducing antigen-specific cellular immune responses. One-way ANOVA statistical analysis was performed using GraphPad Prism 8 software to analyze the significant differences between groups (***p < 0.001 vs. LNP control; **p < 0.01 vs. LNP control; *p < 0.05 vs. LNP control; ###p < 0.001 vs. HPV-M; ##p < 0.01 vs. HPV-M; #p < 0.05 vs. HPV-M).

[0276] 4.2 Flow cytometry detection of HPV16 and 18 E6 and E7-specific T cell responses

[0277] At the same time, the antigen-specific T cell responses induced by the above five HPV vaccines were detected by the same flow cytometry method as in Example 2. The levels of IFN-γ and TNF-α in CD4+ and CD8+ cells were obtained by gating the cells, and the significance analysis of each experimental group and the control group was performed using the One-way ANOVA statistical method. The pairwise comparison of the experimental group and the original sequence HPV-M was performed using the Student's t-test. The results are shown in Figure 5. When the low dose of 5μg was used for two injections, the CD4 in the spleen cells of the four experimental groups of mice with HPV-1, HPV-2, HPV-3 and HPV-4 was significantly higher than that of the control group. + and CD8 + The percentages of IFN-γ and TNF-α in the cells were significantly higher than those in the HPV-M vaccine group, indicating that the vaccination was able to successfully induce an effective immune response. One-way ANOVA statistical analysis was performed using GraphPad Prism 8 software to analyze the significance between the groups (***p<0.001 vs. LNP control; **p<0.01 vs. LNP control; *p<0.05 vs. LNP control; ###p<0.001 vs. HPV-M; ##p<0.01 vs. HPV-M; #p<0.05 vs. HPV-M).

[0278] Example 5 Evaluation of cellular immunity after vaccination of mice with different HPV mRNA vaccines

[0279] Twenty-five SPF-grade female C57BL / 6 mice, aged 6-8 weeks, were randomly divided into seven groups of five mice each. Mice were immunized with the mRNA vaccine three times every two weeks according to the groupings shown in Table 4. Seven days after immunization, mice were sacrificed, spleens were removed, placed on a 70 μm nylon filter, and the cells were thoroughly ground in 2 ml of RPMI-1640 complete medium to prepare a cell suspension. The cells were then counted.

[0280] Table 7. Grouping of mice in the vaccine humoral immune response test

[0281] 5.1 ELISpot detection of HPV16 and 18 E6, E7-specific IFNγ and IL-2 levels

[0282] The same ELISpot method as in Example 2 was used to detect the levels of antigen-specific IFNγ and IL-2 induced by the above four HPV vaccines.

[0283] As shown in Figure 6, after three injections of 12.5 μg, the SFU / 10 6The mean number of IFN-γ and IL-2 spots on splenocytes was greater than that in the negative control group. Compared with the HPV-M vaccine group, the HPV-1, HPV-4, and HPV-5 vaccine groups all significantly increased IFN-γ and IL-2 levels in mouse splenocytes after stimulation with specific peptides, indicating that these three vaccines significantly enhanced antigen-specific cellular immune responses. One-way ANOVA statistical analysis was performed using GraphPad Prism 8 software to analyze the significant differences between groups (***p < 0.001 vs. LNP control; **p < 0.01 vs. LNP control; *p < 0.05 vs. LNP control; ###p < 0.001 vs. HPV-M; ##p < 0.01 vs. HPV-M; #p < 0.05 vs. HPV-M).

[0284] 5.2 Flow cytometry detection of HPV16 and 18 E6 and E7-specific T cell responses

[0285] At the same time, the antigen-specific T cell responses induced by the four HPV vaccines were detected by flow cytometry as in Example 2. The levels of IFN-γ and TNF-α in CD4+ and CD8+ cells were obtained by gating the cells, and the significance between the experimental groups was analyzed using the One-way ANOVA statistical method. As shown in Figure 7, after three injections of 12.5 μg, the CD4+ and CD8+ levels in the spleen cells of the mice in the four experimental groups were significantly higher than those in the control group. + and CD8 + The percentages of IFN-γ and TNF-α in the cells were significantly higher than those in the negative control group. Compared with the HPV-M vaccine group, HPV-1, HPV-4 and HPV-5 significantly increased the CD8 + The percentages of IFN-γ and TNF-α in cells were significantly increased, which further indicated that the three vaccines were able to induce stronger antigen-specific cellular immune responses than the HPV-M vaccine. One-way ANOVA statistical analysis was performed using GraphPad Prism 8 software to analyze the significance between the groups (***p<0.001 vs. LNP control; **p<0.01 vs. LNP control; *p<0.05 vs. LNP control; ###p<0.001 vs. HPV-M; ##p<0.01 vs. HPV-M; #p<0.05 vs. HPV-M).

