Human papilloma virus mRNA vaccine

Abstract

The invention relates to the field of biological medicine, in particular to mRNA designed for human papilloma viruses 16 and 18, a composition, a vaccine preparation and application of the mRNA. The mRNA, the composition and the vaccine preparation can induce potent and long-acting cellular immune response, can induce protective T cell memory, and can resist secondary attack and relapse of viruses; in addition, the growth rate of human papilloma virus positive tumors can be slowed down or regression of the human papilloma virus positive tumors can be mediated. The mRNA, the composition and the vaccine preparation provided by the invention can provide new technical means for treating infection of human papilloma virus type 16, positive cancer or precancerous lesion of the human papilloma virus type 16, infection of human papilloma virus type 18 and positive cancer or precancerous lesion of the human papilloma virus type 18.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an mRNA vaccine for treating human papillomavirus-positive tumors or precancerous lesions. More particularly, the present invention relates to an mRNA vaccine for treating human papillomavirus type 16 and / or human papillomavirus type 18-positive tumors or precancerous lesions. Background Art

[0002] Human papillomavirus (HPV) belongs to the Papillomaviridae family and is a small, non-enveloped, double-stranded circular DNA virus. More than 200 types have been identified so far. Among them, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are considered to be 15 high-risk HPV types. If persistently infected with these high-risk HPV types, they can lead to HPV-related cancers such as cervical cancer, head and neck squamous cell carcinoma (HNSCC), anogenital cancer, and penile cancer.

[0003] Currently, all vaccines approved for marketing against HPV virus are preventive vaccines, such as GlaxoSmithKline's Cervarix, Merck's Gardasil 4 and Gardasil 9, but no HPV therapeutic vaccine has been approved for human use. Although preventive HPV vaccines can prevent most HPV-related cancers, they cannot treat existing HPV infections. In addition, the number of preventive vaccines and insufficient vaccination coverage have greatly affected the prevention of global cancer incidence. Therefore, there is an urgent need for effective new therapies, including therapeutic HPV vaccines, to help treat patients who already have HPV-related malignancies.

[0004] Although there are 15 known high-risk types of HPV, among which HPV 16 is the most common and most carcinogenic high-risk type, about 60% of cervical cancer patients are HPV 16 positive. At present, the types targeted by the HPV therapeutic vaccines that have been developed are mostly HPV 16, and there are also some types 18. The vaccine types include subunit vaccines, nucleic acid vaccines (DNA vaccines and RNA vaccines), live vector vaccines (bacterial vector vaccines and viral vector vaccines) and cell vaccines, etc., but different therapeutic HPV vaccines have their own advantages and disadvantages. No breakthrough has been heard of any vaccine with particularly excellent clinical effects. The field still needs a truly potent, long-lasting, and highly safe therapeutic vaccine for high-risk types such as HPV16. The present application designs a new therapeutic HPV16 mRNA vaccine and evaluates its preclinical anti-tumor efficacy, which will provide new technical means for the treatment of HPV 16-positive cancers and precancerous lesions, and will be expanded to other high-risk types such as HPV 18 on its basis. Summary of the invention

[0005] In a first aspect, the present invention provides a polypeptide or a polypeptide combination, wherein the N-terminus of the N-terminus of the polypeptide in the polypeptide or the polypeptide combination comprises a signal peptide sequence, the C-terminus comprises a transmembrane-intracellular domain sequence of MHC-I, and the polypeptide or the polypeptide combination comprises the amino acid sequence of human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, and / or the amino acid sequence of human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof; or the polypeptide or the polypeptide combination comprises the amino acid sequence of human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, and / or the amino acid sequence of human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof; wherein the human papillomavirus HPV16 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 105 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 105; the human papillomavirus HPV16 E7 protein comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 6; The E6 protein comprises the amino acid sequence shown in SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 131; the human papillomavirus HPV18 E7 protein comprises the amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 79.

[0006] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence set forth in SEQ ID NO: 106, or an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 106; and / or the amino acid sequence set forth in SEQ ID NO: 5 or 78, or an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 5 or 78; or the amino acid sequence set forth in SEQ ID NO: 132, or an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 132; and / or the amino acid sequence set forth in SEQ ID NO: 80, or an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 80.

[0007] In a second aspect, the present invention provides a nucleic acid or a nucleic acid combination, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV16 E6 protein of the present invention or an immunogenic fragment thereof, wherein the polynucleotide is RNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125 and 126, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125 and 126; and / or the nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV16 E7 protein of the present invention or an immunogenic fragment thereof, wherein the polynucleotide is RNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69; or the nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV18 protein of the present invention A polynucleotide encoding an E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151 and 152, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151 and 152; and / or a nucleic acid or a combination of nucleic acids comprises a polynucleotide encoding an E7 protein of the human papillomavirus HPV18 or an immunogenic fragment thereof of the present invention, wherein the polynucleotide is RNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99 and 100, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99 and 100.

[0008] In a third aspect, the present invention provides a nucleic acid or a nucleic acid combination, the nucleic acid or nucleic acid combination comprising a polynucleotide encoding the human papillomavirus HPV16 E6 protein of the present invention or an immunogenic fragment thereof, wherein the polynucleotide is a DNA, the polynucleotide comprising a nucleotide sequence as shown in one of SEQ ID NOs: 128, 129 and 130, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 128, 129 and 130; and / or the nucleic acid or nucleic acid combination comprising a polynucleotide encoding the human papillomavirus HPV16 E7 protein of the present invention or an immunogenic fragment thereof, wherein the polynucleotide is a DNA, the polynucleotide comprising a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76; or the nucleic acid or nucleic acid combination comprising a polynucleotide encoding the human papillomavirus HPV18 A polynucleotide encoding the E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is a DNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156, or a nucleotide sequence having at least 85% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156; and / or a nucleic acid or a combination of nucleic acids comprises a polynucleotide encoding the human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof of the present invention, wherein the polynucleotide is a DNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 102, 103 and 104, or a nucleotide sequence having at least 85% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 102, 103 and 104.

[0009] In a fourth aspect, the present invention provides a nucleic acid or a combination of nucleic acids comprising a polynucleotide encoding the polypeptide or the combination of polypeptides of the present invention.

[0010] In one embodiment, the polynucleotide is RNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 107, 108, 109 and 110, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 107, 108, 109 and 110; and / or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29; or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 133, 134, 135 and 136, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 133, 134, 135 and 136; and / or the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NOs: The nucleotide sequence shown in one of SEQ ID NOs:81, 82, 83 and 84, or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs:81, 82, 83 and 84.

[0011] In one embodiment, the polynucleotide is RNA, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 111, 112, 113 and 114, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 111, 112, 113 and 114; and / or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40; or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 137, 138, 139 and 140, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 137, 138, 139 and 140; and / or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: The nucleotide sequence shown in one of SEQ ID NOs:85, 86, 87 and 88, or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs:85, 86, 87 and 88.

[0012] In one embodiment, the polynucleotide is a DNA, comprising a nucleotide sequence as shown in SEQ ID NO: 115, 116, 117 and 118, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in SEQ ID NO: 115, 116, 117 and 118; and / or a nucleotide sequence as shown in one of SEQ ID NO: 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NO: 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51; or a nucleotide sequence as shown in one of SEQ ID NO: 141, 142, 143 and 144, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NO: 141, 142, 143 and 144; and / or a polynucleotide as shown in SEQ ID NO: The nucleotide sequence shown in one of SEQ ID NOs: 89, 90, 91 and 92, or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs: 89, 90, 91 and 92.

[0013] In one embodiment, the polynucleotide is a DNA, the polynucleotide comprising a nucleotide sequence as shown in one of SEQ ID NOs: 119, 120, 121 and 122, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 119, 120, 121 and 122; and / or the polynucleotide comprising a nucleotide sequence as shown in one of SEQ ID NOs: 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62; or the polynucleotide comprising a nucleotide sequence as shown in one of SEQ ID NOs: 145, 146, 147 and 148, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 145, 146, 147 and 148; and / or the polynucleotide comprising a nucleotide sequence as shown in SEQ ID NOs: The nucleotide sequence shown in one of SEQ ID NOs: 93, 94, 95 and 96, or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs: 93, 94, 95 and 96.

[0014] In a fifth aspect, the present invention provides a composition comprising the polypeptide or polypeptide combination of the present invention.

[0015] In a sixth aspect, the present invention provides a composition comprising a nucleic acid or a combination of nucleic acids of the present invention. In some embodiments, the composition comprises a lipid encapsulating a nucleic acid or a combination of nucleic acids. In some embodiments, the composition comprises lipid nanoparticles or lipid polymer complexes. In some embodiments, the lipid encapsulating the polynucleotide comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol-modified lipid; optionally, the composition further comprises a cationic polymer, wherein the cationic polymer is associated with the nucleic acid or a combination of nucleic acids as a complex, and is co-encapsulated in the lipid to form a lipid polymer complex. In some preferred embodiments, the cationic polymer is protamine. In some embodiments, the cationic lipid comprises a lipid compound of formula (I) or a pharmaceutically acceptable salt thereof, which is as defined herein. In a preferred embodiment, the cationic lipid is SW-II-140-2.

[0016] In a preferred embodiment, the composition comprises 40 mol % SW-II-140-2, 15 mol % DOPE, 43.5 mol % cholesterol and 1.5 mol % DMG-PEG.

[0017] In a seventh aspect, the present invention provides a vaccine preparation comprising the polypeptide or polypeptide combination of the present invention or the composition of the present invention.

