An antigenic molecule, nucleic acid molecule encoding same and uses

Abstract

This application relates to an antigen molecule, the nucleic acid molecule encoding it, and its applications. The structure of the antigen molecule, from N-terminus to C-terminus, includes an XCL1 region, an antigenic region, and a 4-1BBL region. The XCL1 region is an amino acid sequence derived from XCL1, and the 4-1BBL region is an amino acid sequence derived from the extracellular region of 4-1BBL. This antigen molecule exhibits a higher tumor inhibition rate and can effectively delay the onset and growth rate of tumors.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to an antigen molecule and the nucleic acid molecule encoding it, and its applications. Background Technology

[0002] HPV is a major pathogen causing cervical cancer, anogenital cancers (including vulvar cancer, vaginal cancer, anal cancer, and penile cancer), head and neck cancer, and genital warts. Approximately 80% of men and women will experience HPV infection during their lifetime, but most infected individuals can clear the virus naturally with their immune system. However, persistent HPV infection can potentially develop into precancerous lesions or even malignant tumors. Persistent infection with high-risk HPV types is clearly a major contributing factor to cervical intraepithelial neoplasia (CIN) and cervical cancer, especially HPV types 16 and 18, which are closely associated with over 70% of CIN grade 2 / 3 lesions and cervical squamous cell carcinoma (SCC).

[0003] Currently available preventative HPV vaccines (bivalent, quadrivalent, and nonavalent) are effective in preventing HPV infection, but they do not treat patients already infected with HPV. Persistent infection with high-risk HPV viruses for two years can progress to cervical lesions of infertility (HSIL), which can then develop into cervical cancer, anal cancer, vaginal cancer, oropharyngeal cancer, etc. Developing therapeutic HPV vaccines is an ideal strategy for controlling existing HPV infection and treating HPV-related cancers and precancerous lesions. Tumor therapeutic vaccines refer to products that induce or enhance the body's specific active immune response against tumor antigens, thereby achieving therapeutic goals such as controlling and killing tumor cells, eliminating minimal residual disease, and establishing lasting anti-tumor memory. Active tumor immunotherapy has various technical routes, targeting antigen types including tumor-specific antigens (TSA), tumor-associated antigens (TAA), or other antigens that aid in tumor treatment.

[0004] The HPV genome consists of seven early genes (named E1 to E7) and two late genes (L1 and L2). When HPV infects the basal cells of the cervical epithelium, its viral genome tends to integrate with the host cell's genome. Crucially, the proteins E6 and E7 are expressed in almost all cervical cancer cells and are essential for maintaining the disease's phenotypic characteristics. Therefore, they are ideal target proteins for developing therapeutic vaccines. Invention Overview

[0005] This application provides an antigen molecule, a nucleic acid molecule encoding it, and its application. This structure has a higher tumor inhibition rate and can effectively delay the onset and growth rate of tumors.

[0006] The first aspect of this application provides an antigen molecule whose structure, from the N-terminus to the C-terminus, includes an XCL1 region, an antigen region, and a 4-1BBL region, wherein the XCL1 region is an amino acid sequence derived from XCL1, and the 4-1BBL region is an amino acid sequence derived from the extracellular region of 4-1BBL.

[0007] In some embodiments, the antigen molecule further includes a trimerization motif located at the C-terminus of the 4-1BBL region or between the antigen region and the 4-1BBL region.

[0008] In some embodiments, the amino acid sequence derived from XCL1 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36; optionally, the amino acid sequence derived from XCL1 is the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36.

[0009] In some embodiments, the amino acid sequence derived from the extracellular region of 4-1BBL comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37; optionally, the amino acid sequence derived from the extracellular region of 4-1BBL is the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37.

[0010] In some embodiments of the above-mentioned antigen molecule, the trimerizing motif is a Foldon domain derived from the fibritin protein of T4 phage; further optionally, the trimerizing motif comprises the amino acid sequence shown in SEQ ID NO:6, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:6; even more optionally, the trimerizing motif is the amino acid sequence shown in SEQ ID NO:6.

[0011] In some embodiments, the antigen region of the aforementioned antigen molecule comprises an HPV antigen; optionally, the HPV antigen comprises an amino acid sequence derived from HPV16 E7 protein and an amino acid sequence derived from HPV18 E6E7 protein; further optionally, the amino acid sequence derived from HPV16 E7 protein or HPV18 E6E7 protein is a full-length sequence or a truncated sequence; even further optionally, the amino acid sequence derived from HPV16 E7 protein is a full-length sequence, and the amino acid sequence derived from HPV18 E6E7 protein is a fusion protein comprising a full-length sequence derived from HPV18 E6 protein and a truncated version derived from HPV18 E7 protein; even further optionally, the full-length sequence derived from HPV18 E6 protein is the amino acid sequence shown in positions 1-102 of SEQ ID NO:3, and the truncated version derived from HPV18 E7 protein comprises the amino acid sequences shown in positions 113-123 of SEQ ID NO:3 and the amino acid sequences shown in positions 134-148 of SEQ ID NO:3.

[0012] In some embodiments, the amino acid sequence derived from the HPV18 E6E7 protein is the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:3, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:3.

[0013] In some embodiments, the amino acid sequence derived from the HPV16 E7 protein is the full-length sequence of the HPV16 E7 protein. Optionally, the full-length sequence of the HPV16 E7 protein is the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO: 2.

[0014] In some embodiments, the antigen region of the antigen molecule is arranged from the N-terminus to the C-terminus as follows: an amino acid sequence derived from HPV16 E7 protein, an amino acid sequence derived from HPV18 E6E7 protein; or an amino acid sequence derived from HPV18 E6E7 protein, an amino acid sequence derived from HPV16 E7 protein; optionally, the amino acid sequence of the antigen region is as shown in SEQ ID NO:47.

[0015] In some embodiments, the antigen molecule further includes a signal peptide at the N-terminus of the XCL1 region; optionally, the signal peptide is an IgE signal peptide; further optionally, the amino acid sequence of the IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO:1, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:1; even more optionally, the amino acid sequence of the IgE signal peptide is the amino acid sequence shown in SEQ ID NO:1.

[0016] In some embodiments, the antigen molecule described above comprises the following structure from the N-terminus to the C-terminus: an IgE signal peptide - an amino acid sequence derived from XCL1 - an amino acid sequence derived from HPV16 E7 protein - an amino acid sequence derived from HPV18 E6E7 protein - an amino acid sequence derived from the extracellular region of 4-1BBL - a trimerization motif; or, an IgE signal peptide - an amino acid sequence derived from XCL1 - an amino acid sequence derived from HPV16 E7 protein - an amino acid sequence derived from HPV18 E6E7 protein - a trimerization motif - an amino acid sequence derived from the extracellular region of 4-1BBL, wherein: "-" indicates direct linkage via peptide bonds or indirect linkage via one or more amino acids.

[0017] In some embodiments, the antigen molecule described above may include independent linking sequences between different amino acid sequences; optionally, the linking sequences may be selected from one or more of the following: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46.

[0018] In some embodiments, the antigen molecule comprises, from the N-terminus to the C-terminus, an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40; optionally, the antigen molecule comprises, from the N-terminus to the C-terminus, an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 97%, 96%, 97%, 96%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 94%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, The amino acid sequence shown in NO:40.

[0019] A second aspect of this application provides a nucleic acid molecule comprising a polynucleotide sequence encoding the aforementioned antigen molecule.

[0020] In some embodiments, the polynucleotide sequence of the aforementioned nucleic acid molecule is a DNA sequence or an mRNA sequence; optionally, the polynucleotide sequence is a DNA sequence comprising a polynucleotide sequence as shown in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:39 or SEQ ID NO:41, or a polynucleotide sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the polynucleotide sequence shown in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:39 or SEQ ID NO:41.