[0286] Example 6 Antitumor Effects of Different HPV mRNA Vaccines in the TC-1 Mouse Tumor Model

[0287] This study evaluated the pharmacodynamics of different HPV mRNA vaccines in the TC-1 mouse tumor model using the same method as in Example 3. Mice were administered the mRNA vaccines according to the groupings shown in Table 8.

[0288] Table 8 Mouse grouping for HPV-M vaccine efficacy evaluation

[0289] The results are shown in Figure 8. In the TC-1 mouse tumor model, compared to the LNP-treated control group, the average tumor volume of mice treated with each vaccine group was significantly reduced, and the survival of the mice was significantly prolonged. At 22 days after administration, the proportion of mice with tumor-free mice in the HPV-M, HPV-1, HPV-4, and HPV-5 vaccine groups was 30% (3 / 10), 50% (5 / 10), 60% (6 / 10), and 70% (7 / 10), respectively. Therefore, overall, at equivalent doses, the anti-tumor effects of HPV-1, HPV-4, and HPV-5 vaccines were superior to those of the HPV-M vaccine, with HPV-5 being the most effective.

[0290] Example 7 Pharmacodynamic Evaluation of Different HPV mRNA Vaccines

[0291] 6.1 Antitumor Effects of HPV mRNA Vaccine After Intramuscular Injection

[0292] This study evaluated the pharmacodynamics of different HPV mRNA vaccines in the TC-1 mouse tumor model using the same methods as in Example 3. Mice were immunized with the mRNA vaccines three times weekly according to the groupings shown in Table 9 ( Figure 9 ). Tumor size was measured three times weekly, and mouse survival was recorded.

[0293] Table 9 Grouping of mice for HPV-M vaccine efficacy evaluation

[0294] The results are shown in Figure 9. In the TC-1 mouse tumor model, the average tumor volume of mice treated with each vaccine group was significantly reduced compared to mice treated with LNP as a control group. At 22 days after administration, the proportion of mice with tumor disappearance in the HPV-M, HPV-1, HPV-4, and HPV-5 vaccine groups was 40% (4 / 10), 70% (7 / 10), 70% (7 / 10), and 90% (9 / 10), respectively. The survival of mice treated with each vaccine group was also significantly prolonged, with the results showing that the HPV-5 treatment group was significantly superior to the HPV-M treatment group. Therefore, in general, at the same dose, the anti-tumor effects of HPV-1, HPV-4, and HPV-5 vaccines were superior to those of HPV-M vaccines, with HPV-5 being the most effective.

[0295] 6.2 Studies on the Long-term Antitumor Effects and Immune Memory of Different HPV mRNA Vaccines

[0296] Mice with complete tumor regression were selected and re-inoculated with an equal dose of TC-1 tumor cells on the contralateral side of their backs 68 days after the initial administration. Untreated mice were inoculated with tumor cells and served as a control group. Tumor growth and mouse survival were recorded. Cellular immunity was assessed at the endpoint using the same ELISpot and flow cytometry methods as in Example 2.

[0297] The results are shown in Figure 9. Compared with the rapid tumor growth in the control group, all mice administered with HPV mRNA vaccine (except for the HPV-4 group, in which 14.3% (1 / 7) died) had no tumor recurrence and remained completely tumor-free for at least 42 days. As shown in Figure 9, the SFU / 10 6 The average number of IFN-γ and IL-2 spots in splenocytes was greater than that in the negative control group, and the CD4 + and CD8 + The percentages of cells and IFN-γ and TNF-α were higher than those in the negative control group. Among them, HPV-4 and HPV-5 significantly increased CD8 + The percentages of IFN-γ and TNF-α in cells were analyzed. These results indicate that the four vaccines, especially HPV-5, can induce long-term antigen-specific immune memory, providing strong protection against tumor recurrence. One-way ANOVA statistical analysis was performed using GraphPad Prism 8 software to analyze the significance of the differences between the groups (***p < 0.001 vs. LNP control; **p < 0.01 vs. LNP control; *p < 0.05 vs. LNP control; ns, not significant).

