Therapeutic HPV nucleic acid vaccine

Abstract

The invention provides a therapeutic HPV (human papillomavirus) nucleic acid vaccine. The therapeutic HPV nucleic acid vaccine comprises (i) a nucleic acid sequence for encoding an antigen; (ii) a nucleic acid sequence encoding a linker; the linker is selected from at least one of 2A self-cleavage peptide or GS-linked polypeptide; the antigens are early regulatory proteins E6 and E7 of human papilloma virus type 16 or type 18; the connexon is connected with a nucleotide sequence for coding the human papilloma virus early regulatory protein E6 and a nucleotide sequence for coding the human papilloma virus early regulatory protein E7.

Description

Technical Field

[0001] The present invention relates to the technical field of gene drugs, and in particular to a therapeutic HPV nucleic acid vaccine. Background Art

[0002] Therapeutic nucleic acid vaccines mainly include DNA vaccines and RNA vaccines. The principle is to use nucleic acids (DNA or RNA) to encode target antigens. After the nucleic acids are introduced into body cells, the cells will synthesize corresponding antigen proteins according to the genetic information carried by the nucleic acids. These antigen proteins can be recognized by the immune system, thereby activating the body's immune response to achieve the purpose of treating diseases.

[0003] Cancers caused by human papillomavirus (HPV) infection account for about 5% of all cancers. It is estimated that 625,600 women and 69,400 men develop HPV-related cancers every year. Despite progress in vaccination and screening methods for preventing and diagnosing early cancers, the treatment of HPV-related high-risk diseases, especially cervical intraepithelial neoplasia, cervical cancer, and oropharyngeal cancer, etc., are still diseases with a large unmet demand. Among them, HPV is related to the incidence of cervical cancer, and 90% of cervical cancers are closely related to persistent infection with high-risk HPV, among which the correlation of HPV types 16 and 18 is the highest. The therapeutic HPV nucleic acid vaccine is for HPV-infected positive patients, mainly targeting high-risk HPV16 and 18. In view of this, the present invention is proposed. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides the following technical solutions:

[0005] In an alternative embodiment, the present invention provides a therapeutic HPV nucleic acid vaccine, which comprises: (i) a nucleic acid sequence encoding an antigen; (ii) a nucleotide sequence encoding a linker;

[0006] The linker is selected from at least one of 2A self-cleaving peptide or GS linker polypeptide; the antigen is the early regulatory proteins E6 and E7 of human papillomavirus type 16 or 18; the linker connects the nucleic acid sequence encoding the early regulatory protein E6 of human papillomavirus and the nucleic acid sequence encoding the early regulatory protein E7 of human papillomavirus.

[0007] In an alternative embodiment, the antigen is the early regulatory proteins E6 and E7 of human papillomavirus type 16; in an alternative embodiment, the antigen is the early regulatory proteins E6 and E7 of human papillomavirus type 18; in an alternative embodiment, the antigen is the early regulatory protein E6 of human papillomavirus type 16 and the early regulatory protein E7 of human papillomavirus type 18; in an alternative embodiment, the antigen is the early regulatory protein E6 of human papillomavirus type 18 and the early regulatory protein E7 of human papillomavirus type 16.

[0008] In an alternative embodiment, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide.

[0009] In an alternative embodiment, the therapeutic HPV nucleic acid vaccine is an RNA vaccine.

[0010] In an alternative embodiment, the RNA vaccine is selected from mRNA vaccines or circular RNA vaccines.

[0011] In an alternative embodiment, the mRNA vaccine described in the present disclosure comprises mRNA, which consists of a sequence including a 5' cap, 5' UTR, ORF, 3' UTR and 3' poly(A) tail in sequence from the 5' end to the 3' end.

[0012] In an alternative embodiment, the 5' UTR nucleotide sequence is as shown in Seq ID NO.1-3; in an alternative embodiment, the 5' UTR nucleotide sequence is as shown in Seq ID NO.1.

[0013] In an alternative embodiment, the 3' UTR sequence is as shown in Seq ID NO.4-6; in an alternative embodiment, the 3' UTR sequence is as shown in Seq ID NO.4.

[0014] In an alternative embodiment, one or more uridines in the mRNA are replaced with modified nucleosides. In an alternative embodiment, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U). In an alternative embodiment, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0015] In an alternative embodiment, the nucleic acid molecular sequence encoding the early regulatory protein E6 of human papillomavirus type 16 is as shown in Seq ID NO.8, the nucleic acid molecular sequence encoding the early regulatory protein E7 of human papillomavirus type 16 is as shown in Seq ID NO.7, the nucleic acid molecular sequence encoding the early regulatory protein E6 of human papillomavirus type 18 is as shown in Seq ID NO.9, and the nucleic acid molecular sequence encoding the early regulatory protein E7 of human papillomavirus type 18 is as shown in Seq ID NO.10. In an alternative embodiment, the therapeutic HPV nucleic acid vaccine comprises a nucleic acid molecular sequence as shown in Seq ID NO.16 or Seq ID NO.20.

[0016] In an alternative embodiment, the therapeutic HPV nucleic acid vaccine further comprises a nucleic acid sequence encoding a co-stimulatory molecule; the linker connects the nucleic acid sequence encoding the antigen and / or the nucleic acid sequence encoding the co-stimulatory molecule.

[0017] The co-stimulatory molecule is selected from at least one of the B7 family and members of the tumor necrosis factor family; the members of the tumor necrosis factor family are selected from at least one of TNF-β, TNF-α, LT-β, CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, GITRL.