[0021] A third aspect of this application provides a vector comprising the aforementioned nucleic acid molecules.

[0022] In some embodiments, the vector is a plasmid; alternatively, the vector is a pVAX.1 plasmid with the aforementioned nucleic acid molecule inserted into an open reading frame.

[0023] A fourth aspect of this application provides a DNA vaccine comprising the aforementioned nucleic acid molecule or the aforementioned vector.

[0024] The fifth aspect of this application provides a cell comprising the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, or the aforementioned DNA vaccine.

[0025] The sixth aspect of this application provides a pharmaceutical composition comprising the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned carrier, the aforementioned DNA vaccine, or the aforementioned cell, and the pharmaceutical composition further comprising pharmaceutically acceptable excipients.

[0026] The seventh aspect of this application provides the use of the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating HPV16-positive and / or HPV18-positive diseases.

[0027] The eighth aspect of this application provides a method for treating HPV16-positive and / or HPV18-positive diseases, comprising administering an effective amount of the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cells, or the aforementioned pharmaceutical composition to an individual in need of treatment for HPV16-positive and / or HPV18-positive diseases.

[0028] In some embodiments of the above method, the administration is an intramuscular injection followed by an electrical pulse.

[0029] The ninth aspect of this application provides the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cell, or the aforementioned pharmaceutical composition for treating a disease; optionally, the disease for treating is a disease that is HPV16 positive and / or HPV18 positive.

[0030] The tenth aspect of this application provides a drug-device combination kit, comprising the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cells or the aforementioned drug composition, and an electroporator.

[0031] It should be understood that the aspects and embodiments of this application described herein include those described as "comprising," "forming," and "essentially consisting of." The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application. Attached Figure Description

[0032] Figure 1 The graph shows the inhibition results of each group of plasmid DNA vaccines on the volume of tumor cells in mice at different time points after TC-1 tumor cell inoculation in Example 1 of this application.

[0033] Figure 2 The image shows the results of detecting specific T cell responses against HPV epitope peptides produced by lymphocytes using ELISPOT IFN-γ spots in Example 2 of this application; immunization of mice with 10 μg of molecule 16, both after the second and third immunizations, significantly elicited 16E7 and 18E6 specific cellular immunity 7 days later.

[0034] Figure 3A and Figure 3B The graphs show the inhibition results of each group of plasmid DNA vaccines in Example 4 of this application on the volume and tumor weight of TC-1 tumor cells in mice. * represents P≤0.05, ** represents P≤0.01, *** represents P≤0.001, compared with the PVAX1 empty vector group. * / ** / *** all indicate significant differences compared with the PVAX1 group. Invention Details

[0035] the term

[0036] In this application, "XCR1" refers to XC motif chemokine receptor 1. Chemokines and their receptors play a crucial role in the development, localization, and immune response of immune cells; therefore, chemokines can be used to regulate the body's immune response and improve vaccine efficacy. In humans, 23 chemokine receptors are known, among which XCR1 is a specific receptor for XC motif chemokine ligand 1 (XCL1).

[0037] In this application, "4-1BBL" is also known as 4-1BB ligand or CD137L. It is a type II transmembrane protein of the TNF superfamily, mainly found on antigen-presenting cells such as macrophages, B cells, T cells, and dendritic cells (DCs).

[0038] In this application, the "trimerization motif" is used to compensate for the loss of transmembrane domains and / or cytoplasmic domains, restoring the ability of proteins to form homotrimers. Known structures suitable for this function include: the Foldon domain of T4 phage fibritin protein, which is a domain that can cause non-covalent oligomerization of target proteins. It is derived from the C-terminus of T4 phage fibrin and consists of 27 amino acids. The degree of oligomerization formed by non-covalent interactions is relatively high. CPCCPP is similar to a hinge region and is suitable for antibodies and fusion proteins. It facilitates the granulation of trimeric proteins and can further enhance immunogenicity.

[0039] In this application, "signal peptide" has the common definition in the art, and it is usually located at the N-terminus of a protein precursor.

[0040] In this application, "linker sequence" or "linker" refers to a peptide chain in a polypeptide or protein that links two amino acid fragments together. Linker peptides can be flexible linker peptides, allowing the two linked amino acid fragments to have a certain degree of mobility. Common flexible linker peptides consist of Gly and Ser residues ("GS" linker). Besides GS flexible linker peptides, there are other flexible linker peptides, such as (Gly)8, which are known in the art. Linker sequences can also be rigid links, which can be used to completely isolate two linked proteins, maintaining their independent functions. Commonly used rigid linker peptides include α-helix peptides, (XP)n, etc., where P represents proline, X can be any amino acid, preferably Ala, Lys, or Glu, and n represents the number of XP repetitions. Shearable peptides can also be used as linker sequences, such as P2A and T2A. Those skilled in the art can independently adjust and select different linker peptides according to specific application scenarios and the 3D structure requirements of fusion proteins.

[0041] In this application, "N-end direction" and "C-end direction" are used to describe the relative positional relationship between two sequences, and have the conventional meaning understood by those skilled in the art.

[0042] In this application, the percentages of “identity” or “homology”, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5%, refer to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment, and have the conventional meaning understood by those skilled in the art.

[0043] In this application, depending on the context, "variant" can refer to a variant of a protein, polypeptide, or amino acid sequence, or a variant of a nucleic acid molecule or polynucleotide sequence. A "variant" of a protein, polypeptide, or amino acid sequence refers to a sequence or molecule that retains the same or substantially the same biological activity as the original sequence. The variant may be derived from homologous proteins of different mutant strains of the same HPV subtype, or may be a synthetic sequence based on a natural molecule or an existing molecule. In some embodiments, the "variant" of the amino acid sequence differs from the amino acid sequence by at least one amino acid, for example, by at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution may be a conserved amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitution" 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); nonpolar to nonpolar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), and phenylalanine (F, Phe); and acidic to acidic amino acids, such as aspartic acid (D, Asp) and glutamic acid (E, Gln). u); basic amino acids for basic amino acids, such as arginine (R, Arg), histidine (H, His), and lysine (K, Lys); charged amino acids for charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys), and arginine (R, Arg); hydrophobic amino acids for 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). The "variants" of the amino acid sequence may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 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 range 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 any two of the aforementioned values.In this application, a "conservative substitution variant" of a protein, polypeptide, or amino acid sequence refers to a protein or enzyme in which one or more amino acid residues are substituted without altering the overall conformation and function of the protein or enzyme. This includes, but is not limited to, substitution of amino acids in the parent protein's amino acid sequence in the manner described above as "conservative substitution." Therefore, the similarity of two proteins or amino acid sequences with similar functions may vary. For example, similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes that, as determined by BLAST or FASTA algorithms, have more than 60% amino acid identity, preferably more than 75%, most preferably more than 85%, and ideally more than 90%, and possess the same or substantially similar properties or functions as the native or parent protein or enzyme. Those skilled in the art will understand that variants of nucleic acid molecules or polynucleotide sequences encoding proteins include "synonymous mutants," which are nucleic acid molecules or polynucleotide sequences obtained by substituting one or more codons in the nucleic acid molecule or polynucleotide sequence with other codons encoding the same amino acid as the codons.

[0044] Unless otherwise specified, the term "nucleotide" in this application refers not only to naturally occurring ribonucleotide or deoxyribonucleotide monomers, but also to their related structural variants, including derivatives and analogs, which are functionally equivalent in relation to the specific context of use of the nucleotide, unless the context explicitly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moieties of nucleotides include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as the substitution of carbon and nitrogen atoms of the base by other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino oligonucleotides.