[0018] In an eighth aspect, the present invention provides a vaccine preparation comprising the nucleic acid or nucleic acid combination of the present invention or the composition of the present invention.

[0019] In a ninth aspect, the present invention provides an expression vector comprising the nucleic acid or nucleic acid combination of the present invention.

[0020] In a tenth aspect, the present invention provides a host cell comprising the nucleic acid or nucleic acid combination of the present invention or the expression vector of the present invention.

[0021] In an eleventh aspect, the present invention provides a kit comprising a polypeptide or polypeptide combination of the present invention, a nucleic acid or nucleic acid combination of the present invention, a composition of the present invention, or a vaccine preparation of the present invention, and one or more therapeutic agents selected from the following: chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen targeting drugs. In a preferred embodiment, the therapeutic agent is an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0022] In the twelfth aspect, the present invention provides use of a polypeptide or polypeptide combination of the present invention, a nucleic acid or nucleic acid combination of the present invention, a composition of the present invention, a vaccine preparation of the present invention, or a kit of the present invention in the preparation of a medicament for treating human papillomavirus type 16 infection, human papillomavirus type 16-positive cancer or precancerous lesions, human papillomavirus type 18 infection, human papillomavirus type 18-positive cancer or precancerous lesions in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The in vitro expression detection results of prepared mRNA 8#, 10#, 18#, 19#, 22#, 24#, 29#, 31#, 33#, 47#, 50# and 51# are shown.

[0024] Figures 2A-2D It was shown that the SW0128 LPP vaccine formulation induced a cellular immune response. Figure 2A The immunization procedure of C57BL / 6NTac mice is shown; Figure 2B and Figure 2C The figure shows the results of cellular immune response detected by ELISpot after the SW0128LPP vaccine preparations containing 3 μg, 10 μg, and 30 μg mRNA were administered to each group of mice on day 0, and the spleens of the mice were taken on day 6; Figure 2D The graph shows the results of cellular immune response detected by ELISpot after SW0128 LPP vaccine preparations containing 3 μg, 10 μg, and 30 μg mRNA were administered to mice in each group on days 0 and 7, respectively, and spleens of the mice were taken on day 13.

[0025] Figure 3A-3B It was shown that low doses of SW0128 LPP vaccine preparations were able to induce cellular immune responses. Figure 3A The immunization procedure of C57BL / 6NTac mice is shown; Figure 3B The results of cellular immune response induced by SW0128 LPP vaccine preparations under different immunization schedules detected by ELISpot are shown, wherein P is the positive control well and N is the negative control well.

[0026] Figures 4A-4C SW0128 was shown to induce regression of HPV-16 positive TC-1 tumors in C57BL / 6NTac mice. Figure 4A The immunization procedure of C57BL / 6NTac mice is shown; Figure 4B Shows the effect of SW0128 LPP vaccine preparation immunization on tumor volume under different immunization schedules; Figure 4C Shown is the effect of SW0128 LPP vaccine preparation immunization on body weight under different immunization schedules.

[0027] Figure 5 The SW0128 LPP vaccine formulation was shown to induce a reduction in tumor growth rate in C57BL / 6J mice.

[0028] Figures 6A-6C It was shown that the SW0128 LPP vaccine formulation promoted the proliferation of memory T cells. Fig. 6AIt was shown that C57BL / 6NTac mice with complete tumor regression after treatment with the SW0128LPP vaccine formulation could remain tumor-free for a long time after being challenged with tumor cells again; Figure 6B Figure 59 shows the E7 expression of C57BL / 6NTac mice with complete tumor regression after treatment with SW0128 LPP vaccine formulation and C57BL / 6NTac mice without SW0128 LPP vaccine formulation at day 59 (D59) after initial subcutaneous inoculation of TC-1 tumor cells. 49-57 Dextramer + CD8 + Flow cytometry results of T cells; Figure 6C The ELISpot assay results of C57BL / 6NTac mice with complete tumor regression after treatment with the SW0128 LPP vaccine formulation and C57BL / 6NTac mice without treatment with the SW0128 LPP vaccine formulation are shown 93 days (D93) after the initial TC-1 tumor cell inoculation.

[0029] Figures 7A-7D The results show the tumor-suppressive effect of the combined administration of SW0128 LPP vaccine preparation and anti-PD-L1 antibody. Fig. 7A Dosing regimen for C57BL / 6J mice is shown; Figure 7B The tumor volume detection results are shown; Figure 7C Shown is the survival rate of C57BL / 6J mice; Fig.7D Results of analyses of tumor regression are shown.

[0030] Figure 8 The in vitro expression test results of the prepared mRNA-LPP preparations 1801, 1802, 1803 and 1804 are shown.

[0031] Figures 9A-9C LPP vaccine formulations 1801, 1802, 1803, and 1804 were shown to induce cellular immune responses. Fig.9A The immunization schedule for C57BL / 6J mice is shown; Fig. 9B and Fig. 9C The graph shows the results of cellular immune response detected by ELISpot after LPP vaccine preparations 1801, 1802, 1803 and 1804 containing 10 μg of mRNA were administered to mice in each group on day 0 and spleens of the mice were taken on day 7. Specific implementation plan

[0032] General Definitions and Terminology

[0033] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0034] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are terms widely used in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2n d Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Meanwhile, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0035] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising."

[0036] As used herein, the connection term "and / or" between multiple mentioned elements should be understood to include individual and combined options. In other words, "and / or" includes "and" and "or". For example, A and / or B includes A, B and A+B. A, B and / or C include A, B, C and any combination thereof, such as A+B, A+C, B+C and A+B+C. More elements defined by "and / or" are understood in a similar manner and include any one of them and any combination thereof.

[0037] As used herein, the singular forms "a", "an" or "the" include plural references unless the context indicates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0038] Recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each different value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless expressly indicated to the contrary, values ​​or ranges set forth herein are modified by "about", meaning ±20%, ±10%, ±5%, or ±3% of the recited or claimed value or range.

[0039] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0040] As used herein, "nucleic acid combination" may refer to a combination or set comprising more than one nucleic acid. Such nucleic acids may exist separately, for example, in different parts of different compositions or kits or in different vectors, or may exist simultaneously in the same part of the same composition or kit or in the same vector. The nucleic acids in the nucleic acid combination may be the same or different, and may each encode the same polypeptide (part) or may encode different polypeptides (parts).

[0041] As used herein, "polypeptide combination" may refer to a combination or set comprising more than one polypeptide. Such polypeptides may exist separately, for example, in different compositions or different parts of a kit, or may exist simultaneously in the same composition or the same part of a kit. The polypeptides in the polypeptide combination may be the same or different, and may each contain the same amino acid sequence (part) or may contain different amino acid sequences (parts).

[0042] As used herein, the term "polypeptide" refers to a polymer comprising two or more amino acids covalently linked by peptide bonds. A "protein" may comprise one or more polypeptides, wherein the polypeptides interact with each other covalently or non-covalently. Unless otherwise indicated, "polypeptide" and "protein" may be used interchangeably.

[0043] As used herein, the term "wild type" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.

[0044] As used herein, the term "% identity" with respect to a sequence refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window". Optimal alignment can be performed manually, or by means of algorithms known in the art, including but not limited to the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the publicly available BLASTN or BLASTP algorithms available on the website of the National Center for Biotechnology Information (NCBI).

[0045] By determining the number of identical positions corresponding to sequences to be compared, dividing this number by the number of positions compared (for example, the number of positions in the reference sequence), and multiplying this result by 100, obtain % homogeneity. In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the region gives the homogeneity degree. In some embodiments, the homogeneity degree is given to the entire length of the reference sequence. The comparison of determining sequence homogeneity can be performed with tools known in the art, preferably utilizing the best sequence alignment, for example, utilizing Align, utilizing standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend0.5.

[0046] In this article, "nucleotide" includes deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotide" is a constituent substance of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes. "Nucleotide" is usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylic acid containing adenine, "C (c)" refers to deoxycytidine or cytidine containing cytosine, "G (g)" refers to deoxyguanosine or guanylate containing guanine, "U (u)" refers to uridylate containing uracil, and "T (t)" refers to deoxythymidylate containing thymine.

[0047] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.

[0048] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."

[0049] As used herein, "encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA can be used as a template to synthesize polymers and macromolecules in other biological processes, as long as there is a clear nucleotide sequence or a clear amino acid sequence. Therefore, a gene encodes a protein when the gene's mRNA produces a protein in a cell or other biological system through transcription and translation.

[0050] As used herein, the term "untranslated region (UTR)" generally refers to a region (non-coding region) in RNA (such as mRNA) that is not translated into an amino acid sequence, or a corresponding region in DNA.

[0051] As used herein, the term "coding region" refers to a portion of DNA that is capable of transcribing messenger RNA, which is capable of synthesizing the corresponding protein, or a corresponding region in RNA.

[0052] As used herein, the term "host cell" refers to a cell for accepting, maintaining, replicating, expressing a polynucleotide (e.g., a nucleic acid or nucleic acid combination herein) or a vector. In some embodiments, the host cell may be a cell in which a polypeptide or polypeptide combination of the invention is expressed.