[0018] In an alternative embodiment, the members of the tumor necrosis factor family are selected from at least one of CD252, CD154, CD70, TNFSF7, 4-1BBL, GITRL.

[0019] In an alternative embodiment, the members of the tumor necrosis factor family are CD70 and 4-1BBL.

[0020] In an alternative embodiment, the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2.

[0021] In an alternative embodiment, the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, and HHLA2.

[0022] In an alternative embodiment, the member of the B7 family is CD80.

[0023] In an alternative embodiment, the therapeutic HPV nucleic acid vaccine comprises a nucleic acid molecular sequence as shown in Seq ID NO.19.

[0024] In an alternative embodiment, the therapeutic HPV nucleic acid vaccine further comprises a delivery vector encapsulating the aforementioned nucleic acid sequence; in an alternative embodiment, the delivery vector comprises lipid nanoparticles, cationic liposomes or plasmids.

[0025] In an alternative embodiment, the present invention provides an isolated nucleic acid molecule encapsulated in a delivery vector and formulated into the therapeutic HPV nucleic acid vaccine as described above.

[0026] In an alternative embodiment, the present invention provides a biomaterial comprising any one of an expression cassette, a vector, an engineered bacterium or a cell line, and the biomaterial contains or expresses the isolated nucleic acid molecule as described in any one of the foregoing.

[0027] In an alternative embodiment, the present disclosure provides a method comprising administering to a subject an effective amount of at least one of the vaccines, the isolated nucleic acid molecules or the biomaterials as described in any one of the foregoing to induce a cellular immune response or a humoral immune response in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 Shows the in vitro cellular level expression of the samples in Example 2.

[0030] Figure 2 Shows the curve of the change in the average tumor volume of mice of the samples in Example 3.

[0031] Figure 3 Shows the curve of the change in the average tumor volume of mice of the samples in Example 4.

[0032] Figure 4 Shows the curve of the change in the average tumor volume of mice of the samples in Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The implementation of the present invention is not limited to the following examples. Any form of modification and / or change made to the present invention will fall within the protection scope of the present invention. In the present invention, unless otherwise specified, all devices and raw materials can be purchased from the market or are commonly used in this industry. Without special indication, the methods used in the examples are common techniques in the art.

[0034] For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0035] "Sequence identity" between two nucleotide sequences indicates the percentage of identical nucleotides between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0036] The term "% identity" or a similar term refers to the percentage of identical nucleotides or amino acids between the sequences to be compared under optimal alignment. This percentage is purely statistical, and the differences between the two sequences may be (but not necessarily) randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is usually carried out by comparing such sequences relative to a fragment or "comparison window" after optimal alignment to identify local regions of the corresponding sequences.

[0037] The "nucleic acid sequence" and / or "isolated nucleic acid molecule" of the present invention are nucleic acid molecules prepared by means such as genetic engineering. For example, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" of the present invention may or may not exist in nature as a whole. For another example, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" in the present invention may contain fragments of nucleic acid molecules existing in nature or may not contain fragments of nucleic acid molecules existing in nature.

[0038] In an alternative embodiment, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" of the present invention include nucleotide sequences from different organisms (e.g., from different species). For example, in an alternative embodiment, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" contain murine nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences.

[0039] The "nucleic acid sequence" and / or "isolated nucleic acid molecule" of the present invention contain recombinant nucleotides and / or synthetic nucleotides. "Recombinant nucleotides" refer to molecules constructed by ligating nucleotide molecules that can replicate in a vector. "Synthetic nucleotides" refer to molecules amplified or chemically synthesized or synthesized by other means.

[0040] "Nucleic acid sequence" and / or "isolated nucleic acid molecule" can express genes using multiple promoters by multiple open reading frames (ORFs), that is, more than one separate mRNA transcript can be produced from a single "nucleic acid sequence" and / or "isolated nucleic acid molecule". For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first polypeptide / amino acid, and a second promoter can be operably linked to a polynucleotide sequence encoding a second polypeptide / amino acid. Generally, any number of promoters can be used to express any number of polypeptides / amino acids.

[0041] "Nucleic acid sequence" and / or "isolated nucleic acid molecule" can also express genes using one promoter by one open reading frame (ORF), producing one mRNA transcript and translating it into one polypeptide, which is then self-cleaved by enzymes in the cell to produce multiple polypeptides / amino acids.

[0042] The open reading frame (ORF) described in the present invention is a continuous DNA or RNA that starts with a start codon (such as ATG or AUG) and ends with a stop codon (such as, TAA, TAG or TGA, or UAA, UAG or UGA).

[0043] The "therapeutic HPV nucleic acid vaccine" of the present invention introduces nucleic acid (DNA or RNA) encoding the HPV antigen protein into body cells, expresses the antigen protein in the cells, stimulates the body's immune system to produce specific immune responses, including cellular immunity and humoral immunity, so as to achieve the purpose of clearing HPV-infected cells and diseased tissues.

[0044] In an alternative embodiment, the therapeutic HPV nucleic acid vaccine of the present invention is applied to the treatment of HPV-positive tumors.

[0045] The "HPV-positive tumors" of the present invention include cancers caused by HPV infection and / or certain components thereof (especially HPV E6, and / or, HPV E7). Such cancers include, but are not limited to, anogenital cancers, cervical cancers and penile cancers, as well as cancers in the head and neck region, such as cancers in the genital region or the head and neck region.