[0045] In this application, "DNA vaccine" is a novel genetically engineered vaccine that introduces a vector (such as a plasmid) containing a DNA gene sequence encoding a target antigen protein into a host cell (such as through intramuscular injection), expresses the antigen protein through a transcription system, and induces the host to produce an immune response against the antigen protein, thereby achieving the purpose of immunization.

[0046] In this application, the term "about" refers to a typical range of error for various values ​​that is readily known to those skilled in the art. References to a value or parameter "about" herein include embodiments for that value or parameter itself. As used herein, when the term "about" precedes a numerical value, it typically indicates a range of 10% above or below that value. For example, "about 100" encompasses 90 and 110. In some embodiments, when "about" is followed by an integer not greater than 10, "about" includes a decimal portion, the integer being obtained by rounding the values ​​after the decimal point of these decimals. For example, "about 9" includes the endpoints of the range 8.5 to 9.5 and all values ​​in between.

[0047] antigen molecules and nucleic acid molecules

[0048] The first aspect of this application provides an antigen molecule whose structure, from the N-terminus to the C-terminus, includes an XCL1 region, an antigen region, and a 4-1BBL region. The XCL1 region is an amino acid sequence derived from XCL1, and the 4-1BBL region is an amino acid sequence derived from the extracellular region of 4-1BBL. This application, by comparing different antigen molecule structures, has determined a screened antigen molecule structure design. The combination of the XCL and 4-1BBL regions in the antigen molecule structure can improve the DC cell targeting of tumor vaccines and provide the second signal required for T cell activation, thereby improving the effect of tumor-specific T cell killing and elimination of tumor cells. This results in a higher tumor inhibition rate and can effectively delay the onset and growth rate of tumors.

[0049] In some embodiments, the antigen molecule further comprises a trimerization motif located at the C-terminus of the 4-1BBL region or between the antigen region and the 4-1BBL region. That is, the structure of the antigen molecule from the N-terminus to the C-terminus comprises the following structure: an amino acid sequence derived from XCL1 - antigen region - an amino acid sequence derived from the extracellular region of 4-1BBL - trimerization motif; or, an amino acid sequence derived from XCL1 - antigen region - trimerization motif - an amino acid sequence derived from the extracellular region of 4-1BBL, wherein: "-" indicates direct linkage via peptide bonds or indirect linkage via one or more amino acids.

[0050] In some embodiments, the amino acid sequence derived from XCL1 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36; optionally, the amino acid sequence derived from XCL1 is the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:36.

[0051] In some embodiments of the above-mentioned antigen molecule, the amino acid sequence derived from the extracellular region of 4-1BBL comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37; optionally, the amino acid sequence derived from the extracellular region of 4-1BBL is the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:37.

[0052] In some embodiments of the above-mentioned antigen molecule, the trimerizing motif is a Foldon domain derived from the fibritin protein of T4 phage; further optionally, the trimerizing motif comprises the amino acid sequence shown in SEQ ID NO:6, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:6; even more optionally, the trimerizing motif is the amino acid sequence shown in SEQ ID NO:6. The design of the trimerizing motif enables the antigen protein to form a trimer, enhances the immunogenicity of the antigen protein, and causes the 4-1BBL region to trim. 4-1BBL activates downstream signaling pathways in the form of a trimer, generating co-stimulatory signals that induce the proliferation and activity of CD8+ and CD4+ T cells, thereby enhancing immune function.

[0053] In some embodiments, the antigen region of the antigen molecule described above comprises HPV antigen; optionally, the HPV antigen comprises an amino acid sequence derived from HPV16 E7 protein and an amino acid sequence derived from HPV18 E6E7 protein; further optionally, the amino acid sequence derived from HPV16 E7 protein or HPV18 E6E7 protein is a full-length sequence or a truncated sequence; even further optionally, the amino acid sequence derived from HPV16 E7 protein is a full-length sequence, and the amino acid sequence derived from HPV18 E6E7 protein is a fusion protein comprising a full-length sequence derived from HPV18 E6 protein and a truncated version derived from HPV18 E7 protein; even further optionally, the full-length sequence derived from HPV18 E6 protein is the amino acid sequence shown in positions 1-102 of SEQ ID NO:3, and the truncated version derived from HPV18 E7 protein comprises the amino acid sequences shown in positions 113-123 of SEQ ID NO:3 and the amino acid sequences shown in positions 134-148 of SEQ ID NO:3. This application, by using smaller antigen proteins, facilitates the entry of DNA containing the nucleotide sequence encoding the molecule into the cell nucleus, thereby increasing the level of DNA expression of the target protein within the cell. Furthermore, experimental verification shows that the truncated antigen protein still retains its immunogenicity. In this application, the amino acid sequences of HPV16 E7 protein (HPV16E7) and HPV18 E6E7 protein (HPV18E6E7) are known in the art and can be obtained by those skilled in the art through any known protein database such as Unipro, InterPro, and Genbank. It should be understood that in some embodiments, the amino acid sequences of HPV16E7 and HPV18E6E7 described in this application are not limited to those listed herein.

[0054] In some embodiments, the amino acid sequence of the antigen molecule derived from the HPV18 E6E7 protein is the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:3, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:3.

[0055] In some embodiments of the above-mentioned antigen molecule, the amino acid sequence derived from the HPV16 E7 protein is the full-length sequence of the HPV16 E7 protein; optionally, the full-length sequence of the HPV16 E7 protein is the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO: 2.

[0056] In some embodiments of the antigen molecule described above, the antigen region is arranged in the direction from the N-terminus to the C-terminus as follows: an amino acid sequence derived from the HPV16 E7 protein, an amino acid sequence derived from the HPV18 E6 E7 protein; or an amino acid sequence derived from the HPV18 E6 E7 protein, an amino acid sequence derived from the HPV16 E7 protein; optionally, the amino acid sequence of the antigen region is as shown in SEQ ID NO:47.

[0057] In some embodiments of the antigen molecule described above, the N-terminus of the XCL1 region further comprises a signal peptide; optionally, the signal peptide is an IgE signal peptide; further optionally, the amino acid sequence of the IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO:1, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:1; even more optionally, the amino acid sequence of the IgE signal peptide is the amino acid sequence shown in SEQ ID NO:1.

[0058] In some embodiments, the antigen molecule described above comprises the following structure from the N-terminus to the C-terminus: an IgE signal peptide - an amino acid sequence derived from XCL1 - an amino acid sequence derived from HPV16 E7 protein - an amino acid sequence derived from HPV18 E6E7 protein - an amino acid sequence derived from the extracellular region of 4-1BBL - a trimerization motif; or, an IgE signal peptide - an amino acid sequence derived from XCL1 - an amino acid sequence derived from HPV16 E7 protein - an amino acid sequence derived from HPV18 E6E7 protein - a trimerization motif - an amino acid sequence derived from the extracellular region of 4-1BBL, wherein: "-" indicates direct linkage via peptide bonds or indirect linkage via one or more amino acids.

[0059] In some embodiments of the above-mentioned antigen molecules, there are also independent linking sequences between different amino acid sequences; optionally, the linking sequences are selected from one or more of the following: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46.

[0060] In some embodiments, the antigen molecule comprises, from the N-terminus to the C-terminus, an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40; optionally, the antigen molecule comprises, from the N-terminus to the C-terminus, an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 97%, 96%, 97%, 96%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 38, or SEQ ID NO: 40, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 94%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, The amino acid sequence shown in NO:40.

[0061] A second aspect of this application provides a nucleic acid molecule comprising a polynucleotide sequence encoding the aforementioned antigen molecule.