[0298] Example 8 Evaluation of the anti-tumor effect of the combination of HPV-5 mRNA vaccine and PD-L1 antibody

[0299] This study evaluated the anti-tumor effect of the combination of HPV-5 mRNA vaccine and PD-L1 antibody in the TC-1 mouse tumor model using the same method as in Example 3. Mice were administered mRNA vaccine and PD-L1 antibody according to the grouping shown in Table 10, where PD-L1 antibody (10F.9G2) and rabbit isotype IgG2b (LTF-2) were purchased from BioXcell, and the dosage was 10 mg / kg by intraperitoneal injection. The specific medication regimen is shown in Figure 10. The tumor size was measured three times a week and the survival of the mice was recorded. Tumor growth curves and survival curves were drawn based on the measurement results, and the relative tumor proliferation rate (T / C (%)) and the synergistic efficiency of the drug combination were calculated by relative tumor volume (RTV). The specific calculation formula is as follows: RTV=V t / V0, where V tand V0 are the tumor volumes measured at a certain time point (dt) and at the time of administration (d0) in different cages, respectively. RTV / C RTV )×100%, where T RTV / C RTV The RTV values ​​of the experimental group and the control group are respectively. The synergistic efficiency (%) of the two groups of drugs A and B = (A T / C ×B T / C / AB T / C )×100%, where A T / C 、B T / C and AB T / C These are the T / C values ​​for drug group A, drug group B, and drug AB combination group, respectively.

[0300] Table 10: Mouse grouping for HPV-5 vaccine efficacy evaluation

[0301] The results, shown in Figure 10, show that in the TC-1 mouse tumor model, the PBS control group and the PD-L1 antibody monotherapy group exhibited rapid tumor growth and shorter survival. Compared to PBS, the HPV-5 monotherapy group (3 μg), and especially the HPV-5 combined with PD-L1 antibody group, significantly inhibited tumor growth and significantly prolonged mouse survival.

[0302] Table 11 Analysis of tumor growth data in combination with HPV-5 and PD-L1 antibodies in the mouse TC-1 tumor model

[0303] The tumor data on the 16th day after administration were calculated, and the results are shown in Table 11. The synergistic efficiencies of HPV-5 (0.3 μg) and HPV-5 (3 μg) combined with PD-L1 antibodies were 2.91 and 19.60, respectively (when the value is >1, the two have a synergistic effect), indicating that the HPV-5 mRNA vaccine and PD-L1 antibody have a good anti-tumor synergistic effect.

[0304] The sequences used in the above examples of the present application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of the embodiments of the present application and are not intended to limit the present application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When representing RNA sequences, "T" represents uridine.

[0305] Sequence Listing:

Claims

1. A polynucleotide molecule comprising at least a coding sequence of an HPV antigen polypeptide, wherein the antigen polypeptide comprises at least: 1) amino acid sequence A, and amino acid sequence B; 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B; 3) amino acid sequence B, and amino acid sequence A; or 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A; in, The amino acid sequence A comprises at least SEQ ID NO: 1-4 or variants thereof from the N-terminus to the C-terminus, and the amino acid sequences shown in the SEQ ID NO are directly connected in sequence or connected in sequence through a connecting peptide; The amino acid sequence B comprises at least SEQ ID NO: 5-8 or variants thereof from the N-terminus to the C-terminus, and the amino acid sequences shown in the SEQ ID NOs are directly connected in sequence or are connected in sequence through a connecting peptide; The amino acid sequence C contains the HPV E2 antigen sequence, Preferably, the variants are conservative substitution variants.

2. The polynucleotide molecule according to claim 1, wherein the HPV E2 antigen sequence is SEQ ID NO: 9 or a variant thereof.

3. The polynucleotide molecule according to claim 1 or 2, wherein the connecting peptide comprises 1, 2 or more amino acid residues.