[0046] Human papillomavirus (HPV) is a double-stranded DNA virus. Persistent infection with certain high-risk HPVs is an important factor in the development of cancers such as cervical cancer. The genomes of high-risk HPVs (such as HPV16, 18, etc.) can integrate into the host cell genome. The high-risk HPV E6 protein can bind to and degrade human p53 protein. The loss of human p53 protein leads to disorders in cell cycle regulation, causing cells to evade normal apoptosis programs. The high-risk HPV E7 protein can bind to human retinoblastoma protein (pRb), inactivating pRb, thereby releasing the E2F transcription factor and promoting cells to enter the S phase for DNA replication. Abnormal cell proliferation may ultimately lead to carcinogenesis.

[0047] The human papillomavirus early regulatory protein E6 or E7 described in the present invention can be derived from any human papillomavirus, particularly any high-risk human papillomavirus (HPV) type, such as HPV type 16, 18, 31, 33, 45, or 58. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 protein described herein is derived from different human papillomavirus types. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 is derived from the same HPV type. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 is derived from HPV type 16. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 is derived from HPV18. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 is derived from HPV type 31. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 is derived from HPV type 33. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 is derived from HPV45. In an alternative embodiment, the human papillomavirus early regulatory protein E6 or E7 is derived from HPV type 58.

[0048] In an alternative embodiment, the "human papillomavirus early regulatory protein E6" described in the present invention includes the full-length, truncated form, or immunogenic variant of the human papillomavirus early regulatory protein E6.

[0049] In an alternative embodiment, the "human papillomavirus early regulatory protein E7" described in the present invention includes the full-length, truncated form, or immunogenic variant of the human papillomavirus early regulatory protein E7.

[0050] In an alternative embodiment, the amino acid sequence of the early regulatory protein E6 of human papillomavirus type 16 is as shown in UniprotP03126; the amino acid sequence of the early regulatory protein E7 of human papillomavirus type 16 is as shown in UniprotP03129; the nucleotide sequence of the early regulatory protein E6 of human papillomavirus type 16 is as shown in Seq ID NO.8; the nucleotide sequence of the early regulatory protein E7 of human papillomavirus type 16 is as shown in Seq ID NO.7.

[0051] In an alternative embodiment, the amino acid sequence of the early regulatory protein E6 of human papillomavirus type 18 is as shown in UniprotP06463; the amino acid sequence of the early regulatory protein E7 of human papillomavirus type 18 is as shown in UniprotP06788; the nucleotide sequence of the early regulatory protein E6 of human papillomavirus type 18 is as shown in Seq ID NO.9; the nucleotide sequence of the early regulatory protein E7 of human papillomavirus type 18 is as shown in Seq ID NO.10.

[0052] The "costimulatory molecule polypeptide" of the present invention refers to cell surface molecules and their ligands involved in costimulatory signal transduction, which provide necessary costimulatory signals for the full activation of T (or B) cells. "Costimulatory molecule polypeptides" include CD28, ICOS, CTLA-4, members of the B7 family, members of the tumor necrosis factor family, etc. The main biological functions of "costimulatory molecule polypeptides" include enhancing T cell activation and immune responses, and also participating in the regulation of tumor immune responses. For example, the binding of members of the B7 family to CD28 on T cells can generate positive signals, thereby enhancing immune responses; while the binding of CTLA-4 to members of the B7 family can generate negative signals, blocking the T cell activation brought by CD28 and downregulating immune responses.

[0053] In an alternative embodiment, the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2.

[0054] In an alternative embodiment, the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, and HHLA2.

[0055] In an alternative embodiment, the member of the B7 family is CD80.

[0056] In an alternative embodiment, the tumor necrosis factor family member is selected from at least one of TNF-β, TNF-α, LT-β, CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, GITRL.

[0057] In an alternative embodiment, the tumor necrosis factor family member is selected from at least one of CD252, CD154, CD70, TNFSF7, 4-1BBL, GITRL.

[0058] In an alternative embodiment, the tumor necrosis factor family members are CD70 and 4-1BBL.

[0059] In an alternative embodiment, the therapeutic HPV nucleic acid vaccine further comprises a nucleic acid sequence encoding a co-stimulatory molecule; the linker links the nucleic acid sequence encoding the antigen and / or the nucleic acid sequence encoding the co-stimulatory molecule. In an alternative embodiment, the therapeutic HPV nucleic acid vaccine comprises the nucleic acid molecular sequence shown in Seq ID NO.19.

[0060] 2A peptides (2A self-cleaving peptides) are peptide fragments 18 to 22 amino acid residues in length that can induce the self-cleavage of recombinant proteins containing 2A peptides within cells.

[0061] In an alternative embodiment, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide.

[0062] In an alternative embodiment, the amino acid sequence of the F2A peptide is as shown in Seq ID NO.11; the amino acid sequence of the P2A peptide is as shown in Seq ID NO.12; the amino acid sequence of the T2A peptide is as shown in Seq ID NO.13; the amino acid sequence of the E2A peptide is as shown in Seq ID NO.14.

[0063] In an alternative embodiment, the GS sequence comprises (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q (where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5).

[0064] In an alternative embodiment, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" comprises, in an alternative embodiment, at least one open reading frame nucleotide sequence encoding at least one co-stimulatory molecule polypeptide and an open reading frame nucleotide sequence encoding a 2A self-cleaving peptide; in an alternative embodiment, at least one open reading frame nucleotide sequence encoding at least one co-stimulatory molecule polypeptide is linked by at least one open reading frame nucleotide sequence encoding a cleavable 2A self-cleaving peptide.

[0065] In an alternative embodiment, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" is selected from DNA and / or RNA.