[0062] In some embodiments, the polynucleotide sequence of the aforementioned nucleic acid molecule is a DNA sequence; optionally, it comprises a polynucleotide sequence as shown in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:39, or SEQ ID NO:41, or a polynucleotide sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the polynucleotide sequence shown in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:39, or SEQ ID NO:41. It can be any molecule composed of ribonucleotides, deoxyribonucleotides, and / or nucleotide analogs, as long as its polynucleotide sequence has the same characteristics as the nucleic acid molecule described in this application. Therefore, the polynucleotide sequence can be a DNA sequence or an RNA sequence. It should be understood that the DNA sequence and DNA molecule described in this application may contain one or more ribonucleotides, as long as most of the nucleotides therein are deoxyribonucleotides and function as DNA molecules or DNA sequences; similarly, the RNA sequence and RNA molecule described in this application may also contain one or more deoxyribonucleotides, as long as most of the nucleotides therein are ribonucleotides and function as RNA molecules or RNA sequences.

[0063] Vectors, DNA vaccines and cells

[0064] A third aspect of this application provides a vector comprising the aforementioned nucleic acid molecules.

[0065] In some embodiments, the vector described above is a plasmid; alternatively, the vector is a pVAX.1 plasmid with the aforementioned nucleic acid molecule inserted into the open reading frame.

[0066] The fourth aspect of this application provides a DNA vaccine comprising the aforementioned nucleic acid molecule or the aforementioned vector. A vector (such as a plasmid) containing a DNA gene sequence encoding a target antigen protein in the DNA vaccine can be obtained by inserting the DNA gene sequence encoding the target antigen protein into a backbone plasmid, for example, inserting a polynucleotide sequence as shown in SEQ ID NO:8 into the pVAX.1 plasmid to obtain a target plasmid. In this application, the mechanism of action of the therapeutic HPV vaccine is as follows: firstly, the DNA vaccine is delivered to antigen-presenting cells, and then the antigen-presenting cells present E6 / E7 antigen epitopes through histocompatibility complexes I and II on their surface, respectively, activating CD8. + Cytotoxic T cells and CD4 + Helper T cells; in CD4 +With the assistance of helper T cells, activated HPV antigen-specific CD8 + T cells recognize and kill cells already infected with HPV. In this application, the amino acid sequence derived from the HPV18 E6E7 protein is a truncated version of the HPV18 E6E7 protein amino acid sequence, resulting in a smaller plasmid and improving nuclear translocation efficiency.

[0067] The fifth aspect of this application provides a cell comprising the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, or the aforementioned DNA vaccine.

[0068] Pharmaceutical Composition

[0069] A sixth aspect of this application provides a pharmaceutical composition comprising the aforementioned antigen molecule, nucleic acid molecule, carrier, DNA vaccine, or cell, and further comprising pharmaceutically acceptable excipients. Optionally, the pharmaceutical composition may further comprise one or more other active compounds as needed for a specific indication of treatment. The active ingredients in the pharmaceutical composition, such as antigen molecules, nucleic acid molecules, or carriers containing antigen or nucleic acid molecules, can be placed in a suitable drug delivery system. When the pharmaceutical composition or pharmaceutical article comprises two or more active ingredients, the active ingredients can be mixed with each other or separated from each other, for example, coexisting in the same delivery medium or existing separately in different delivery mediums. Optionally, the pharmaceutically acceptable excipients may be pharmaceutically acceptable carriers, excipients, or stabilizers, appearing in the form of aqueous solutions, lyophilized or other dried formulations. The pharmaceutically acceptable carriers, excipients, or stabilizers, which are non-toxic to subjects at the doses and concentrations used, include buffers (such as phosphates, citrates, histidine, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives, low molecular weight (less than about 10 amino acid residues) peptides, 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; and nonionic surfactants (such as TWEEN). TM PLURONICS TM Or polyethylene glycol).

[0070] Uses and treatment methods

[0071] The seventh aspect of this application provides the use of the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating HPV16-positive and / or HPV18-positive diseases.

[0072] The eighth aspect of this application provides a method for treating HPV16-positive and / or HPV18-positive diseases, comprising administering an effective amount of the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cells, or the aforementioned pharmaceutical composition to an individual in need of treatment for HPV16-positive and / or HPV18-positive diseases.

[0073] In some embodiments of the above method, the administration is an electrical pulse following an intramuscular injection.

[0074] The ninth aspect of this application provides the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cell, or the aforementioned pharmaceutical composition for treating a disease; optionally, the disease for treating is a disease that is HPV16 positive and / or HPV18 positive.

[0075] Drug-device combination kit

[0076] The tenth aspect of this application provides a drug-device combination kit, comprising the aforementioned antigen molecule, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned DNA vaccine, the aforementioned cells or the aforementioned drug composition, and an electroporator.

[0077] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments.

[0078] Example 1: Preliminary screening for HPV DNA vaccine plasmid construction

[0079] To overcome the weakness of low immunogenicity in DNA vaccines, an attempt was made to enhance their immunogenicity by adding vaccine components, such as the following components: FLT3L, CD40L, IL-12, IL-15, IL-2, IFN-α, XCL1, CCL5, OX40L, 4-1BBL, ICOSL, CD80, and CD40. Based on preliminary experiments, XCL1, FLT3L, and 4-1BBL were selected for vaccine molecule construction, and the effects of different component combinations on vaccine immunogenicity were further compared. In this embodiment, the subsequent HPV DNA vaccine adopts the design approach shown in Table 1:

[0080] Table 1. Construction of HPV DNA vaccine plasmids

[0081]

[0082] In constructing A1, A2, A3, and A4: the amino acid sequence of the IgE signal peptide is shown in SEQ ID NO:1; the amino acid sequence of the XCL1 region is shown in SEQ ID NO:4; the amino acid sequence of the FLT3L region is shown in SEQ ID NO:9; the amino acid sequence of the antigen region is shown in SEQ ID NO:47, which is composed of the full-length sequence of HPV16 E7 protein and the truncated amino acid sequence of HPV18 E6E7 protein through a linker sequence. The full-length sequence of HPV16 E7 protein is shown in SEQ ID NO:2, and the truncated amino acid sequence of HPV18 E6E7 protein is shown in SEQ ID NO:3 (a fusion protein derived from the full-length sequence of HPV18 E6 protein and the truncated form of HPV18 E7 protein; the full-length sequence of HPV18 E6 protein is the amino acid sequence shown in positions 1-102 of SEQ ID NO:3, and the truncated form of HPV18 E7 protein includes the amino acid sequence shown in positions 113-123 of SEQ ID NO:3 and SEQ ID NO:47). The amino acid sequence of NO:3 (positions 134-148) and the amino acid sequence of the 4-1BBL region are shown in SEQ ID NO:5; the amino acid sequence of the trimer motif is shown in SEQ ID NO:6.

[0083] After optimizing the codons corresponding to the fusion proteins in Table 1 according to mammalian cell preferences, the fusion proteins were constructed into the multiple cloning site region of the PVAX1 vector to ensure their expression with the correct codon sequence (the amino acid sequence of the fusion protein and its codon-optimized nucleotide sequence are shown in Table 1). A1, A2, A3, and A4 were single-plasmid constructions, while the positive control A5 was a dual-plasmid construction (the nucleotide sequence encoding the IgE signal peptide-HPV16E6E7 structure and the nucleotide sequence encoding the IgE signal peptide-HPV18E6E7 structure were constructed into the multiple cloning site region of the PVAX1 vector, with a mass ratio of 1:1 for the two plasmids in A5). The PVAX1 vectors obtained after constructing A1, A2, A3, and A4 into the PVAX1 vector were used for tumor suppression experiments. The A5 dual-plasmid system served as the positive control, and the PVAX1 vector without exogenous gene loading served as the negative control. The experimental method is as follows:

[0084] Female C57 mice, 6-8 weeks old and weighing 18±2g, were grouped according to Table 1 (n=8). After extracting the plasmids obtained above, each group of mice was inoculated with 4×10^5 TC-1 tumor cells (mouse lung epithelial cells, Hefei Wanwu Biotechnology Co., Ltd.) expressing the HPV16E6E7 gene on day 0. On days 3, 6, and 10, the mice were administered the drug once via intramuscular injection followed by electroporation. Each group was injected with the DNA plasmids corresponding to Table 1, with each injection dose being 10ug / mouse. The tumor volume was measured on days 3, 6, 10, 13, 15, 17, 20, and 22. On day 22, the mice were sacrificed, and the tumors were harvested and weighed.