[0053] As used herein, "antigen" refers to a molecule that can cause an acquired immune response in the body after entering the body. This immune response may involve the production of antibodies, or specific immunogenic active cells, or both. Those skilled in the art will understand that any macromolecule, including almost all proteins or peptides, can be used as an antigen. Furthermore, antigens can come from recombinant or genomic DNA or RNA. Those skilled in the art will understand that any DNA or RNA herein, their nucleotide sequence or partial nucleotide sequence can encode a protein that can cause acquired immunity in the body. Furthermore, those skilled in the art will understand that an antigen does not need to encode the full-length nucleotide sequence of a gene alone. Obviously, the present invention includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences form different mixtures to induce the occurrence of a response. Furthermore, those skilled in the art will understand that antigens do not need to be completely encoded by a gene. Obviously, antigens can be generated synthetically or derived from biological samples. Biological samples include but are not limited to tissue samples, tumor samples, cells or biological fluids.

[0054] As used herein, "antibody" refers to a protective protein produced by the body due to antigen stimulation. It is an immunoglobulin produced by B lymphocytes. The monomer of an antibody is a Y-shaped molecule consisting of four polypeptide chains. It includes two identical heavy chains and two identical light chains, which are connected by disulfide bonds. Each heavy chain is 50kDa, each light chain is 25kDa, and there is a disulfide bond between the light and heavy chains. Its uniqueness lies in its high affinity and specificity for binding partners.

[0055] As used herein, "vaccine" refers to a composition comprising an active ingredient (e.g., a polypeptide or polynucleotide of the present invention) that can induce an immune response in a vaccinated subject by vaccination. As used herein, "mRNA cancer vaccine" provides a unique therapeutic alternative to peptide-based vaccines or DNA vaccines. When an mRNA cancer vaccine is delivered to a cell, the mRNA will be processed into polypeptides within the cell, which are further processed into immunosensitive fragments that can stimulate an immune response against a tumor. The cancer vaccines described herein include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment that can induce an immune response to cancer).

[0056] As used herein, an "aliphatic" group is a non-aromatic group in which the carbon atoms are linked in a chain, and may be saturated or unsaturated.

[0057] As used herein, the term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms. 1 -C 12 Alkyl" or "C 1-12 "Alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising 1 to 12 carbon atoms. As used herein, the term "alkoxy" refers to an alkyl group as described herein, which is attached to the remainder of the molecule through an oxygen atom. The term "alkylene" refers to a divalent group formed by the corresponding alkyl group losing one hydrogen atom. The term "C 1 -C 12 "Alkylene" or "C 1-12 "Alkylene" refers to an optionally substituted straight or branched chain alkylene group comprising 1 to 12 carbon atoms.

[0058] As used herein, the term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. 2 -C 12 "Alkenyl" or "C 2-12 "Alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising 2 to 12 carbon atoms and at least one carbon-carbon double bond. Alkenyl may comprise one, two, three, four or more carbon-carbon double bonds.

[0059] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0060] As used herein, the term "carbocycle" refers to a monocyclic or polycyclic non-aromatic system comprising one or more rings consisting of carbon atoms. 3-8"Carbocycle" means a carbon ring including 3-8 carbon atoms. The carbocycle may include one or more carbon-carbon double bonds or triple bonds. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and the like. As used herein, when the carbocycle is saturated (i.e., does not contain unsaturated bonds), it may also refer to the corresponding cycloalkyl group. Unless otherwise specifically stated, the carbocycles described herein refer to unsubstituted and substituted, i.e., optionally substituted carbocycles.

[0061] As used herein, the term "heterocycle" refers to a monocyclic or polycyclic system including one or more rings and including at least one heteroatom. The heteroatom can be, for example, a nitrogen, oxygen, phosphorus or sulfur atom. The heterocycle can include one or more double bonds or triple bonds and can be non-aromatic. Examples of heterocycles include, but are not limited to, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl and piperidinyl. The heterocycle can contain, for example, 3-10 atoms (non-hydrogen), i.e., 3-10 yuan heterocycles (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 yuan), wherein one or more atoms are heteroatoms (e.g., N, O, S or P). When the heterocycle is saturated (i.e., without unsaturated bonds), it can also refer to the corresponding heterocycloalkyl. Unless otherwise specifically stated, the heterocycle described herein refers to two types of unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles.

[0062] As used herein, the term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. 6 -C 10 The alkylaryl group may have 6-10 carbon atoms, for example 6, 7, 8, 9, 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

[0063] As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. The heteroaryl group may have 5-10 ring atoms (5-10 membered heteroaryl), including 5, 6, 7, 8, 9 or 10 members, particularly 5 or 6 membered heteroaryl groups. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.

[0064] As used herein, the term "interrupted by one or more groups" means that the one or more groups exist on the carbon chain, and the rest of the carbon chain is connected to both ends of the one or more groups.

[0065] Unless otherwise specifically stated, the groups described herein (e.g., R 1-R 7 Any of, such as alkyl, alkylene, alkenyl, aryl, amino, etc.) may be optionally substituted. The optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chloro, bromo, fluoro, or iodo), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxy, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyls (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, wherein X is a halogen selected from bromine, fluorine, chlorine, and iodine), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals (e.g., -C(OR) 2 R"", wherein each OR is the same or different alkoxy and R"" is alkyl or alkenyl), phosphate (e.g. P(O) 4 3- ), thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O) 2 OH), thioaldehyde (e.g. -C(S)H), sulfate (e.g. S(O) 4 2- ), sulfonyl (e.g. -S(O) 2 -), amides (e.g. -C(O)NR 2 or -N(R)C(O)R), azido (e.g. -N 3 ), nitro (e.g. -NO 2 ), cyano (e.g. -CN), isocyano (e.g. -NC), acyloxy (e.g. -OC(O)R), amino (e.g. -NR 2 , NRH or -NH 2 ), carbamoyl (e.g. -OC(O)NR 2 、-OC(O)NRH or -OC(O)NH 2 ), sulfonamides (e.g. -S(O) 2 NR 2 、-S(O) 2 NRH, -S(O) 2 NH 2 、-N(R)S(O) 2 R, -N(H)S(O) 2 R, -N(R)S(O) 2 H, -N(H)S(O) 2 H) and C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 6 -C 10An aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocycle. In any of the foregoing, each R can independently be a substituent as defined herein, such as an alkyl, alkoxy, alkylene, halogen, carbocycle, heterocycle, aryl, heteroaryl, alkenyl. In some embodiments, the substituent itself can be further substituted by, for example, one, two, three, four, five, or six substituents as defined herein. For example, an alkyl can be further substituted by one, two, three, four, five, or six substituents as described herein.

[0066] As used herein, the term "compound" is intended to include isotopic compounds of the depicted structures. "Isotope" refers to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus, such as deuterium isotopes. For example, isotopes of hydrogen include tritium and deuterium. Additionally, the compounds, salts, or complexes of the present invention can be prepared in combination with a solvent or water molecules to form solvates and hydrates by conventional methods.

[0067] The term "optionally" or "optionally (e.g., optionally substituted)" means that the subsequently described event may or may not occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl can be substituted or unsubstituted, and the description includes substituted alkyl radicals and unsubstituted alkyl radicals.

[0068] It should be understood that when chemical groups are written in a specific order, unless otherwise stated, the reverse order is also encompassed. For example, in M 1 defined by the general formula -C(O)NH- as -(R) i -(M1) k -(R) m -(i.e., -(R) i -C(O)-NH-(R) m -), unless otherwise stated, also encompasses compounds where M 1 is -NHC(O)- (i.e., -(R) i -NHC(O)-(R) m -).

[0069] As used herein, "lipid component" is a component of a composition that includes one or more lipids. For example, a lipid component can include one or more cationic lipids, pegylated lipids, structural lipids, or helper lipids.

[0070] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, salts, materials, compositions, and / or dosage forms that are within the scope of reasonable medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0071] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is altered by converting an existing acid or base moiety into its salt form (e.g., by reacting a free basic group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like; and non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent, or in a mixture of the two; non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred.

[0072] Signal peptide sequence and MHC-I transmembrane-intracellular domain sequence

[0073] As used herein, the term "signal peptide (SP) sequence" refers to the signal peptide fragment of MHC-I, which helps the target antigen to be distributed to the cell vesicle structure. As used herein, the term "MHC class I trafficking domain (MITD) sequence" refers to the amino acid sequence of the transmembrane region and cytoplasmic region of MHC-I. It has been reported that adding the SP sequence to the N-terminus of the antigen and the MITD sequence to the C-terminus helps to improve the presentation of MHC class I and class II epitopes in human and mouse dendritic cells (DCs) and improve the efficiency of antigen presentation (see, for example, Kreiter S, et al., Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals. J Immunol. 2008 Jan 1; 180 (1): 309-18.).

[0074] As used herein, "epitope (also called antigenic determinant)" is a part of an antigen that is recognized by the immune system (particularly by antibodies, B cells or T cells) in the appropriate context. Epitopes include B cell epitopes and T cell epitopes. B cell epitopes are peptide sequences necessary for recognition by B cells that produce specific antibodies. B cell epitopes refer to specific regions of an antigen that are recognized by antibodies. The part of an antibody that binds to the epitope is called a paratope. Based on the structure and the interaction with the paratope, an epitope can be a conformational epitope or a linear epitope. A linear or continuous epitope is defined by the primary amino acid sequence of a specific region of a protein. The sequences that interact with the antibody are located successively in adjacent positions on the protein, and the epitope can generally be simulated by a single peptide. A conformational epitope is an epitope defined by the conformational structure of a natural protein. These epitopes can be continuous or discontinuous, that is, the components of the epitope can be located on different parts of the protein that are close to each other in the folded natural protein structure.