[0066] In an alternative embodiment, based on the provided mRNA open reading frame sequence, a person of ordinary skill in the art will be able to obtain the corresponding circular RNA open reading frame sequence and, in accordance with the content of published documents such as CN202180048567.4, prepare a complete circular RNA sequence capable of encoding the same amino acid sequence. In an alternative embodiment, based on the provided mRNA sequence, a person of ordinary skill in the art will be able to obtain the corresponding DNA sequence (e.g., uracil converted to thymine). Similarly, based on the provided DNA sequence, a person of ordinary skill in the art will obtain the corresponding RNA sequence (e.g., thymine converted to uracil). In an alternative embodiment, based on the provided RNA or DNA sequence, a person of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.

[0067] In an alternative embodiment, the DNA is selected from linear DNA and / or circular DNA.

[0068] In an alternative embodiment, the RNA is selected from mRNA or circular RNA.

[0069] In an alternative embodiment, the mRNA described in the present disclosure consists of a sequence comprising a 5' cap, 5' UTR, ORF, 3' UTR, and 3' poly(A) tail in sequence from the 5' end to the 3' end.

[0070] In alternative embodiments, the nucleotide molecule can optimize the mRNA sequence by sequence optimization means to improve the properties related to the expression efficacy after in vivo administration: for example, improving mRNA stability, increasing the translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding of the expressed protein or preventing its misfolding, reducing the toxicity of the expression product, reducing the cell death caused by the expression product, increasing and / or reducing protein aggregation, to obtain an mRNA with improved properties. The purposes of sequence optimization also include: optimizing the formulation and delivery characteristics of the nucleotide-based therapeutic agent while maintaining the structural and functional integrity; overcoming the expression threshold; increasing the expression rate; half-life and / or protein concentration; optimizing protein localization; and avoiding adverse biological responses such as immune responses and / or degradation pathways. The sequence optimization means include: (1) codon optimization according to the codon frequency in a specific organ and / or host organism to ensure proper folding and appropriate expression; (2) adjusting the G / C content to increase mRNA stability or reduce the secondary structure; (3) minimizing tandem repeat codons or base runs that may damage the gene construct or expression; (4) customizing the transcription and translation control regions; (5) reducing or eliminating the problematic secondary structure within the polynucleotide.

[0071] In alternative embodiments, the 5'-end cap is selected from ARCA, mCAP, dmCAP, m7G(5'')ppp(5'')(2''OMeA)pG, tmCAP, m7(3''OMeG)(5'')ppp(5'')(2''OMeA)pG, m7(3''OMeG)(5'')ppp(5'')(2''OMeG)pG, dmCAP or m7G(5'')ppp(5'')(2''OMeG)pG;

[0072] In alternative embodiments, the 5'-end cap is m7Gppp(5')(2'-OMeA)pG.

[0073] In alternative embodiments, the length of the 5'-UTR is preferably 10 to 200 nucleotides.

[0074] In alternative embodiments, the length of the 5'-UTR is 15 to 100 nucleotides.

[0075] In alternative embodiments, the 5'-UTR nucleotide sequence is as shown in Seq ID NO.1 to 3.

[0076] In alternative embodiments, the 5'-UTR nucleotide sequence is as shown in Seq ID NO.1.

[0077] In alternative embodiments, the 3'-UTR sequence is as shown in Seq ID NO.4 to 6.

[0078] In an alternative embodiment, the 3'UTR sequence is as shown in Seq ID NO.4.

[0079] In an alternative embodiment, one or more uridines in the mRNA are replaced with modified nucleosides.

[0080] In an alternative embodiment, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0081] In an alternative embodiment, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0082] In an alternative embodiment, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0083] In an alternative embodiment, the components of the lipid nanoparticles include at least one of a protonatable cationic lipid, a structural lipid, a helper lipid, and a surfactant.

[0084] In an alternative embodiment, by weight parts, the components of the lipid nanoparticles include: 40 - 60 parts of a protonatable cationic lipid, 20 - 40 parts of a structural lipid, 10 - 30 parts of a helper lipid, and 0.5 - 5 parts of a surfactant.

[0085] In an alternative embodiment, the protonatable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA.

[0086] In an alternative embodiment, the structural lipid includes cholesterol and / or a cholesterol derivative.

[0087] In an alternative embodiment, the helper lipid includes at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0088] In an alternative embodiment, the surfactant includes at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0089] In an alternative embodiment, the liposome nanoparticles comprise 20% to 50% cationic lipid by mole percentage, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 20% to 50% DOPG, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 5% to 20% cholesterol, for example, but not limited to, 5%, 10%, 15% or 20%; and 1% to 5% PEG-DMG, for example, but not limited to, 1%, 2%, 3%, 4% or 5%.

[0090] In an alternative embodiment, the liposome nanoparticles comprise 20% to 50% cationic lipid by mole percentage, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 20% to 50% DSCP, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 5% to 20% cholesterol, for example, but not limited to, 5%, 10%, 15% or 20%; and 1% to 5% PEG-DMG, for example, but not limited to, 1%, 2%, 3%, 4% or 5%.

[0091] In an alternative embodiment, the liposome nanoparticles comprise 50% Dlin-MC3-DMA, 10% DOPG, 38.5% cholesterol and 1.5% PEG-DMG by mole percentage.

[0092] In an alternative embodiment, the liposome nanoparticles comprise 50% Dlin-MC3-DMA, 10% DSCP, 38.5% cholesterol and 1.5% PEG-DMG by mole percentage.