[0085] The results of the tumor suppression test are as follows Figure 1 As shown in Table 2. In Table 2, comparing the results of A1 and A2, and A3 and A4, it can be seen that adding an amino acid sequence derived from XCL1 to the construction resulted in a better tumor-suppressing effect than adding an amino acid sequence derived from FLT3L. Comparing constructions A2 and A3, it can be seen that adding a trimerizing motif and an amino acid sequence derived from 4-1BBL to the construction enhanced the tumor-suppressing effect in both tumor volume inhibition rate and tumor weight inhibition rate. Figure 1 It can be seen that before day 7 after the three vaccinations, the tumor volume in all groups showed an increasing trend, and the difference between the groups was small. However, after day 7 of the three vaccinations, the tumor volume in group A5 (positive control group) decreased slowly, while the tumor volume in group A3 decreased rapidly, which was better than that in group A5. By day 22, the tumor volume in group A3 had decreased by 89.7%, indicating that the tumor cells in group A3 were not only inhibited in their growth, but the existing tumor cells were also gradually eliminated.

[0086] Table 2. Results of the tumor suppression test

[0087]

[0088] Note: *P≤0.05, **P≤0.01, ***P≤0.001, compared with PVAX1 unloaded group.

[0089] Tumor weight inhibition rate: TWI% = (1 - tumor weight in the drug-treated group / tumor weight in the empty-load group) × 100%

[0090] Tumor volume inhibition rate: TGI% = [1-(TV Xn -TV X0 ) / (TV Mn -TV M0 )]×100%, of which: TV Xn TV is the average tumor volume on day n in the treatment group. X0 The mean tumor volume on day 0 in the treatment group, TV Mn It is the average tumor volume on day n in the empty group, TV M0 This is the average tumor volume on day 0 of the empty-group tumor. Tumor volume = 0.5 × A × B 2 A represents the long diameter of the tumor, and B represents the short diameter of the tumor.

[0091] Example 2: Effect of DNA vaccine on HPV-specific T-cell immune response.

[0092] Female C57 mice, 6-8 weeks old and weighing 18±2g, were grouped as shown in Table 3. Plasmids were constructed using A3 from Example 1 and administered intramuscularly to the mice. A pulsed current was then used to alter the permeability of mouse muscle cells, allowing the plasmid to enter the cells. Mice in each group were immunized three times on days 0, 7, and 14, with each injection dose being 5ug / mouse. Mice were sacrificed on day 7 after each immunization, and splenic lymphocytes were isolated. A synthetic HPV epitope peptide library (HPV) was used... A truncated peptide library of HPV16E7 or HPV18E6 is used. The HPV16E7 truncated peptide library is designed as follows: starting from the first amino acid at the N-terminus of the full-length HPV16E7 protein, 15 amino acids are taken as peptide 1 for synthesis; then, stepping 7 amino acids from the N-terminus to the C-terminus, 15 amino acids are taken starting from the eighth amino acid at the N-terminus as peptide 2 for synthesis; and so on, until the 15 amino acids at the C-terminus of the full-length HPV16E7 protein are covered. HPV18E6 (e.g., SEQ ID) The truncated peptide library (amino acids 19-167 in NO:34) is synthesized in the same manner. After stimulating spleen lymphocytes with either a negative control medium or a negative control medium, the specific T-cell response against HPV epitope peptides produced by spleen lymphocytes is detected. The specific method is as follows: 1) After mixing cells and stimulants, add them to well plates and incubate the well plates in a 37℃, 5% CO2 incubator for 12-48 hours. Remove the cells from the well plates and wash the cells with PBS; 2) Dilute the detection antibody (commercially available Anti-mouse IFN-γmAb) with PBS containing 0.5% fetal bovine serum. 1) Add 100 μl of the enzyme-labeled reagent streptavidin-alkaline phosphatase to each well with PBS at a concentration of 1 μg / ml. Incubate the plate at room temperature for 2 hours, then wash with PBS. 2) Dilute the enzyme-labeled reagent streptavidin-alkaline phosphatase with PBS containing 0.5% fetal bovine serum at a ratio of 1:1000. Add 100 μl of the enzyme-labeled reagent to each well. Incubate the plate at room temperature for 1 hour, then wash with PBS. 3) Filter the substrate solution (commercially available BCIP / NBT-plus) through a 0.45 μm filter. Add 100 μl of the substrate solution to each well until obvious spots appear. 4) Rinse with tap water to stop the colorimetric reaction. After the plate is dried, check the readings on an ELISA spectrometer or dissecting microscope.

[0093] Table 3. Mouse grouping

[0094]

[0095] Experimental results are as follows Figure 2As shown: Compared with the blank control group, after stimulation with the HPV16E7 or HPV18E6 truncated peptide library, the spleen lymphocytes of mice after the first, second and third vaccinations produced a large number of spots in the experimental group, which showed significant differences compared with the blank control group. This indicates that after DNA vaccine injection, mouse spleen lymphocytes can produce specific T cell responses against HPV16E7 and HPV18E7 epitope peptides.

[0096] Example 3: Further Construction of HPV DNA Vaccine Plasmid

[0097] Based on the selected XCL1, 4-1BBL, and trimerizing motifs, HPV DNA vaccine plasmids were further screened and constructed, using the design approach shown in Table 4 below:

[0098] Table 4. Construction of HPV DNA vaccine plasmids

[0099]

[0100]

[0101] After optimizing the codons corresponding to the fusion proteins in Table 4 according to mammalian cell preferences, they were constructed into the multiple cloning site region of the PVAX1 vector so that the fusion proteins could be expressed in the correct codon sequence (the amino acid sequence of the fusion proteins and their codon-optimized nucleotide sequences are shown in Table 4). The optimized codons of each element are consistent with those in Example 1.

[0102] The expression of each constructed plasmid in in vitro mammalian 293F cells was verified. The experimental methods are as follows: 1) Observe the growth status of 293F cells. When the cell viability is above 90%, the cells are diluted to 1×10⁻⁶. 61) Dispense 1.5 ml of plasmid DNA per well into 6-well plates and place them in a cell culture incubator for later use; 2) Plasmid DNA dilution: Take a sterile 1.5 ml EP tube, add 300 μl of Opti MEM medium and 6 μg of plasmid DNA to each tube, and mix well; 3) Preparation of Lipo3000 reagent: Add 300 μl of Opti MEM, 30 μl of Lipo3000 and 12 μl of P3000 to a sterile 1.5 ml EP tube and mix well; 4) Add the diluted plasmid obtained in step 2 to the Lipo3000 reagent prepared in step 3, and let stand at room temperature for 15 min to allow the plasmid DNA and liposomes to fully form a DNA-liposome complex; 5) Add the mixed DNA-liposome complex to the cells obtained in step 1 and mix gently; 6) Place in a CO2 incubator and shake to culture. Collect the cell supernatant after 48 h, perform Western blotting, and take pictures using a High-sig ECL Western Blot substrate. Experimental results showed that each plasmid could be expressed normally in mammalian cells in vitro.