[0075] The term "domain" or "region" refers to a specific portion of an amino acid sequence that can be preferably linked to a specific function or structure. The polypeptide of the MHC-II molecule has two domains, α1, α2 and β1, β2, a transmembrane region and a cytoplasmic region, respectively. The α chain of the MHC-I molecule has three domains, α1, α2 and α3, a transmembrane region and a cytoplasmic region. The term "transmembrane region" refers to the portion of the protein that substantially accounts for the portion present in the cell membrane and is preferably used to anchor the protein in the membrane.

[0076] The term "major histocompatibility complex (MHC)" refers to a gene complex that occurs in all vertebrates. MHC proteins or molecules function in signaling between lymphocytes and antigen presenting cells in normal immune responses. Human MHC, also known as HLA or human leukocyte antigen, is located on chromosome 6 and includes MHC-I and MHC-II.

[0077] The term "MHC-I" or "MHC class I" refers to the major histocompatibility complex class I proteins or genes. Within the human MHC-I region, there are the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, CD1a, CD1b, and CD1c subregions. MHC class I proteins are present on the surface of almost all cells, including most tumor cells. MHC-I proteins are loaded with antigens, which are usually derived from endogenous proteins or pathogens present within the cell, and then presented to cytotoxic T lymphocytes (CTLs). T cell receptors are able to recognize and bind peptides complexed with MHC-I class molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that is able to bind to a specific MHC / peptide complex. MHC class I molecules primarily mediate the presentation of endogenous antigens.

[0078] The alpha chain of MHC-I is a glycoprotein with a molecular weight of about 44 kDa. It can be divided into three functional regions: the external region, the transmembrane region and the cytoplasmic region. The external region is divided into three domains, namely α1, α2 and α3. The transmembrane region spans the lipid bilayer of the plasma membrane. It consists of 23 usually hydrophobic amino acid residues that are arranged in an alpha helix. The cytoplasmic region, that is, the part facing the cytoplasm and connected to the transmembrane region, is usually 32 amino acid residues in length and is able to interact with elements of the cytoskeleton.

[0079] The term "MHC-II" or "MHC class II" refers to major histocompatibility complex class II proteins or genes. MHC class II proteins are mainly expressed on antigen presenting cells such as B cells, monocytes, macrophages, and dendritic cells. MHC class II molecules mainly mediate the presentation of exogenous antigens, and they present exogenous antigen polypeptide molecules to Th cells (helper T cells).

[0080] The exact number of amino acids in different MHC molecule domains or regions depends on differences between mammalian species and gene classes within species. The skilled artisan will appreciate that function may be maintained if less than the complete amino acid sequence of a selected domain or region is used.

[0081] The term "MHC / peptide complex" relates to a non-covalent complex of a binding domain of an MHC class I or MHC class II molecule and an MHC class I or MHC class II binding peptide.

[0082] Human papillomavirus

[0083] As used herein, the term "human papillomavirus (HPV)" belongs to the family Papillomaviridae and is a small, non-enveloped double-stranded circular DNA virus with a diameter of 52-55nm, which usually infects humans. More than 200 types have been identified so far. Among them, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are considered to be high-risk HPVs. This DNA virus is about 8kb and can be mainly divided into three regions from a structural perspective, the early protein coding region (E region), the late protein coding region (L region), and the upstream regulatory region (i.e., non-coding region). The early proteins E1, E2, E4, E5, E6, and E7 encoded in the E region play an important role in the processes of viral replication, transcription, translation, and cell transformation, among which E6 and E7 proteins are the most important oncogenic proteins. Although both high-risk HPV and low-risk HPV contain E6 and E7 proteins, the activity of E6 and E7 proteins in low-risk HPV is not sufficient to induce the development of preneoplastic lesions and cancer, while the activity of E6 and E7 proteins in high-risk HPV is closely related to the occurrence and development of cancer. For example, the E6 protein of HPV16 can inhibit apoptosis by degrading p53, and the E7 protein can inhibit cell cycle progression by degrading pRB (see, for example, Ki, EY, Park, JS Natural History of Human Papillomavirus Infection. Curr Obstet Gynecol Rep 3, 123–127 (2014).).

[0084] HPV type 16 is the most common and carcinogenic mucosal high-risk HPV. About 60% of cervical cancer patients are infected with HPV type 16. At present, most of the HPV therapeutic vaccines studied are designed to target HPV type 16, followed by HPV type 18, which accounts for the second largest proportion of HPV-related tumor patients, and the main targets are its E6 protein or E7 protein. As used herein, the HPV type 16 E6 protein is shown in NCBI accession number AAA46939.1, and the HPV type 16 E7 protein is shown in NCBI accession number AAA46940.1; the HPV type 18 E6 protein is shown in GenBank accession number CAA28664.1, and the HPV type 18 E7 protein is shown in GenBank accession number CAA28665.1.

[0085] Peptide or combination of peptides

[0086] In one general aspect, the invention provides a polypeptide or a combination of polypeptides.

[0087] In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus E6 protein or an immunogenic fragment thereof. In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus E7 protein or an immunogenic fragment thereof. In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus E6 protein or an immunogenic fragment thereof and the amino acid sequence of a human papillomavirus E7 protein or an immunogenic fragment thereof.

[0088] In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof. In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof. In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof and the amino acid sequence of human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof.

[0089] In some embodiments, the polypeptide or polypeptide combination comprises the amino acid sequence of the human papillomavirus HPV18 E6 protein or its immunogenic fragment. In some embodiments, the polypeptide or polypeptide combination comprises the amino acid sequence of the human papillomavirus HPV18 E7 protein or its immunogenic fragment. In some embodiments, the polypeptide or polypeptide combination comprises the amino acid sequence of the human papillomavirus HPV18 E6 protein or its immunogenic fragment and the amino acid sequence of the human papillomavirus HPV18 E7 protein or its immunogenic fragment.

[0090] As used herein, "immunogenicity" mainly refers to the ability to induce an immune response to itself or related proteins (such as therapeutic proteins) or to cause immune-related events, that is, the ability of an antigen to stimulate specific immune cells, activate, proliferate, and differentiate immune cells, and ultimately produce immune effector substances such as antibodies and sensitized lymphocytes. "Immunogenic fragment" refers to any part of a full-length polypeptide or polypeptide antigen that is less than the full length but retains the ability to induce an immune response to the polypeptide or polypeptide antigen.

[0091] In some embodiments, the human papillomavirus HPV16 E6 protein comprises the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the human papillomavirus HPV16 E7 protein comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the human papillomavirus HPV16 E6 protein comprises the amino acid sequence shown in SEQ ID NO:105, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:105, and the human papillomavirus HPV16 E7 protein comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:6.

[0092] In some embodiments, the human papillomavirus HPV18 E6 protein comprises the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence of SEQ ID NO: 131. In some embodiments, the human papillomavirus HPV18 E7 protein comprises the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the human papillomavirus HPV18 E6 protein comprises the amino acid sequence shown in SEQ ID NO:131, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:131, and the human papillomavirus HPV18 E7 protein comprises the amino acid sequence shown in SEQ ID NO:79, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:79.

[0093] In some embodiments, the N-terminus of the polypeptide or polypeptide combination comprises a signal peptide sequence. In some embodiments, the C-terminus of the polypeptide or polypeptide combination comprises a transmembrane-intracellular domain sequence of MHC-I. In some embodiments, the N-terminus of the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises a transmembrane-intracellular domain sequence of MHC-I.

[0094] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and the human papillomavirus HPV16 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 105 or an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 105.

[0095] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and the human papillomavirus HPV18 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 131 or an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 131.

[0096] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof and the amino acid sequence of human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I; and, wherein the human papillomavirus HPV16 E6 protein comprises the amino acid sequence shown in SEQ ID NO:105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:105, and the human papillomavirus HPV16 E7 protein comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:6.

[0097] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV18 E6 protein or its immunogenic fragment and the amino acid sequence of human papillomavirus HPV18 E7 protein or its immunogenic fragment, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I; and, wherein the human papillomavirus HPV18 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131, and the human papillomavirus HPV18 E7 protein comprises the amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 79.

[0098] In a preferred embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and the human papillomavirus HPV16 E7 protein comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO:6.

[0099] In a preferred embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and the human papillomavirus HPV18 E7 protein comprises the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO:79.

[0100] In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO:7 (herein, this signal peptide sequence is also referred to as SP sequence or sec1.0). In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO:77 (herein, this signal peptide sequence is also referred to as sec2.0). In some embodiments, the transmembrane-intracellular segment domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO:7 or 77; and the transmembrane-intracellular segment domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO:8. In a preferred embodiment, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO:7; and the transmembrane-intracellular segment domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO:8.

[0101] In some embodiments, the polypeptide further comprises one or more linkers.

[0102] The linker may comprise an amino acid sequence of any length, in particular an amino acid sequence of 1-50, preferably 1-30, such as 1-10 amino acid residues. Exemplary linkers may include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S) or a combination thereof, such as GGAS, GGGS, GGGSG or (G4S)n, wherein n is an integer of 1-30, preferably 1-10. The linker may also be a hinge region or a functional equivalent thereof. Other suitable linkers may be organic compounds or polymers generally suitable for use in pharmaceutical proteins, including but not limited to polyethylene glycol.

[0103] In a preferred embodiment, the linker is a GS linker. In one embodiment, the linker comprises the amino acid sequence shown in SEQ ID NO: 9 or 10.