[0093] In an alternative embodiment, the present disclosure provides a method for preparing a vaccine according to any one of the foregoing embodiments, characterized in that it comprises: mixing the "nucleic acid sequence" and / or "isolated nucleic acid molecule" with a delivery formulation to form a vaccine; the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0094] In an alternative embodiment, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" comprises at least 2 independent nucleotide sequences, and the method for preparing the vaccine comprises: mixing each independent nucleotide with a delivery formulation respectively, and then mixing the delivery formulations encapsulating each independent nucleotide at a mass ratio of (10 to 1):(1 to 10) to form a vaccine.

[0095] In an alternative embodiment, the mass ratio of the delivery formulation wrapping each independent nucleotide segment is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0096] In an alternative embodiment, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" comprise at least two independent nucleotide sequences, and the method for preparing the vaccine includes: mixing each independent nucleotide segment at a mass ratio of (10-1):(1-10), and then mixing the nucleotide mixture with a delivery formulation respectively to form a vaccine.

[0097] In an alternative embodiment, the mass ratio of each independent nucleotide segment is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0098] In an alternative embodiment, the "independent nucleotide" of the present invention means that there is no direct or indirect connection between the nucleotide sequence and other sequences.

[0099] In an alternative embodiment, the "nucleic acid sequence" and / or "isolated nucleic acid molecule" is mRNA.

[0100] In an alternative embodiment, in the method for preparing the vaccine, mRNA is dissolved in a buffer to obtain an aqueous phase, and each lipid component of the liposome nanoparticles is measured and dissolved in an organic solvent to obtain an organic phase. After mixing the aqueous phase and the organic phase, the organic phase is removed to obtain the vaccine.

[0101] In an alternative embodiment, the volume ratio of the aqueous phase to the organic phase is 1:(2-4), preferably 1:3.

[0102] In an alternative embodiment, the buffer includes citrate buffer or sodium acetate, preferably citrate buffer.

[0103] In an alternative embodiment, the pH of the buffer is 3-7, preferably 4.

[0104] In an alternative embodiment, the concentration of mRNA in the aqueous phase is 0.05 mg / mL - 0.5 mg / mL, preferably 0.1 mg / mL.

[0105] In an alternative embodiment, the organic solvent is selected from C1-C4 lower alcohols, preferably absolute ethanol.

[0106] In an alternative embodiment, the concentration of the lipid component in the organic phase is 5 mg / mL to 7 mg / mL, preferably 6 mg / mL.

[0107] In an alternative embodiment, a microfluidic device is used to mix the aqueous phase and the organic phase, and the organic solvent is filtered by tangential flow filtration;

[0108] Preferably, the flow rate of the microfluidic device is > 3 ml / min, more preferably 12 mL / min.

[0109] In an alternative embodiment, after mixing, a concentration step is further included, and the final concentration of mRNA in the concentration step is 50 μg / mL to 200 μg / mL, preferably 100 μg / mL.

[0110] In an alternative embodiment, the diameter of the lipid nanoparticles is less than about 200 nm. In an alternative embodiment, the diameter of the lipid nanoparticles is less than less than about 150 nm. In an alternative embodiment, the diameter of the lipid nanoparticles is less than 100 nm. In an alternative embodiment, the diameter of the lipid nanoparticles is about 55 nm to about 90 nm.

[0111] In an alternative embodiment, the present disclosure provides a method, the method comprising administering to a subject an effective amount of at least one of the vaccines described in any one of the foregoing or the isolated nucleic acid molecules described in any one of the foregoing to induce a cellular immune response or a humoral immune response in the subject.

[0112] In an alternative embodiment, the method comprises administering to a subject at least two effective amounts of at least one of the vaccines described in any one of the foregoing or the isolated nucleic acid molecules described in any one of the foregoing.

[0113] In an alternative embodiment, the time interval between the first administration and the second administration in the at least two administrations is not less than 14 days.

[0114] In an alternative embodiment, the subject has a weakened immune system; in an alternative embodiment, the age of the subject is not higher than 5 years old or not lower than 65 years old.

[0115] In an alternative embodiment, the effective amount in the present disclosure is as low as 40 μg, 30 μg, 25 μg, 20 μg, 15 μg, 10 μg, 5 μg, 3 μg or 1 μg of at least one of the vaccines described in any one of the foregoing or the isolated nucleic acid molecules described in any one of the foregoing.

[0116] Example 1: Process for preparing LNP from RNA

[0117] A therapeutic HPV nucleic acid vaccine (the delivery vector is a lipid nanoparticle), wherein the lipid nanoparticle comprises 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol, and 1% PEG-DMG in terms of molar percentage.

[0118] The preparation method is as follows:

[0119] (a) Dissolve the isolated nucleic acid molecule in a citrate buffer solution with a pH of 4, and adjust the concentration to 0.1 mg / ml to obtain an aqueous phase.

[0120] (b) Dissolve Dlin-MC3-DMA, DOPG, cholesterol, and PEG-DMG in anhydrous ethanol according to the formula amount, and adjust the concentration of the lipid components in the organic phase to 6 mg / mL to obtain an organic phase.

[0121] (c) Mix the aqueous phase in step (a) and the organic phase in step (b) at a volume ratio of 1:3 using a microfluidic device, and mix at a flow rate of 12 mL / min. Immediately dilute the mixture 100-fold with a PBS solution with a pH of 7.4, and use tangential flow filtration (TFF) to remove the ethanol component in the solution, and then concentrate to a concentration of 55 μg / ml of mRNA in the system to obtain a therapeutic HPV nucleic acid vaccine (the delivery vector is a lipid nanoparticle).