4. The polynucleotide molecule according to claim 1 or 2, wherein the connecting peptide consists of 2-10 amino acid residues, preferably, the amino acid residues are glycine, serine and / or alanine residues, and further preferably, the connecting peptide consists of two alanine residues.

5. The polynucleotide molecule of claim 1, wherein the HPV antigen polypeptide comprises SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity thereto. 6 . The polynucleotide molecule according to claim 1 , further comprising a coding sequence of an immunostimulatory factor or a functional domain thereof on the 5′ end of the coding sequence of the HPV antigen polypeptide. 7 . The polynucleotide molecule according to claim 6 , wherein the immunostimulatory factor is Flt3L.

8. The polynucleotide molecule according to claim 7, wherein the polypeptide sequence of the immunostimulatory factor comprises at least the amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

10.

9. The polynucleotide molecule according to any one of claims 1 to 8, further comprising a secretory signal peptide coding sequence, preferably, the secretory signal peptide coding sequence is located at the 5' end of the HPV antigen polypeptide coding sequence.

10. The polynucleotide molecule according to claim 9, wherein the secretory signal peptide is tPA-SP.

11. The polynucleotide molecule of claim 10, wherein the secretory signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

11.

12. The polynucleotide molecule according to any one of claims 9 to 11, comprising the coding sequences of the secretory signal peptide, the immunostimulatory factor and the HPV antigen polypeptide linked sequentially from the 5' end to the 3' end.

13. The polynucleotide molecule according to any one of claims 1 to 12, which is DNA, RNA, or a hybrid of DNA and RNA.

14. The polynucleotide molecule according to any one of claims 1 to 13, comprising any polynucleotide sequence selected from SEQ ID NOs: 39 to 54, or consisting of any polynucleotide sequence selected from SEQ ID NOs: 39 to 54, or encoded by any polynucleotide sequence selected from SEQ ID NOs: 39 to 54.

15. The polynucleotide molecule according to any one of claims 1-14, further comprising a 5'UTR structure, preferably, the 5'UTR structure comprises at least a polynucleotide sequence as shown in SEQ ID NO:22 or SEQ ID NO:25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:22 or SEQ ID NO:

25.

16. The polynucleotide molecule according to any one of claims 1-15, further comprising a 3'UTR structure, preferably, the 3'UTR structure comprises at least a polynucleotide sequence as shown in SEQ ID NO:23 or SEQ ID NO:26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:23 or SEQ ID NO:

26.

17. According to the polynucleotide molecule according to any one of claims 1-16, the mRNA further comprises a polyA tail, preferably, the polyA tail sequence comprises at least 50, at least 60 or at least 100 A nucleotides; preferably, the polyA tail comprises at least the polynucleotide sequence as shown in SEQ ID NO:24 or SEQ ID NO:27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:24 or SEQ ID NO:

27.

18. The polynucleotide molecule according to any one of claims 1 to 17, which is an mRNA molecule.

19. The polynucleotide molecule according to any one of claims 1-18, wherein the mRNA molecule sequence comprises the polynucleotide sequence shown in SEQ ID NO:54, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

54.

20. The polynucleotide molecule according to any one of claims 1-19, wherein part or all of the uridine in the mRNA molecule is chemically modified uridine, preferably, the chemically modified uridine is 1-methyl-pseudouridine.

21. The polynucleotide molecule according to any one of claims 1-19, wherein the mRNA further comprises a 5' cap structure, preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

22. A delivery system comprising the polynucleotide molecule according to any one of claims 1-21.

23. The delivery body of claim 22, wherein the delivery body is a lipid nanoparticle (LNP).

24. The delivery system of claim 23, wherein the LNP comprises ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids, Preferably, the content of ionizable lipids is 35 mol%-65 mol%, the content of the sum of phospholipids and cholesterol is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%; Preferably, the content of ionizable lipids is 40 mol%-50 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%; Preferably, the content of ionizable lipid is 48 mol%-50 mol%, the content of phospholipid is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipid is 1.5 mol%-2.5 mol%.

25. A pharmaceutical composition or pharmaceutical preparation comprising a polynucleotide molecule according to any one of claims 1-21 or a delivery body according to claims 22-24.

26. The pharmaceutical composition or pharmaceutical preparation according to claim 25, further comprising an immunostimulatory factor and / or an adjuvant.