[0104] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:106 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:106.

[0105] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:5 or 78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:5 or 78.

[0106] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:106, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:106; and the amino acid sequence shown in SEQ ID NO:5 or 78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:5 or 78.

[0107] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:132 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:132.

[0108] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:80 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:80.

[0109] In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:132, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:132; and the amino acid sequence shown in SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:80.

[0110] In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, the amino acid sequences shown in SEQ ID NOs: 7, 9, 105, 10 and 8. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO: 106.

[0111] In one embodiment, the polypeptide or polypeptides in the combination of polypeptides comprise, from N-terminus to C-terminus, the amino acid sequences shown in SEQ ID NOs: 7, 9, 6, 10 and 8. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:5.

[0112] In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, the amino acid sequences shown in SEQ ID NO: 77, 9, 6, 10 and 8. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO: 78.

[0113] In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, the amino acid sequences shown in SEQ ID NOs: 7, 9, 131, 10 and 8. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO: 132.

[0114] In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, the amino acid sequences shown in SEQ ID NOs: 7, 9, 79, 10 and 8. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO: 80.

[0115] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 105 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 105. The amino acid sequence shown in SEQ ID NO: 105 is the amino acid sequence of the E6 protein of human papillomavirus type 16.

[0116] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 6 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 6. The amino acid sequence shown in SEQ ID NO: 6 is the amino acid sequence of the E7 protein of human papillomavirus type 16.

[0117] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 131 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 131. The amino acid sequence shown in SEQ ID NO: 131 is the amino acid sequence of the E6 protein of human papillomavirus type 18.

[0118] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 79 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 79. The amino acid sequence shown in SEQ ID NO: 79 is the amino acid sequence of the E7 protein of human papillomavirus type 18.

[0119] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 106. The amino acid sequence shown in SEQ ID NO: 106 is the amino acid sequence of the E6 protein of human papillomavirus type 16, which comprises a signal peptide sequence (sec1.0) at the N-terminus and a transmembrane-intracellular domain sequence of MHC-I at the C-terminus.

[0120] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 5 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 5. The amino acid sequence shown in SEQ ID NO: 5 is the amino acid sequence of the E7 protein of human papillomavirus type 16, which comprises a signal peptide sequence (sec1.0) at the N-terminus and a transmembrane-intracellular domain sequence of MHC-I at the C-terminus.

[0121] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 78 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 78. The amino acid sequence shown in SEQ ID NO: 78 is the amino acid sequence of the E7 protein of human papillomavirus type 16, which comprises a signal peptide sequence (sec2.0) at the N-terminus and a transmembrane-intracellular domain sequence of MHC-I at the C-terminus.

[0122] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 132 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 132. The amino acid sequence shown in SEQ ID NO: 132 is the amino acid sequence of the E6 protein of human papillomavirus type 18, which comprises a signal peptide sequence (sec1.0) at the N-terminus and a transmembrane-intracellular domain sequence of MHC-I at the C-terminus.

[0123] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 80 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 80. The amino acid sequence shown in SEQ ID NO: 80 is the amino acid sequence of the E7 protein of human papillomavirus type 18, which comprises a signal peptide sequence (sec1.0) at the N-terminus and a transmembrane-intracellular domain sequence of MHC-I at the C-terminus.

[0124] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO:105 or 106 and the amino acid sequence shown in one of SEQ ID NO:5, 6 and 78.

[0125] In some embodiments, the polypeptide or polypeptide combination encoded by the polynucleotide of the present invention comprises the amino acid sequence shown in SEQ ID NO:131 or 132 and the amino acid sequence shown in SEQ ID NO:79 or 80.

[0126] The polypeptide or polypeptide combination encoded by the polynucleotide of the present invention can be used as a polypeptide antigen to induce a protective immune response against human papillomavirus infection in a subject.

[0127] Nucleic acid or combination of nucleic acids

[0128] On the other hand, the present invention provides nucleic acids or nucleic acid combinations encoding polypeptides or polypeptide combinations described herein. In some embodiments, nucleic acids or nucleic acid combinations include polynucleotides encoding polypeptides or polypeptide combinations described herein. Nucleic acids (or polynucleotides) can be single-stranded or double-stranded. Nucleic acids (or polynucleotides) include but are not limited to DNA, cDNA, RNA (e.g., mRNA), recombinantly produced and chemically synthesized nucleic acids (or polynucleotides). Nucleic acids (or polynucleotides) or nucleic acid combinations of the present invention may be contained in a vector. Nucleic acids (or polynucleotides) or nucleic acid combinations of the present invention may include naturally occurring, synthetic and modified nucleotides. In some embodiments, nucleic acids (or polynucleotides) or nucleic acid combinations of the present invention may be used to express polypeptides or polypeptide combinations described herein in cells to provide polypeptide antigens. In some embodiments, polypeptide antigens can induce an immune response to human papillomavirus type 16 in suitable subjects. In some embodiments, polypeptide antigens can induce an immune response to human papillomavirus type 18 in suitable subjects.

[0129] Nucleic acid (or polynucleotide) or nucleic acid combination may comprise one or more segments (nucleotide fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments). Nucleic acid (or polynucleotide) or nucleic acid combination may comprise a segment encoding a polypeptide of interest (e.g., a polypeptide and polypeptide antigen described herein). In a specific embodiment, nucleic acid (or polynucleotide) or nucleic acid combination may comprise a coding sequence of a polypeptide of interest and a regulatory sequence (including but not limited to transcription and translation regulatory sequences). In some embodiments, the regulatory sequence comprises one or more of the following: a promoter sequence, a 5' untranslated region (5'UTR) sequence, a 3' untranslated region (3'UTR) sequence, and a poly (A) sequence.

[0130] Edit Code sequence

[0131] As used herein, "coding sequence" or "coding region sequence" refers to a nucleotide sequence in a polynucleotide that can be used as a template for synthesizing a nucleotide sequence having a determined nucleotide sequence (e.g., tRNA and mRNA) or a determined amino acid sequence in a biological process. The coding sequence can be a DNA sequence or an RNA sequence. If the mRNA corresponding to the DNA sequence (including the coding strand identical to the mRNA sequence and the template strand complementary thereto) is translated into a polypeptide in a biological process, it can be considered that the DNA sequence or mRNA sequence encodes the polypeptide.

[0132] As used herein, "codon" refers to three consecutive nucleotide sequences (also known as triplet codes) in a polynucleotide, which encode a specific amino acid. Synonymous codons (codons encoding the same amino acid) are used at different frequencies in different species, which is called "codon preference". It is generally believed that for a given species, the coding sequence using its preferred codon can have higher translation efficiency and accuracy in the species expression system. Therefore, polynucleotides can be "codon optimized", that is, the codons in the polynucleotides are changed to reflect the codons preferred by the host cell, and preferably the amino acid sequence encoded by it is not changed. It will be understood by those skilled in the art that due to the degeneracy of codons, the polynucleotides of the present invention may include such coding sequences, which are different from the coding sequences described herein (e.g., having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homogeneity with the coding sequences described herein) but encode the same amino acid sequence.

[0133] In some embodiments, the nucleic acid or nucleic acid combination of the present invention comprises the coding sequence of a polypeptide antigen as described herein. In some embodiments, the nucleic acid or nucleic acid combination of the present invention comprises the coding sequence of a polypeptide as described herein. In some embodiments, the nucleic acid or nucleic acid combination of the present invention comprises a nucleotide sequence complementary to the coding sequence of a polypeptide antigen as described herein. In some embodiments, the nucleic acid or nucleic acid combination of the present invention comprises a nucleotide sequence complementary to the coding sequence of a polypeptide as described herein. In some embodiments, the coding sequence comprises a start codon at its 5' end and a stop codon at its 3' end. In some embodiments, the coding sequence comprises an open reading frame (ORF) as described herein.

[0134] In some embodiments, the coding sequences of the present invention encode any of the polypeptide antigens described above.

[0135] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which comprises: (1) the amino acid sequence set forth in SEQ ID NO:105; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:105; (3) an immunogenic fragment of the amino acid sequence set forth in SEQ ID NO:105; or (4) an immunogenic fragment of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:105.

[0136] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises a SP sequence (SEQ ID NO:7) and a MITD sequence (SEQ ID NO:8), and the polypeptide antigen comprises: (1) an amino acid sequence as set forth in SEQ ID NO:106; (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as set forth in SEQ ID NO:106; (3) an immunogenic fragment of the amino acid sequence as set forth in SEQ ID NO:106; or (4) an immunogenic fragment of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as set forth in SEQ ID NO:106.

[0137] In one embodiment, the coding sequence of the present invention encodes a polypeptide or a combination of polypeptides, which comprises the amino acid sequence of a polypeptide antigen as described above.

[0138] In one embodiment, the coding sequence (coding region) of a polypeptide or polypeptide combination described herein comprises a nucleotide sequence comprising: (1) a nucleotide sequence of one of SEQ ID NOs: 124, 125, 126, 107, 108, 109 and 110; (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence as set forth in one of SEQ ID NOs: 124, 125, 126, 107, 108, 109 and 110; (3) a nucleotide sequence as set forth in one of SEQ ID NOs: 128, 129, 130, 115, 116, 117 and 118; or (4) a nucleotide sequence as set forth in one of SEQ ID NOs: 128, 129, 130, 115, 116, 117 and 118. The nucleotide sequence shown in one of NO:128, 129, 130, 115, 116, 117 and 118 has a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

[0139] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which comprises: (1) the amino acid sequence set forth in SEQ ID NO:6; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:6; (3) an immunogenic fragment of the amino acid sequence set forth in SEQ ID NO:6; or (4) an immunogenic fragment of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:6.