[0122] Example 2

[0123] In this experimental example, the expression of the isolated nucleic acid at the in vitro cell level was detected. The specific method is as follows: Digest the 293 cells cultured for more than 24 hours and seed them into a 6-well plate. After culturing at 37 °C for 24 hours, observe the cell state under a microscope. When the cell confluence reaches more than 80%, mRNA transfection can be carried out. Transfect the corresponding mRNA into 293 cells using the lipofectamine2000 kit (transfect 5 μg of mRNA per well), and the specific operation refers to the kit instructions.

[0124] After adding the mRNA, continue to culture at 37 °C for 24 hours, lyse the HEK293 cells, and specifically detect the target protein by SDS-PAGE immunoblotting with a loading amount of 10 μg of total protein.

[0125] In this example, an antibody against the early regulatory protein E7 of HPV type 16 was used as the primary antibody and a goat anti-rabbit-HRP antibody was used as the secondary antibody for incubation, and then color development was carried out. When analyzing the detection results of the protein expression level, the internal reference GADPH was used for standardized quantification, and at the same time, cells not transfected with mRNA were set as negative controls to compare the protein amounts expressed after transfection of different isolated nucleic acids into cells.

[0126] In this example, samples LRIO-207 (the sequence of the isolated nucleic acid is as shown in Seq ID NO.16) and 087 (the sequence of the isolated nucleic acid is as shown in Seq ID NO.17) were used to detect the expression at the in vitro cell level.

[0127] In addition to the above open reading frame sequence, sample LRIO-207 also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil of the RNA is replaced by 5' pseudouracil.

[0128] In addition to the above open reading frame sequence, sample 087 also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in Seq ID NO.2), a 3' UTR (as shown in Seq ID NO.20), and a 3' polyA tail of 124 adenine nucleotides; and the uracil of the RNA is replaced by 5' pseudouracil.

[0129] The connection form between the functional regions of the isolated nucleic acid molecule in sample LRIO-207 is: E7…P2A…E6.

[0130] The connection form between the functional regions of the isolated nucleic acid molecule in sample 087 (refer to GreenLeaf Pharma 2023-HPV16 E6 E7-based mRNA vaccine istherapeutic in mice bearing aggressive HPV-positive lesions) is: signal peptide - E6m…Furin…E7m.

[0131] The above-mentioned "connection form between the functional regions" describes the order of the functional regions of the isolated nucleic acid molecule from the 5' end to the 3' end; for the functional regions connected by "-", not only the encoded nucleotide sequences are connected respectively, but also the amino acid sequences are connected respectively; for the functional regions connected by “…”, the encoded nucleotide sequences are connected respectively, and the amino acid sequences are not connected.

[0132] For example, the connection form "E7…P2A…E6" between the functional regions in the co-stimulatory molecule polypeptide expressed by sample LRIO-207 means that sample LRIO-207 is a polynucleotide sequence, and the functional regions of the polypeptide expressed by it (which can also be expressed as "the amino acid sequence encoded by sample LRIO-207") from the 3' end to the 5' end are E7, P2A, and E6 respectively.

[0133] The expression of sample LRIO-207 and sample 087 at the in vitro cell level is shown in Figure 1The results showed that the nucleic acid molecules isolated from sample LRIO-207 and sample 087 were able to express the early regulatory protein E7 of human papillomavirus type 16, but the expression level of the early regulatory protein E7 of human papillomavirus type 16 in sample LRIO-207 was higher than that in sample 087.

[0134] Example 3

[0135] C57BL / 6 mice (female, 5 - 6 weeks old, average body weight 15 - 20 g, purchased from Zhuhai Biosino Biotechnology Co., Ltd.) were selected for in vivo anti-tumor efficacy evaluation.

[0136] The experimental mice were subcutaneously inoculated with TC-1 tumor cells. When the average tumor volume reached about 100 mm 3 ³, the mice were randomly divided into 3 groups according to the tumor volume, and the coefficient of variation (CV) of the tumor volume was ≤ 1 / 3. The day of grouping was defined as D0. The three groups were respectively denoted as the NC group (injected with empty LNP), the LRIO-207 group, and the 087 group, with 6 mice in each group.

[0137] On the 0th, 7th, and 14th days of the experiment, each group was intramuscularly injected with 5 μg / mouse, and the tumor volume was detected 2 - 3 times per week after administration.

[0138] Among them, the LRIO-207 group was injected with lipid nanoparticles containing the isolated nucleic acid molecule (therapeutic HPV nucleic acid vaccine). The preparation method was referred to Example 1, and the nucleotide sequence of the isolated nucleic acid molecule was shown in Example 2. The lipid nanoparticles injected into the 087 group were prepared according to the method described in Example 1, and the nucleotide sequence was shown in Example 2.

[0139] The experimental results are shown in Figure 2 As shown. Compared with the NC group, the other experimental groups could all inhibit tumor growth; and the effect of the LRIO-207 group on inhibiting tumor growth was better than that of the 087 group.

[0140] Example 4

[0141] CD80 and CD70 humanized mice (female, 5 - 6 weeks old, average body weight 15 - 20 g, purchased from Shanghai Model Organisms Center, Inc.) were selected for in vivo anti-tumor efficacy evaluation.

[0142] The experimental mice were subcutaneously inoculated with TC-1 tumor cells. When the average tumor volume reached about 100 mm³, the mice were randomly divided into 3 groups according to the tumor volume, and the coefficient of variation (CV) of the tumor volume was ≤ 1 / 3. The day of grouping was defined as D0. The three groups were respectively denoted as the NC group (injected with empty LNP), the LRIO-207 group, and the LRIO-118 group, with 6 mice in each group.