27. A pharmaceutical composition or pharmaceutical product according to claim 26, wherein the immunostimulatory factor is selected from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, Pre-IFNα-2β, MIP-α, STING, HSP70, immune checkpoint inhibitors (preferably PD-1 inhibitors or PD-L1 inhibitors).

28. The pharmaceutical composition or pharmaceutical product according to claim 27, wherein the immunostimulatory factor is a protein or a nucleic acid molecule encoding the protein, preferably, the nucleic acid molecule is mRNA.

29. According to the pharmaceutical composition or pharmaceutical product of claim 27, the immune checkpoint inhibitor is preferably one or more of pembrolizumab, nivolumab, tilelizumab, toripalimab, sintilimab, carrelizumab, penampalimab, sepalimab, envolimab, sugemalimab, slulizumab, putalimab, and adebelimumab; further preferably, the PD-1 inhibitor is pembrolizumab.

30. The pharmaceutical composition or pharmaceutical preparation according to any one of claims 25-29, which is an mRNA vaccine.

31. A method for treating or preventing a disease associated with HPV infection, comprising administering to an individual a polynucleotide molecule according to any one of claims 1-21, a delivery vehicle according to any one of claims 22-24, or a pharmaceutical composition or pharmaceutical product according to any one of claims 25-29.

32. The method according to claim 31, wherein the HPV infection-related disease is cervical cancer.

33. The method according to claim 31, wherein the HPV infection-related disease is cervical precancerous lesions; Preferably, the HPV infection-related disease is high-grade cervical intraepithelial lesion; Preferably, the HPV infection-related disease is high-grade squamous intraepithelial lesion (HSIL); Preferably, the HPV infection-related disease is cervical intraepithelial neoplasia (CIN) grade 2 or grade 3 (CIN2 / 3); Preferably, the HPV infection-related disease is low-grade LSIL intraepithelial neoplasia (CIN 1).

34. The method according to claim 31, wherein the HPV infection-related disease is HPV infection-related cancer; Preferably, the HPV infection-related disease is cervical adenocarcinoma in situ (AIS); Preferably, the HPV infection-related disease is head and neck squamous cell carcinoma (HNSCC); Preferably, the HPV infection-related disease is oropharyngeal squamous cell carcinoma; Preferably, the HPV infection-related disease is one of anal cancer, vaginal cancer, vulvar cancer and penile cancer.

35. The method according to claim 31, wherein the HPV infection-related disease is a disease associated with human papillomavirus (HPV) type 16 and / or type 18; Preferably, the HPV infection-related disease is human papillomavirus (HPV) type 16 and / or type 18-related grade 2 or grade 3 cervical intraepithelial neoplasia (CIN2 / 3); Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV18-related cervical cancer; Preferably, the HPV infection-related disease is head and neck squamous cell carcinoma associated with human papillomavirus (HPV) type 16 and / or type 18; Preferably, the HPV infection-related disease is anal cancer associated with human papillomavirus (HPV) 16 and / or HPV18; Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV18-related vaginal cancer; Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV18-related vulvar cancer; Preferably, the HPV infection-related disease is human papillomavirus (HPV) 16 and / or HPV18-related penile cancer.

36. The method of any one of claims 31-35, wherein the administration is intratumoral, perilymphatic, or intramuscular injection.

37. The method of any one of claims 31-35, further comprising administering to the individual an immunostimulatory factor, chemotherapy, radiotherapy, and / or targeted therapy.

38. The method according to claim 37, wherein the immunostimulatory factor is selected from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, Pre-IFNα-2β, MIP-α, STING, HSP70, immune checkpoint inhibitors (preferably PD-1 inhibitors or PD-L1 inhibitors).

39. The method according to claim 38, wherein the immune checkpoint inhibitor is preferably one or more of pembrolizumab, nivolumab, tislelizumab, toripalimab, sintilimab, carrelizumab, penampalimab, sepalimab, envolimab, sugemalimab, slulizumab, putalimab, and adebelimumab; further preferably, the PD-1 inhibitor is pembrolizumab.

40. The method according to any one of claims 38-39, wherein the immunostimulatory factor is a protein or a nucleic acid molecule encoding the protein, preferably, the nucleic acid molecule is mRNA.

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