[0140] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises a SP sequence (SEQ ID NO:7) and a MITD sequence (SEQ ID NO:8), and the polypeptide antigen comprises: (1) an amino acid sequence as set forth in SEQ ID NO:5; (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as set forth in SEQ ID NO:5; (3) an immunogenic fragment of the amino acid sequence as set forth in SEQ ID NO:5; or (4) an immunogenic fragment of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as set forth in SEQ ID NO:5.

[0141] In one embodiment, the coding sequence of the present invention encodes a polypeptide or a combination of polypeptides, which comprises the amino acid sequence of a polypeptide antigen as described above.

[0142] In one embodiment, the coding sequence (coding region) of a polypeptide or polypeptide combination described herein comprises a nucleotide sequence, the nucleotide sequence comprising: (1) a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 65, 66, 1, 22, 23, 25 and 26; (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 65, 66, 1, 22, 23, 25 and 26; (3) a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 72, 73, 3, 44, 45, 47 and 48; or (4) a nucleotide sequence as shown in one of SEQ ID NOs: 19, 20, 21, 22, 23, 25 and 26. The nucleotide sequence shown in one of NO:18, 42, 70, 72, 73, 3, 44, 45, 47 and 48 has a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

[0143] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises a sec2.0 sequence (SEQ ID NO:77) and a MITD sequence (SEQ ID NO:8), and the polypeptide antigen comprises: (1) an amino acid sequence as set forth in SEQ ID NO:78; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as set forth in SEQ ID NO:78; (3) an immunogenic fragment of the amino acid sequence as set forth in SEQ ID NO:78; or (4) an immunogenic fragment of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as set forth in SEQ ID NO:78.

[0144] In one embodiment, the coding sequence of the present invention encodes a polypeptide or a combination of polypeptides, which comprises the amino acid sequence of a polypeptide antigen as described above.

[0145] In one embodiment, the coding sequence (coding region) of a polypeptide or polypeptide combination described herein comprises a nucleotide sequence comprising: (1) a nucleotide sequence as set forth in one of SEQ ID NOs: 64, 67, 68, 69, 21, 24, 27, 28, and 29; (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence as set forth in one of SEQ ID NOs: 64, 67, 68, 69, 21, 24, 27, 28, and 29; (3) a nucleotide sequence as set forth in one of SEQ ID NOs: 71, 74, 75, 76, 43, 46, 49, 50, and 51; or (4) a nucleotide sequence as set forth in one of SEQ ID NOs: 71, 74, 75, 76, 43, 46, 49, 50, and 51. The nucleotide sequence shown in one of NO:71, 74, 75, 76, 43, 46, 49, 50 and 51 has a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

[0146] In a preferred embodiment, the coding sequence of the polypeptide or polypeptide combination described herein comprises a nucleotide sequence, the nucleotide sequence comprising: (1) the nucleotide sequence of SEQ ID NO:1; (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence of SEQ ID NO:1; (3) the nucleotide sequence of SEQ ID NO:3; or (4) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence of SEQ ID NO:3.

[0147] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which comprises: (1) the amino acid sequence set forth in SEQ ID NO:131; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:131; (3) an immunogenic fragment of the amino acid sequence set forth in SEQ ID NO:131; or (4) an immunogenic fragment of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:131.

[0148] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises a SP sequence (SEQ ID NO:7) and a MITD sequence (SEQ ID NO:8), and the polypeptide antigen comprises: (1) an amino acid sequence as set forth in SEQ ID NO:132; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as set forth in SEQ ID NO:132; (3) an immunogenic fragment of the amino acid sequence as set forth in SEQ ID NO:132; or (4) an immunogenic fragment of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as set forth in SEQ ID NO:132.

[0149] In one embodiment, the coding sequence of the present invention encodes a polypeptide or a combination of polypeptides, which comprises the amino acid sequence of a polypeptide antigen as described above.

[0150] In one embodiment, the coding sequence (coding region) of a polypeptide or polypeptide combination described herein comprises a nucleotide sequence, the nucleotide sequence comprising: (1) a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151, 152, 133, 134, 135 and 136; (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151, 152, 133, 134, 135 and 136; (3) a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155, 156, 141, 142, 143 and 144; or (4) a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155, 156, 141, 142, 143 and 144. The nucleotide sequence shown in one of NO:154, 155, 156, 141, 142, 143 and 144 has a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.

[0151] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which comprises: (1) the amino acid sequence set forth in SEQ ID NO:79; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:79; (3) an immunogenic fragment of the amino acid sequence set forth in SEQ ID NO:79; or (4) an immunogenic fragment of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:79.

[0152] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises a SP sequence (SEQ ID NO:7) and a MITD sequence (SEQ ID NO:8), and the polypeptide antigen comprises: (1) an amino acid sequence as set forth in SEQ ID NO:80; (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as set forth in SEQ ID NO:80; (3) an immunogenic fragment of the amino acid sequence as set forth in SEQ ID NO:80; or (4) an immunogenic fragment of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as set forth in SEQ ID NO:80.

[0153] In one embodiment, the coding sequence of the present invention encodes a polypeptide or a combination of polypeptides, which comprises the amino acid sequence of a polypeptide antigen as described above.

[0154] In one embodiment, the coding sequence (coding region) of a polypeptide or polypeptide combination described herein comprises a nucleotide sequence comprising: (1) a nucleotide sequence as set forth in one of SEQ ID NOs: 98, 99, 100, 81, 82, 83, and 84; (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence as set forth in one of SEQ ID NOs: 98, 99, 100, 81, 82, 83, and 84; (3) a nucleotide sequence as set forth in one of SEQ ID NOs: 102, 103, 104, 89, 90, 91, and 92; or (4) a nucleotide sequence as set forth in one of SEQ ID NOs: 103, 104, 89, 90, 91, and 92. The nucleotide sequence shown in one of NO:102, 103, 104, 89, 90, 91 and 92 has a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity. In one embodiment, the coding sequence (coding region) of a polypeptide or polypeptide combination described herein comprises a nucleotide sequence comprising: a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125, 126, 107, 108, 109 and 110 and a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68, 69, 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29; or a nucleotide sequence as shown in one of SEQ ID NOs: 128, 129, 130, 115, 116, 117 and 118 and a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75, 76, 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51.

[0155] In one embodiment, the coding sequence (coding region) of a polypeptide or polypeptide combination described herein comprises a nucleotide sequence comprising: a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151, 152, 133, 134, 135 and 136 and a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99, 100, 81, 82, 83 and 84; or a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155, 156, 141, 142, 143 and 144 and a nucleotide sequence as shown in one of SEQ ID NOs: 102, 103, 104, 89, 90, 91 and 92.

[0156] RNA

[0157] In some embodiments, the nucleic acid or nucleic acid combination of the present invention is RNA. In some embodiments, the polynucleotide of the present invention is RNA. As used herein, the definition of "RNA" covers single-stranded, double-stranded, linear and circular RNA. The RNA of the present invention can be chemically synthesized, recombinantly produced and in vitro transcribed RNA. In one embodiment, the RNA of the present invention is used to express the polypeptide or polypeptide combination of the present invention in a host cell. In one embodiment, the RNA of the present invention is single-stranded RNA. In one embodiment, the RNA of the present invention is an in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription using a DNA template by RNA polymerase (e.g., as described herein).

[0158] In some embodiments, the RNA of the present invention is a messenger RNA (mRNA). In general, the mRNA may comprise a 5'-UTR sequence, a coding sequence of a polypeptide (e.g., a polypeptide or polypeptide combination of the present invention), a 3'-UTR sequence, and an optional poly (A) sequence. The mRNA may be produced, for example, by in vitro transcription or chemical synthesis. In one embodiment, the mRNA of the present invention is obtained by in vitro transcription using a DNA template by an RNA polymerase (e.g., T7 RNA polymerase). In one embodiment, the mRNA of the present invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3'-UTR, and (4) an optional poly (A) sequence. The 5'-UTR, coding sequence, 3'-UTR, and poly (A) sequence are as described herein. In one embodiment, the mRNA of the present invention is a nucleoside-modified mRNA. In one embodiment, the mRNA of the present invention comprises an optional 5' cap.

[0159] In some embodiments, the RNA of the present invention comprises the coding sequence of a polypeptide antigen as described herein. In some embodiments, the RNA of the present invention comprises the coding sequence of a polypeptide as described herein. In some embodiments, the RNA of the present invention comprises the coding sequence of a polypeptide combination as described herein.

[0160] In some embodiments, the RNA of the present invention further comprises structural elements that help to improve the stability and / or translation efficiency of the RNA, including but not limited to a 5' cap, a 5'-UTR, a 3'-UTR, and a poly(A) sequence.

[0161] As used herein, the term "untranslated region (UTR)" generally refers to a region (non-coding region) in RNA (such as mRNA) that is not translated into an amino acid sequence, or a corresponding region in DNA. Generally, the UTR located at the 5' end (upstream) of the open reading frame (start codon) can be referred to as the 5' untranslated region 5'-UTR; the UTR located at the 3' end (downstream) of the open reading frame (stop codon) can be referred to as the 3'-UTR. In the presence of a 5' cap, the 5'-UTR is located downstream of the 5' cap, for example, directly adjacent to the 5' cap. In a specific embodiment, an optimized "Kozak sequence" can be included in the 5'-UTR, for example, near the start codon, to improve translation efficiency. In the presence of a poly (A) sequence, the 3'-UTR is located upstream of the poly (A) sequence, for example, directly adjacent to the poly (A) sequence.