[0143] On the 0th, 7th, and 14th days of the experiment, each mouse in each group was administered 20 μg by intramuscular injection, and the tumor volume was measured 2-3 times a week after administration.

[0144] Among them, the samples LRIO-207 group and the samples LRIO-118 group were injected with lipid nanoparticles containing isolated nucleic acid molecules (therapeutic HPV nucleic acid vaccine), and the preparation method is shown in Example 1. The nucleotide sequence of the isolated nucleic acid molecule contained in the samples LRIO-207 group is shown in Example 2. The nucleotide sequence of the isolated nucleic acid molecule contained in the samples LRIO-118 group is shown in Seq ID NO.19.

[0145] In addition to the above open reading frame sequences, the isolated nucleic acids contained in the samples LRIO-207 group and the samples LRIO-118 group also include a 5' cap (m7Gppp(5')), a 5' UTR (such as Seq ID NO.1), a 3' UTR (such as Seq ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil of the RNA is replaced with 5' pseudouracil.

[0146] The connection form between the functional regions of the isolated nucleic acid molecule in the samples LRIO-118 is: CD80-E7…P2A…E6…T2A…CD70.

[0147] The experimental results are shown in Figure 3 Compared with the NC group, all other experimental groups could inhibit tumor growth; the samples LRIO-118 group had a better inhibitory effect on tumor growth than the samples LRIO-207 group.

[0148] Example 5

[0149] C57BL / 6 mice (female, 5-6 weeks old, average weight 15-20 g, purchased from Zhuhai Biosino Biotechnology Co., Ltd.) were selected for in vivo anti-tumor efficacy evaluation.

[0150] The experimental mice were subcutaneously inoculated with TC-1 tumor cells. When the average tumor volume reached about 100 mm 3 , the mice were randomly divided into four groups according to the tumor volume, and the coefficient of variation (CV) of the tumor volume was ≤1 / 3. The day of grouping was defined as D0. In the anti-PD-L1 antibody experiment, the four groups were respectively designated as the NC group (injected with empty LNP), the LRIO-207 group, the LRIO-207+α-PD-L1 group, and the α-PD-L1 group, with 6 mice in each group. In the anti-PD-1 antibody experiment, the four groups were respectively designated as the NC group (injected with empty LNP), the LRIO-207 group, the LRIO-207+α-PD-1 group, and the α-PD-1 group, with 6 mice in each group.

[0151] On day 0, day 7, and day 14 of the experiment, the LRIO-207 group, the LRIO-207 + α-PD-1 group, and the LRIO-207 + α-PD-L1 group were intramuscularly injected with the sample LRIO-207 at a dose of 5 μg per mouse. On day 0, day 3, day 7, day 10, day 14, and day 17 of the experiment, the α-PD-1 group, the α-PD-L1 group, the LRIO-207 + α-PD-1 group, and the LRIO-207 + α-PD-L1 group were intraperitoneally injected with anti-PD-1 or anti-PD-L1 antibody at a dose of 200 μg per mouse. After administration, the tumor volume was detected 2-3 times a week.

[0152] Among them, the sample LRIO-207 group was injected with lipid nanoparticles containing the isolated nucleic acid molecule (therapeutic HPV nucleic acid vaccine). The preparation method is shown in Example 1, and the nucleotide sequence of the isolated nucleic acid molecule is shown in Example 2. The anti-PD-1 antibody and the anti-PD-L1 antibody were purchased from BioXCell.

[0153] The experimental results are shown in Figure 4 as follows. Compared with the NC group, all other experimental groups could inhibit tumor growth; the LRIO-207 + α-PD-1 group had a better effect on inhibiting tumor growth than the LRIO-207 group and the α-PD-1 group used alone; the LRIO-207 + α-PD-L1 group had a better effect on inhibiting tumor growth than the LRIO-207 group and the α-PD-L1 group used alone.

[0154] Example 6

[0155] In this experimental example, the expression of the isolated nucleic acid at the in vitro cell level was detected. The specific method was as follows: The 293 cells cultured for more than 24 hours were digested and seeded into 6-well plates. After culturing at 37 °C for 24 hours, the cell status was observed under a microscope. When the cell confluence reached more than 80%, mRNA transfection could be carried out. The corresponding mRNA was transfected into 293 cells using the lipofectamine2000 kit (5 μg of mRNA was transfected into each well), and the specific operation referred to the kit instructions.

[0156] After adding the mRNA, the cells were continuously cultured at 37 °C for 24 hours. The HEK293 cells were lysed, and the target protein was specifically detected by SDS-PAGE immunoblotting with a loading amount of 10 μg of total protein.

[0157] In this example, the early regulatory protein E6 antibody against murine HPV type 16 was used as the primary antibody and the goat anti-mouse-HRP antibody was used as the secondary antibody for incubation, and then color development was carried out. When analyzing the detection results of the protein expression level, the internal reference GADPH was used for standardized quantification, and at the same time, the cells not transfected with mRNA were set as negative controls to compare the protein amounts expressed after transfection of different isolated nucleic acids into cells.

[0158] In this embodiment, in addition to the open reading frame sequence, the samples for detecting the expression at the in vitro cell level further include a 5' cap (m7Gppp(5')), a 5' UTR, a 3' UTR, and a 3' polyA tail of 100 adenine nucleotides; and the uracil of the RNA is replaced with 5' pseudouracil. The sequence conditions of each sample are described in Table 1.