[0103] Example 4: Growth inhibition of TC-1 tumor cells by DNA vaccine in mice

[0104] Female C57 mice, 6-8 weeks old and weighing 18±2g, were grouped according to Table 4 (n=8). After extracting the plasmids obtained in Example 3, each group of mice was inoculated with 4*10^5 TC-1 tumor cells (mouse lung epithelial cells) expressing the HPV16E6E7 gene on day 0. On day 3 (3 days before inoculation), day 4, and day 11, the mice were immunized by intramuscular injection followed by electroporation. Each group was injected with the DNA plasmids corresponding to Table 4. Each group of mice was injected a total of 3 times. The tumor volume was observed on day 7, day 10, day 12, day 15, and day 18, and the tumor weight was observed on day 18.

[0105] The results are shown in Table 5. Figure 3A and 3B As shown, immunization with HPV plasmid DNA vaccine can effectively delay the onset and growth rate of tumors, and has a certain effect in preventing tumor occurrence and inhibiting tumor progression. Animal experiments with control groups revealed that the vaccines constructed from No. 16 and No. 16-2 had the best efficacy, with no significant difference between the two groups. These two groups showed a significant difference from the other control groups starting around day 10 (3 days after the second immunization), with slower tumor growth.

[0106] Table 5. Growth inhibition of TC-1 tumor cells by DNA vaccines in mice.

[0107]

[0108] *P≤0.05, **P≤0.01, ***P≤0.001, compared with PVAX1 unloaded group

[0109] Sequence List:

[0110] The amino acid sequence of the IgE signal peptide in SEQ ID NO:1;

[0111] MKRELLCVLLLCGLAFPLPDQGIHGRFRR

[0112] SEQ ID NO:2 Full-length amino acid sequence of HPV16 E7 protein;

[0113] HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

[0114] SEQ ID NO:3 Shortened amino acid sequence of HPV18 E6E7 protein;

[0115] ARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCA

[0116] SEQ ID NO:4 is derived from the amino acid sequence of mouse XCL1;

[0117] VGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTG

[0118] SEQ ID NO:5 is an amino acid sequence derived from the extracellular region of mouse 4-1BBL;

[0119] RTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE

[0120] SEQ ID NO:6 is derived from the trimerized motif amino acid sequence of T4 phage;

[0121] GYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCP

[0122] The amino acid sequence constructed from SEQ ID NO:7 A3

[0123] MKRELLCVLLLCGLAFPLPDQGIHGRFRRVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWEGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCP

[0124] The nucleotide sequence encoding A3 construct of SEQ ID NO:8;

[0125]

[0126] SEQ ID NO:9 is the amino acid sequence derived from the extracellular domain of murine FLT3L;

[0127] GTPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATELPEPRPRQ

[0128] The amino acid sequence of construct A1 of SEQ ID NO:10;

[0129] MKRELLCVLLLCGLAFPLPDQGIHGRFRRGTPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATELPEPRPRQGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCA

[0130] The amino acid sequence of construct A2 of SEQ ID NO:11;

[0131] MKRELLCVLLLCGLAFPLPDQGIHGRFRRVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCA

[0132] Amino acid sequence of SEQ ID NO:12 A4 construct;

[0133] MKRELLCVLLLCGLAFPLPDQGIHGRFRRGTPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATELPEPRPRQGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCPRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE

[0134] Amino acid sequence of HPV16 E6E7 in the construction of SEQ ID NO:13 A5;

[0135] MDWTWILFLVAAATRVHSFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

[0136] The nucleotide sequence encoding the A1 construct of SEQ ID NO:14;

[0137]

[0138] SEQ ID NO:15 encodes the nucleotide sequence constructed from A2;

[0139]

[0140] SEQ ID NO:16 encodes the nucleotide sequence constructed from A4;

[0141]

[0142] The nucleotide sequence of HPV 16 E6E7 in the construction of A5 encoded by SEQ ID NO:17;

[0143] ATGGACTGGACTTGGATTCTCTTTCTCGTGGCCGCTGCAACCCGAGTACATTCTTTTCAGGACCCACAGGAGAGTGGCAGAAAACTCCCCCAGCTTTGTACTGAGCTGCAGACAACCATTCATGACATTATCTTGGAGTGCGTTTACTGCAAGCAGCAGCTGCTGCGCAGAGAGGTCTATGACCGGGACCTGTGCATAGTGTACAGAGATGGCAATCCCTACGCCGTATGCGATAAGTGTCTGAAGTTCTACTCCAAAATCTCCGAATATCGCCACTATTGCTATAGCCTGTATGGGACCACCCTCGAGCAGCAATACAATAAGCCCCTGTGTGATCTGCTGATACGCTGCATTAATTGTCAGAAGCCATTGCAGAGGCACCTGGACAAAAAGCAAAGGTTCCACAATATAAGAGGCCGCTGGACCGGAAGATGCATGAGTTGTTGCAGATCTTCCCGGACAAGACGCGAGACCCAACTTAGGGGTCGGAAGAGACGGAGCCATGGAGACACCCCTACGTTGCATGAATACATGCTTGATCTGCAGCCAGAGACAACGGATCTCTATGGTTATGGACAACTGAACGACTCCTCAGAAGAGGAGGATGAGATTGACGGACCAGCTGGGCAAGCAGAGCCTGACCGAGCTCACTACAATATAGTGACGTTCTGCTGCAAGTGCGACAGCACACTTAGACTTTGTGTGCAGAGTACCCATGTCGACATCAGGACCCTGGAAGATCTGTTGATGGGTACTCTGGGAATAGTGTGTCCGATCTGCTCTCAAAAGCCG

[0144] The amino acid sequence of C1 construction of SEQ ID NO:18;

[0145] MKRELLCVLLLCGLAFPLPDQGIHGRFRRVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCP

[0146] Amino acid sequence of C2 construct; SEQ ID NO:19

[0147] MKRELLCVLLLCGLAFPLPDQGIHGRFRRHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWEGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCP

[0148] Amino acid sequence of C3 construct; SEQ ID NO:20

[0149] MKRELLCVLLLCGLAFPLPDQGIHGRFRRHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCP

[0150] Amino acid sequence of C4 construct; SEQ ID NO:21

[0151] MKRELLCVLLLCGLAFPLPDQGIHGRFRRHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCA

[0152] Amino acid sequence of C5 construct; SEQ ID NO:22

[0153] MKRELLCVLLLCGLAFPLPDQGIHGRFRRHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCPRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE

[0154] Amino acid sequence of C6 construct; SEQ ID NO:23

[0155] MKRELLCVLLLCGLAFPLPDQGIHGRFRRVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE

[0156] Amino acid sequence of C7 construct; SEQ ID NO:24

[0157] MKRELLCVLLLCGLAFPLPDQGIHGRFRRHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE

[0158] The amino acid sequence constructed from 16 - 2 of SEQ ID NO:25;

[0159] MKRELLCVLLLCGLAFPLPDQGIHGRFRRVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCPRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE

[0160] The nucleotide sequence encoding the C1 construct of SEQ ID NO:26;

[0161]

[0162] SEQ ID NO:27 encodes the nucleotide sequence constructed from C2;

[0163]

[0164] SEQ ID NO:28 encodes the nucleotide sequence constructed from C3;