[0162] In some embodiments, the RNA comprises a 5'-UTR. In a preferred embodiment, the 5'-UTR comprises the nucleotide sequence shown in SEQ ID NO: 11. In some embodiments, the RNA comprises a 3'-UTR. In a preferred embodiment, the 3'-UTR comprises the nucleotide sequence shown in SEQ ID NO: 12. In some embodiments, the RNA comprises a 5'-UTR and a 3'-UTR. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence shown in SEQ ID NO: 11, and the 3'-UTR comprises the nucleotide sequence shown in SEQ ID NO: 12.

[0163] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to a nucleotide sequence comprising continuous or discontinuous adenylic acid. The poly(A) sequence is typically located at the 3' end of the RNA, such as the 3' end (downstream) of the 3'-UTR. In some embodiments, the poly(A) sequence does not contain nucleotides other than adenylic acid at its 3' end. The poly(A) sequence can be transcribed from the coding sequence of the DNA template by a DNA-dependent RNA polymerase during the preparation of the IVT-RNA, or can be linked to the free 3' end of the IVT-RNA, such as the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).

[0164] In some embodiments, the RNA comprises a poly(A) sequence. In one embodiment, the poly(A) sequence comprises continuous adenylic acid. In one embodiment, the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95 or 100 and up to 120, 150, 180, 200, 300 adenylic acid. In one embodiment, the poly(A) sequence comprises at least 50 nucleotides. In one embodiment, the poly(A) sequence comprises at least 80 nucleotides. In one embodiment, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 70, 80, 90, 100, 120 or 150 nucleotides. In one embodiment, the continuous adenylic acid sequence in the poly(A) sequence is interrupted by a sequence comprising U, C or G nucleotides. In a specific embodiment, the poly(A) sequence comprises the nucleotide sequence shown in SEQ ID NO:15.

[0165] As used herein, the term "5' cap" generally refers to an N7-methylguanosine structure (also known as "m7G cap", "m7Gppp-") attached to the 5' end of an mRNA via a 5' to 5' triphosphate bond. The 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or can be attached to the RNA post-transcriptionally using a capping enzyme.

[0166] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125, and 126. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence as shown in one of SEQ ID NOs: 124, 125, and 126.

[0167] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69.

[0168] In one embodiment, the polynucleotide comprises a nucleotide sequence as set forth in one of SEQ ID NOs: 124, 125 and 126, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as set forth in one of SEQ ID NOs: 124, 125 and 126; and, the polynucleotide comprises a nucleotide sequence as set forth in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as set forth in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69.

[0169] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of a transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:106, or an amino acid sequence that has at least 95% identity to the amino acid sequence shown in SEQ ID NO:106, wherein the polynucleotide is RNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:107, 108, 109, 110, 111, 112, 113 and 114, or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:107, 108, 109, 110, 111, 112, 113 and 114.

[0170] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 107 or 111. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 108 or 112. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 109 or 113. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 110 or 114.

[0171] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises a human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of the transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 95% identity with the amino acid sequence as shown in SEQ ID NO:5, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NO:1, 22, 23, 25, 26, 2, 33, 34, 36 and 37 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in one of SEQ ID NO:1, 22, 23, 25, 26, 2, 33, 34, 36 and 37.

[0172] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or 2. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:22 or 33. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:23 or 34. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:25 or 36. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:26 or 37.

[0173] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises the amino acid sequence of the transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:78 or an amino acid sequence that has at least 95% identity to the amino acid sequence shown in SEQ ID NO:78, wherein the polynucleotide is RNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:21, 24, 27, 28, 29, 32, 35, 38, 39 and 40 or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:21, 24, 27, 28, 29, 32, 35, 38, 39 and 40.

[0174] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:21 or 32. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:24 or 35. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:27 or 38. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:28 or 39. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:29 or 40.

[0175] In a preferred embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or 2.

[0176] In one embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 107, 108, 109, 110, 111, 112, 113 and 114, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 107, 108, 109, 110, 111, 112, 113 and 114; and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 1, 21, 22, 23, 24, 25, 26, 27, 28, 29, 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: The nucleotide sequence shown in one of NO:1, 21, 22, 23, 24, 25, 26, 27, 28, 29, 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.

[0177] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151, and 152. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence as shown in one of SEQ ID NOs: 150, 151, and 152.

[0178] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99, and 100. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence as shown in one of SEQ ID NOs: 98, 99, and 100.

[0179] In one embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151 and 152, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151 and 152; and, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99 and 100, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99 and 100.

[0180] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of a transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:132, or an amino acid sequence that has at least 95% identity to the amino acid sequence shown in SEQ ID NO:132, wherein the polynucleotide is RNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:133, 134, 135, 136, 137, 138, 139 and 140, or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:133, 134, 135, 136, 137, 138, 139 and 140.

[0181] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 133 or 137. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 134 or 138. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 135 or 139. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 136 or 140.

[0182] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises a human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of the transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises an amino acid sequence as shown in SEQ ID NO: 80 or an amino acid sequence having at least 95% identity with the amino acid sequence as shown in SEQ ID NO: 80, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NO: 81, 82, 83, 84, 85, 86, 87 and 88 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in one of SEQ ID NO: 81, 82, 83, 84, 85, 86, 87 and 88.

[0183] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 80, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 81 or 85. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 80, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 83 or 87. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 80, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 84 or 88.

[0184] In a preferred embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:80, and the polynucleotide RNA comprises the nucleotide sequence shown in SEQ ID NO:82 or 86.

[0185] In one embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 133, 134, 135, 136, 137, 138, 139 and 140, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 133, 134, 135, 136, 137, 138, 139 and 140; and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87 and 88, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to ... The nucleotide sequence shown in one of NO:81, 82, 83, 84, 85, 86, 87 and 88 has a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0186] Modified nucleotides

[0187] In some embodiments, the nucleotides in the RNA (e.g., mRNA) of the present invention can be naturally occurring nucleotides (e.g., naturally occurring ribonucleotides) and modified nucleotides. Modified nucleotides can be, for example, nucleotides that are not present in naturally occurring RNA, such as non-standard nucleotides or deoxynucleotides. Modification of nucleotides can occur on nucleosides, such as on ribose moieties and / or nucleobase moieties. Modified nucleotides can be incorporated during transcription (e.g., in vitro transcription) or added during RNA chemical synthesis.

[0188] In one embodiment, the RNA is modified by comprising one or more modified nucleosides. In one embodiment, the RNA is modified by replacing one or more uracils with modified uridines. In one embodiment, the modified uridines include 1-methyl pseudouracil, pseudouracil, 5-methyl-uracil or a combination thereof.

[0189] Examples of modified uridines may include, but are not limited to, 1-methyluridine, 1-methyl-pseudouridine, 3-methyl-uridine, 3-methyl-pseudouridine, 2-methoxy-uridine, 5-methoxy-uridine, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine, 5-Methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyl-uridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl-4-thio-pseudouridine, 5-methyl- 2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)uridine 2'-O-methyl-uridine, 5-(2-methoxycarbonylmethyl)-2-thio-uridine, α-thio-uridine, 2'-O-methyl-uridine, 5,2'-O-dimethyl-uridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyl-uridine, 5-methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carbamoylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethyl-2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2'-O-methyl-uridine, 1-thio-uridine, 5-(2-methoxycarbonylvinyl)uridine and 5-[3-(1-E-propenylamino)uridine.

[0190] In one embodiment, the RNA (e.g., mRNA) of the present invention is modified by comprising one or more modified nucleobases. In one embodiment, the modified nucleobases include modified cytosine, modified uracil, or a combination thereof. In one embodiment, the modified uracil is independently selected from pseudouracil, 1-methyl-pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the modified cytosine is independently selected from 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof. In one embodiment, the ratio of modified nucleobases in the RNA of the present invention is 10%-100%, that is, the RNA of the present invention can be modified by replacing 10%-100% of the nucleobases therein with modified nucleobases.

[0191] In some embodiments, RNA (e.g., mRNA) of the present invention is modified by replacing one or more uracils with modified uracils. In one embodiment, the modified uracils include 1-methyl pseudouracil, pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the modified uracils include pseudouracils. In one embodiment, the modified uracils include 5-methyl-uracils. In one embodiment, the modified uracils include 1-methyl-pseudouracils.

[0192] In one embodiment, RNA is modified by replacing at least one uracil with a modified uracil. In one embodiment, RNA is modified by replacing all uracils with modified uracils. In one embodiment, the ratio of modified uracils in RNA is 10%-100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In one embodiment, the ratio of modified uracils in RNA is 20%-100%. In one embodiment, 20%-100% of uracils in the RNA are replaced by 1-methyl pseudouracil. In a preferred embodiment, 100% of uracils in RNA are replaced by 1-methyl pseudouracil.

[0193] 1-Methyl-pseudouridine has the following structure:

[0194]

[0195] In a specific embodiment, the mRNA of the present invention comprises the nucleotide sequence of any one of SEQ ID NO:2, 32, 33, 34, 35, 36, 37, 38, 39, 40, 85, 86, 87, 88, 111, 112, 113, 114, 137, 138, 139 and 140, and wherein 100% of uracil is replaced by 1-methylpseudouracil.