[0159] Table 1

[0160] Sample number Open reading frame sequence 5’UTR sequence 3’UTR sequence Relative expression level LRIO-207 Seq ID NO.16 Seq ID NO.1 Seq ID NO.3 1 LRIO-208 Seq ID NO.16 Seq ID NO.1 Seq ID NO.18 0.6 LRIO-209 Seq ID NO.20 Seq ID NO.1 Seq ID NO.4 0.7 LRIO-210 Seq ID NO.16 Seq ID NO.2 Seq ID NO.4 0.7 LRIO-211 Seq ID NO.16 Seq ID NO.3 Seq ID NO.5 0.7 LRIO-211 Seq ID NO.16 Seq ID NO.3 Seq ID NO.6 0.7

Claims

1. A therapeutic HPV nucleic acid vaccine, characterized in that: The therapeutic HPV nucleic acid vaccine comprises: (i) a nucleic acid sequence encoding an antigen; (ii) a nucleic acid sequence encoding a linker; The linker is selected from at least one of a 2A self-cleaving peptide or a GS-linked polypeptide; the antigens are early regulatory proteins E6 and E7 of human papillomavirus type 16 or type 18; the linker connects a nucleic acid sequence encoding the human papillomavirus early regulatory protein E6 and a nucleic acid sequence encoding the human papillomavirus early regulatory protein E7.

2. The therapeutic HPV nucleic acid vaccine according to claim 1, characterized in that: The antigens are early regulatory proteins E6 and E7 of human papillomavirus type 16; Preferably, the antigens are early regulatory proteins E6 and E7 of human papillomavirus type 18; Preferably, the antigen is the early regulatory protein E6 of human papillomavirus type 16 and the early regulatory protein E7 of human papillomavirus type 18; Preferably, the antigens are early regulatory protein E6 of human papillomavirus type 18 and early regulatory protein E7 of human papillomavirus type 16; Preferably, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide.

3. The therapeutic HPV nucleic acid vaccine according to claim 1, characterized in that: The therapeutic HPV nucleic acid vaccine is an RNA vaccine; Preferably, the RNA vaccine is selected from an mRNA vaccine or a circular RNA vaccine; Preferably, the mRNA vaccine comprises mRNA, and the mRNA consists of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail from the 5' end to the 3' end.

4. The therapeutic HPV nucleic acid vaccine according to claim 3, characterized in that: The 5'UTR nucleotide sequence is as shown in Seq ID NO.1 to 3; preferably, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1; Preferably, the 3'UTR sequence is as shown in Seq ID NO.4 to 6; Preferably, the 3'UTR sequence is as shown in Seq ID NO.4; Preferably, one or more uridines in the mRNA are replaced with modified nucleosides; Preferably, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U); Preferably, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

5. The therapeutic HPV nucleic acid vaccine according to claim 1, characterized in that: The nucleic acid molecule sequence encoding the early regulatory protein E6 of human papillomavirus type 16 is shown in Seq ID NO.8, the nucleic acid molecule sequence encoding the early regulatory protein E7 of human papillomavirus type 16 is shown in Seq ID NO.7, the nucleic acid molecule sequence encoding the early regulatory protein E6 of human papillomavirus type 18 is shown in Seq ID NO.9, and the nucleic acid molecule sequence encoding the early regulatory protein E7 of human papillomavirus type 18 is shown in Seq ID NO.10; Preferably, the therapeutic HPV nucleic acid vaccine comprises a nucleic acid molecule sequence as shown in Seq ID NO.16 or Seq ID NO.

20.

6. The therapeutic HPV nucleic acid vaccine according to claim 1, characterized in that: The therapeutic HPV nucleic acid vaccine further comprises a nucleic acid sequence encoding a co-stimulatory molecule; the linker connects the nucleic acid sequence encoding an antigen, and / or the nucleic acid sequence encoding a co-stimulatory molecule; Preferably, the co-stimulatory molecule is selected from at least one of the B7 family and the tumor necrosis factor family members; preferably, the tumor necrosis factor family member is selected from at least one of TNF-β, TNF-α, LT-β, ​​CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, and GITRL; Preferably, the tumor necrosis factor family member is selected from at least one of CD252, CD154, CD70, TNFSF7, 4-1BBL, and GITRL; Preferably, the tumor necrosis factor family members are CD70 and 4-1BBL; Preferably, the B7 family member is selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and HHLA2; Preferably, the B7 family member is selected from at least one of CD80, CD86, B7-DC, PD-L1 and HHLA2; Preferably, the B7 family member is CD80; Preferably, the therapeutic HPV nucleic acid vaccine comprises the nucleic acid molecule sequence shown in Seq ID NO.

19.

7. The therapeutic HPV nucleic acid vaccine according to claim 1, characterized in that: The therapeutic HPV nucleic acid vaccine also includes a delivery vector that encapsulates the nucleic acid sequence; in an optional embodiment, the delivery vector includes lipid nanoparticles, cationic liposomes or plasmids.

8. An isolated nucleic acid molecule, characterized in that The isolated nucleic acid molecule is mixed with a delivery vector to prepare the therapeutic HPV nucleic acid vaccine according to any one of claims 1 to 7.

9. A biomaterial, characterized in that: The biological material comprises any one of an expression cassette, a vector, an engineered bacterium or a cell line, and the biological material contains or expresses the isolated nucleic acid molecule according to claim 8.

10. A method, characterized in that The method comprises administering to a subject an effective amount of at least one of the vaccines of claims 1 to 7, the isolated nucleic acid molecules of claim 8, or the biological material of claim 9 to induce a cellular immune response or a humoral immune response in the subject.

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