[0165] ATGAAAAGAGAGCTCCTTTGTGTGTTGCTTCTTTGTGGGCTGGCCTTTCCACTGCCAGACCAAGGTATTCATGGGCGCTTCAGAAGGCACGGAGATACACCGACGCTCCACGAGTATATGCTGGACTTGCAACCTGAAACTACAGACCTGTACGGATATGGTCAGCTCAATGATTCATCCGAGGAGGAGGATGAGATCGACGGCCCAGCCGGACAGGCTGAGCCAGACCGCGCCCACTATAACATCGTGACCTTTTGCTGCAAGTGCGACAGTACCCTTCGGCTTTGCGTGCAGAGTACTCACGTTGACATCCGAACTTTGGAGGACCTGCTCATGGGGACATTGGGAATTGTGTGTCCCATCTGTTCCCAAAAGCCGGGAGGAGGCGGATCAGGAGGAGGAGGGTCCGGTGGAGGAGGTAGCGCACGCTTTGAAGATCCAACCCGCTCTGGATACAAACTCCCAGACCTGTGCACGGAGCTTAATACCTCCCTGCAGGACATCGAAATCACCTGCGTTTACTGTAAAACCGTCCTGGAGCTGACCGAAGTCTTTGAGAAGGACCTCTTCGTGGTCTACCGGGATTCCATCCCGCACGCCGCTTGTCATAAATGCATCGATTTTTACTCCAGGATTCGCGAGCTTAGGCATTACAGCGATAGCGTGTACGGTGATACACTCGAAAAATTGACCAATACCGGGCTTTACAACCTCCTCATTCGATGCTTGGGCGGTGGTGGTAGTGGAGGTGGCGGCAGTAAGGCTACATTGCAGGATATAGTGCTCCACTTGGGCGGAGGCGGGTCTGGCGGAGGAGGAAGCCAGCTGTTTCTGAATACCCTTAGTTTTGTTTGTCCTTGGTGCGCTGGATATATCCCTGAAGCCCCAAGGGATGGGCAAGCCTATGTCAGGAAAGACGGGGAGTGGGTGTTGCTGAGTACCTTCCTTTGCCCACCCTGTCCT

[0166] The nucleotide sequence encoding the C4 construct of SEQ ID NO:29;

[0167] ATGAAAAGAGAGCTCTTGTGCGTGTTGCTGCTGTGTGGGCTGGCCTTTCCGCTGCCTGACCAAGGTATCCATGGCCGCTTTAGGAGACACGGAGATACACCCACCCTCCACGAGTACATGCTGGATCTCCAGCCAGAGACCACCGATCTGTACGGCTACGGGCAGCTCAACGACAGCTCAGAGGAGGAAGACGAGATAGATGGCCCCGCTGGGCAGGCTGAACCAGATAGGGCCCATTATAATATCGTCACCTTTTGCTGCAAGTGTGACAGTACCCTCAGATTGTGCGTACAGAGCACACACGTTGATATAAGGACTCTCGAAGATTTGTTGATGGGAACCCTGGGGATTGTGTGCCCCATCTGCTCACAAAAACCTGGAGGGGGAGGATCAGGCGGAGGAGGTAGCGGTGGTGGTGGTTCAGCAAGGTTCGAAGACCCAACCCGCAGCGGCTATAAGCTGCCTGACCTTTGCACAGAGCTCAACACCTCTCTGCAGGACATCGAGATAACGTGTGTCTATTGCAAGACAGTGCTGGAGTTGACGGAGGTGTTTGAAAAAGATCTGTTCGTTGTTTACCGCGATAGCATCCCCCATGCAGCCTGTCATAAGTGCATCGACTTCTACTCTCGGATTAGGGAGCTGAGGCACTATAGTGACAGTGTTTATGGTGACACCCTCGAGAAACTGACAAACACTGGCCTGTATAATTTGCTGATAAGATGCCTTGGGGGAGGTGGATCAGGGGGTGGTGGTTCCAAAGCCACCTTGCAAGACATTGTGCTTCACCTGGGAGGGGGAGGCAGTGGAGGTGGAGGTTCTCAGCTGTTCCTCAACACCTTGTCTTTTGTGTGCCCCTGGTGTGCC

[0168] The nucleotide sequence encoding the C5 construct of SEQ ID NO:30;

[0169]

[0170] SEQ ID NO:31 encodes the nucleotide sequence constructed from C6;

[0171]

[0172] SEQ ID NO:32 encodes the nucleotide sequence constructed from C7;

[0173]

[0174] SEQ ID NO:33 encodes the nucleotide sequence constructed from 16-2.

[0175]

[0176] The amino acid sequence of HPV18E6E7 in the construction of SEQ ID NO:34 A5;

[0177] MDWTWILFLVAAATRVHSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLLRHLNEKRRFHNI AGHYRGQCHSCCNRARQERLQRRRETQVRGRKRRSHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLAFQQLFLNTLSFVCPWCASQQ

[0178] SEQ ID NO:35 encodes the nucleotide sequence of HPV18E6E7 in the A5 construction;

[0179] ATGGACTGGACATGGATATTGTTTTTGGTTGCTGCCGCCACCCGCGTCCACTCTGCCAGATTTGAGGATCCAACTCGGTCTGGGTATAAGTTGCCGGACCTTTGTACCGAGCTCAACACGTCTCTGCAAGACATCGAAATCACGTGCGTGTACTGTAAGACCGTCCTGGAACTGACTGAGGTGTTTGAAAAGGACCTGTTCGTGGTGTATCGGGATAGCATCCCACATGCGGCTTGCCACAAATGCATTGACTTCTATTCCCGCATTCGCGAATTGCGCCATTATTCCGACTCTGTGTATGGGGATACCCTGGAAAAGCTGACCAACACAGGCCTCTACAACCTGTTGATCAGGTGCCTGAGGTGCCAGAAGCCATTGCTGCGACATCTCAACGAAAAGCGGAGGTTTCACAACATCGCAGGCCATTACCGAGGTCAGTGTCATTCTTGCTGCAACAGGGCCAGGCAGGAAAGGCTCCAGCGGCGAAGAGAAACACAAGTAAGAGGGCGCAAGCGCAGGTCTCACGGTCCGAAGGCCACACTCCAAGACATCGTCCTTCATCTCGAGCCACAGAACGAGATACCAGTCGATCTGCTGGGCCACGGACAACTGTCCGATAGCGAGGAGGAGAACGATGAAATTGACGGAGTGAACCATCAGCATCTCCCAGCAAGGCGCGCCGAACCCCAGAGGCACACGATGCTCTGTATGTGTTGCAAGTGCGAGGCCCGGATTGAACTGGTCGTGGAATCCAGCGCCGACGATCTGAGGGCTTTTCAACAACTCTTTCTGAATACCCTGAGCTTTGTGTGTCCTTGGTGCGCTAGCCAGCAA

[0180] The amino acid sequence of SEQ ID NO:36 derived from human XCL1:

[0181] VGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNMIQTKPTGTQQSTNTAVTLTG

[0182] SEQ ID NO:37 is an amino acid sequence derived from the extracellular region of human 4-1BBL.

[0183] ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE

[0184] SEQ ID NO:38 Human-derived molecular amino acid sequence:

[0185] MKRELLCVLLLCGLAFPLPDQGIHGRFRRVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTGGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCP

[0186] SEQ ID NO: 39 Human - sourced molecular nucleotide sequence:

[0187]

[0188] Full amino acid sequence of SEQ ID NO:40 human molecule:

[0189] MKRELLCVLLLCGLAFPLPDQGIHGRFRRVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTGGGGGSGGGGSGGGGSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCAEAAAKEAAAKEAAAKGYIPEAPRDGQAYVRKDGEWVLLSTFLCPPCPACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0190] Nucleotide sequence of SEQ ID NO:41 human molecule:

[0191]

[0192] SEQ ID NO:42 Linkage Sequence

[0193] GGGGSGGGGSGGGGS

[0194] SEQ ID NO:43 Linkage Sequence

[0195] EAAAKEAAAKEAAAK

[0196] SEQ ID NO:44 Linkage Sequence

[0197] APAPAPAPAPAPAP

[0198] SEQ ID NO:45 Linkage Sequence

[0199] GGGKRGGGKK

[0200] SEQ ID NO:46 Linkage Sequence

[0201] GSGATNFSLLKQAGDVEENPGP

[0202] The amino acid sequence of the antigen region in SEQ ID NO:47

[0203] HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGGSGGGGSGGGGSARFEDPTRSGYKLPDLCT ELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLGGGGSGGGGSKATLQDIVLHLGGGGSGGGGSQLFLNTLSFVCPWCA

Claims

1. An antigen molecule, the structure of which, in the direction from N-terminus to C-terminus, includes an XCL1 region, an antigen region and a 4-1BBL region, wherein the XCL1 region is an amino acid sequence derived from XCL1 and the 4-1BBL region is an amino acid sequence derived from the extracellular region of 4-1BBL.

2. The antigen molecule according to claim 1, wherein the structure of the antigen molecule further comprises a trimerizing motif, the trimerizing motif being located at the C-terminus of the 4-1BBL region or located between the antigen region and the 4-1BBL region.

3. The antigen molecule according to claim 1, wherein the amino acid sequence derived from XCL1 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:35, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:35, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:35; optionally, the amino acid sequence derived from XCL1 is the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:

35.

4. The antigen molecule according to claim 1, wherein the amino acid sequence derived from the extracellular region of 4-1BBL comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:36, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:36, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:36; optionally, the amino acid sequence derived from the extracellular region of 4-1BBL is the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:

36.

5. The antigen molecule according to claim 1, wherein the trimerizing motif is a Foldon domain derived from the fibritin protein of T4 phage; further optionally, the trimerizing motif comprises an amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:6; even more optionally, the trimerizing motif is an amino acid sequence as shown in SEQ ID NO:

6.

6. The antigen molecule according to claim 1, wherein the antigen region comprises HPV antigen; optionally, the HPV antigen comprises an amino acid sequence derived from HPV16 E7 protein and an amino acid sequence derived from HPV18 E6E7 protein; further optionally, the amino acid sequence derived from HPV16 E7 protein or HPV18 E6E7 protein is a full-length sequence or a truncated sequence; even further optionally, the amino acid sequence derived from HPV16 E7 protein is a full-length sequence, and the amino acid sequence derived from HPV18 E6E7 protein is a fusion protein comprising a full-length sequence derived from HPV18 E6 protein and a truncated version derived from HPV18 E7 protein; even further optionally, the full-length sequence derived from HPV18 E6 protein is the amino acid sequence shown in positions 1-102 of SEQ ID NO:3, and the truncated version derived from HPV18 E7 protein comprises the amino acid sequences shown in positions 113-123 of SEQ ID NO:3 and the amino acid sequences shown in positions 134-148 of SEQ ID NO:

3.

7. The antigen molecule according to claim 6, wherein the amino acid sequence derived from the HPV18 E6E7 protein is the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:3, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:

3.

8. The antigen molecule according to claim 6, wherein the amino acid sequence derived from the HPV16 E7 protein is the full-length sequence of the HPV16 E7 protein; optionally, the full-length sequence of the HPV16 E7 protein is the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:

2.

9. The antigen molecule according to claim 6, wherein the antigen region is arranged in the direction from the N-terminus to the C-terminus as follows: an amino acid sequence derived from HPV16 E7 protein and an amino acid sequence derived from HPV18 E6E7 protein; or an amino acid sequence derived from HPV18 E6E7 protein and an amino acid sequence derived from HPV16 E7 protein; optionally, the amino acid sequence of the antigen region is as shown in SEQ ID NO:

47.

10. The antigen molecule according to claim 1, wherein the antigen molecule further comprises a signal peptide in the N-terminal direction of the XCL1 region; optionally, the signal peptide is an IgE signal peptide; further optionally, the amino acid sequence of the IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO:1, or is an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5, or is a conserved substitution variant of the amino acid sequence shown in SEQ ID NO:1; even more optionally, the amino acid sequence of the IgE signal peptide is the amino acid sequence shown in SEQ ID NO:

1.

11. The antigen molecule according to claim 10, comprising the following structure from the N-terminus to the C-terminus: an IgE signal peptide - an amino acid sequence derived from XCL1 - an amino acid sequence derived from HPV16 E7 protein - an amino acid sequence derived from HPV18 E6E7 protein - an amino acid sequence derived from the extracellular region of 4-1BBL - a trimerization motif; or, an IgE signal peptide - an amino acid sequence derived from XCL1 - an amino acid sequence derived from HPV16 E7 protein - an amino acid sequence derived from HPV18 E6E7 protein - a trimerization motif - an amino acid sequence derived from the extracellular region of 4-1BBL.

12. The antigen molecule according to claim 10, wherein the different amino acid sequences further include mutually independent linking sequences; optionally, the linking sequences are selected from one or more of the following: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:

45.

13. The antigen molecule according to claim 10, comprising, from the N-terminus to the C-terminus, an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 37 or SEQ ID NO: 39, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 37 or SEQ ID NO: 39, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 37 or SEQ ID NO: 39; optionally, the antigen molecule comprising, from the N-terminus to the C-terminus, an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 37 or SEQ ID NO: 39, or an amino acid sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 37 or SEQ ID NO: 39, or a conserved substitution variant of the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO The amino acid sequence shown in NO:37 or SEQ ID NO:

39.

14. A nucleic acid molecule comprising a polynucleotide sequence encoding an antigen molecule as described in any one of claims 1-13.

15. The nucleic acid molecule according to claim 14, wherein the polynucleotide sequence is a DNA sequence or an mRNA sequence; optionally, the polynucleotide sequence is a DNA sequence comprising a polynucleotide sequence as shown in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:38 or SEQ ID NO:40, or a polynucleotide sequence having at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the polynucleotide sequence shown in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:38 or SEQ ID NO:

40.

16. A vector comprising the nucleic acid molecule of claim 14 or 15.

17. The vector according to claim 16, wherein the vector is a plasmid; optionally, the vector is a pVAX.1 plasmid with an open reading frame containing the nucleic acid molecule according to claim 14 or 15.

18. A DNA vaccine comprising the nucleic acid molecule of claim 14 or 15, or the vector of claim 16 or 17.

19. A cell comprising: an antigen molecule as described in any one of claims 1-13, a nucleic acid molecule as described in claim 14 or 15, a vector as described in claim 16 or 17, or a DNA vaccine as described in claim 18.

20. A pharmaceutical composition comprising an antigen molecule as described in any one of claims 1-13, a nucleic acid molecule as described in claim 14 or 15, a carrier as described in claim 16 or 17, a DNA vaccine as described in claim 18, or a cell as described in claim 19, the pharmaceutical composition further comprising pharmaceutically acceptable excipients.

21. Use of an antigen molecule according to any one of claims 1-13, a nucleic acid molecule according to claim 14 or 15, a vector according to claim 16 or 17, a DNA vaccine according to claim 18, a cell according to claim 19, or a pharmaceutical composition according to claim 20, in the preparation of a medicament for treating HPV16-positive and / or HPV18-positive diseases.

22. A method of treating HPV16-positive and / or HPV18-positive diseases, comprising administering to an individual in need of treatment for HPV16-positive and / or HPV18-positive diseases an effective amount of an antigen molecule as described in any one of claims 1-13, a nucleic acid molecule as described in claim 14 or 15, a vector as described in claim 16 or 17, a DNA vaccine as described in claim 18, a cell as described in claim 19, or a pharmaceutical composition as described in claim 20.

23. The method of claim 22, wherein the administration is an intramuscular injection followed by an electrical pulse.

24. A drug-device combination kit comprising an antigen molecule as described in any one of claims 1-13, a nucleic acid molecule as described in claim 14 or 15, a vector as described in claim 16 or 17, a DNA vaccine as described in claim 18, a cell as described in claim 19, or a drug composition as described in claim 20, and an electroporator.

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