[0196] DNA

[0197] In some embodiments, nucleic acid or nucleic acid combination of the present invention is DNA. In some embodiments, polynucleotide of the present invention is DNA. Such DNA can be, for example, a DNA template for in vitro transcription of RNA of the present invention or a DNA vaccine for expressing polypeptide antigens in a host cell. DNA can be double-stranded, single-stranded, linear and circular DNA.

[0198] The DNA template can be provided in a suitable transcription vector. In general, the DNA template can be a double-stranded complex comprising a nucleotide sequence identical to the coding sequence described herein (coding strand) and a nucleotide sequence complementary to the coding sequence described herein (template strand). As known to those skilled in the art, the DNA template can include a DNA sequence corresponding to a promoter, a 5'-UTR, a coding sequence, a 3'-UTR, and an optional poly (A) sequence. The promoter can be a promoter available for a suitable RNA polymerase (particularly a DNA-dependent RNA polymerase) known to those skilled in the art, including but not limited to promoters of SP6, T3, and T7 RNA polymerases. In some embodiments, the DNA comprises a T7 promoter. In some embodiments, the T7 promoter comprises the nucleotide sequence of SEQ ID NO:157. In some embodiments, the 5'-UTR, the coding sequence, the 3'-UTR, and the poly (A) sequence in the DNA template are corresponding sequences or complementary to the RNA described herein. The polynucleotides as DNA vaccines can be provided in a plasmid vector (e.g., a circular plasmid vector).

[0199] In some embodiments, the DNA polynucleotides of the present invention comprise the coding sequence of a polypeptide antigen as described herein. In some embodiments, the DNA polynucleotides of the present invention comprise the coding sequence of a polypeptide as described herein. In some embodiments, the DNA polynucleotides of the present invention comprise the coding sequence of a polypeptide combination as described herein. In some embodiments, the DNA polynucleotides of the present invention comprise the DNA sequence corresponding to (1) a T7 promoter, (2) a 5'-UTR, (3) a coding sequence, (4) a 3'-UTR, and (5) an optional poly (A) sequence as described herein.

[0200] In some embodiments, the DNA polynucleotide comprises a DNA sequence corresponding to the 5'-UTR. In a preferred embodiment, the DNA sequence corresponding to the 5'-UTR comprises the nucleotide sequence of SEQ ID NO:13. In some embodiments, the DNA polynucleotide comprises a DNA sequence corresponding to the 3'-UTR. In a preferred embodiment, the DNA sequence corresponding to the 3'-UTR comprises the nucleotide sequence of SEQ ID NO:14. In some embodiments, the DNA polynucleotide comprises DNA sequences corresponding to the 5'-UTR and the 3'-UTR. In a specific embodiment, the DNA sequence corresponding to the 5'-UTR comprises the nucleotide sequence of SEQ ID NO:13, and the DNA sequence corresponding to the 3'-UTR comprises the nucleotide sequence of SEQ ID NO:14.

[0201] In some embodiments, the DNA polynucleotide comprises a DNA sequence corresponding to the poly(A) sequence. In one embodiment, the DNA sequence corresponding to the poly(A) sequence comprises consecutive deoxyadenylic acids. In one embodiment, the DNA sequence corresponding to the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95 or 100 and up to 120, 150, 180, 200, 300 deoxyadenylic acids. In one embodiment, the consecutive deoxyadenylic acid sequence in the DNA sequence corresponding to the poly(A) sequence is interrupted by a sequence comprising T, C or G nucleotides. In one embodiment, the DNA sequence corresponding to the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:16.

[0202] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:128, 129 and 130. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:128, 129 and 130.

[0203] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA, and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76.

[0204] In one embodiment, the polynucleotide comprises a nucleotide sequence as set forth in one of SEQ ID NOs: 128, 129 and 130, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence as set forth in one of SEQ ID NOs: 128, 129 and 130; and, the polynucleotide comprises a nucleotide sequence as set forth in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence as set forth in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76.

[0205] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of a transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:106, or an amino acid sequence that has at least 95% identity to the amino acid sequence shown in SEQ ID NO:106, wherein the polynucleotide is DNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:115, 116, 117, 118, 119, 120, 121 and 122, or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:115, 116, 117, 118, 119, 120, 121 and 122.

[0206] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 115 or 119. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 116 or 120. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 117 or 121. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 106, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 118 or 122.

[0207] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises a human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of the transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 95% identity with the amino acid sequence as shown in SEQ ID NO:5, wherein the polynucleotide is DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NO:3, 44, 45, 47, 48, 4, 55, 56, 58 and 59 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in one of SEQ ID NO:3, 44, 45, 47, 48, 4, 55, 56, 58 and 59.

[0208] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:3 or 4. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:44 or 55. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:45 or 56. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:47 or 58. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:48 or 59.

[0209] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises the amino acid sequence of the transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:78 or an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:78, wherein the polynucleotide is DNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs:43, 46, 49, 50, 51, 54, 57, 60, 61 and 62 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in one of SEQ ID NOs:43, 46, 49, 50, 51, 54, 57, 60, 61 and 62.

[0210] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:43 or 54. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:46 or 57. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:49 or 60. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:50 or 61. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:51 or 62.

[0211] In a preferred embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:3 or 4.

[0212] In one embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 115, 116, 117, 118, 119, 120, 121 and 122, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 115, 116, 117, 118, 119, 120, 121 and 122; and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 3, 43, 44, 45, 46, 47, 48, 49, 50, 51, 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: The nucleotide sequence shown in one of NO:3, 43, 44, 45, 46, 47, 48, 49, 50, 51, 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.

[0213] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155, and 156. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence as shown in one of SEQ ID NOs: 154, 155, and 156.

[0214] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 102, 103, and 104. In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence as shown in one of SEQ ID NOs: 102, 103, and 104.

[0215] In one embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156; and, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 102, 103 and 104, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 102, 103 and 104.

[0216] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of a transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:132 or an amino acid sequence that has at least 95% identity to the amino acid sequence shown in SEQ ID NO:132, wherein the polynucleotide is DNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:141, 142, 143, 144, 145, 146, 147 and 148 or a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:141, 142, 143, 144, 145, 146, 147 and 148.

[0217] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 141 or 145. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 142 or 146. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 143 or 147. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 132, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 144 or 148.

[0218] In one embodiment, the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination of the present invention, the polypeptide or polypeptide combination comprises a human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof, the N-terminus of the polypeptide in the polypeptide or polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises an amino acid sequence of the transmembrane-intracellular domain sequence of MHC-I. In one embodiment, the polypeptide or polypeptide combination comprises an amino acid sequence as shown in SEQ ID NO: 80 or an amino acid sequence having at least 95% identity with the amino acid sequence as shown in SEQ ID NO: 80, wherein the polynucleotide is DNA, and the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO: 89, 90, 91, 92, 93, 94, 95 and 96 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in SEQ ID NO: 89, 90, 91, 92, 93, 94, 95 and 96.

[0219] In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 80, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 89 or 93. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 80, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 91 or 95. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO: 80, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 92 or 96.

[0220] In a preferred embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence shown in SEQ ID NO:80, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:90 or 94.

[0221] In one embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 141, 142, 143, 144, 145, 146, 147 and 148, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 141, 142, 143, 144, 145, 146, 147 and 148; and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95 and 96, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence as shown in one of SEQ ID NOs: The nucleotide sequence shown in one of NO:89, 90, 91, 92, 93, 94, 95 and 96 has a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0222] Composition

[0223] In one aspect, the invention provides a composition comprising a polypeptide or a combination of polypeptides of the invention.

[0224] In yet another aspect, the present invention also provides a composition comprising a nucleic acid or nucleic acid combination of the present invention. In one embodiment, the composition is used to provide therapeutic immunity for human papillomavirus infection or human papillomavirus positive cancer in a subject. In some embodiments, the composition comprises a DNA polynucleotide of the present invention. In some embodiments, the composition comprises an RNA polynucleotide of the present invention. In one embodiment, the RNA is an in vitro transcribed RNA. In one embodiment, the RNA is an mRNA.

[0225] In some embodiments, the composition comprises a nucleic acid or a nucleic acid combination as described herein and a lipid encapsulating the nucleic acid or nucleic acid combination. The composition is a lipid delivery carrier, and the lipid can encapsulate the nucleic acid or nucleic acid combination of the present invention to form nanoparticles, thereby delivering it to an organism.

[0226] As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic portion and optionally also a hydrophilic portion. Lipids are generally insoluble in water but soluble in many organic solvents. Typically, amphipathic lipids comprising a hydrophobic portion and a hydrophilic portion can be organized into a lipid bilayer structure in an aqueous environment, for example, in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, steroids, and cholesterol esters, etc.

[0227] As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle having a uniform lipid core, which is a particle formed by lipids, and the lipid components undergo intermolecular interactions to form a nanostructured entity. Nucleic acids or combinations of nucleic acids (such as mRNA or combinations of mRNA) are encapsulated in the lipids.

[0228] Particularly preferred nucleic acid compositions can be, for example, lipid nanoparticles (LNPs) and lipid polyplexes (LPPs) as described herein. Methods for preparing such compositions can be as described herein. LPPs are particles having a core-shell structure, wherein the nucleic acid is contained in the polyplex, and the polyplex itself is encapsulated in a biocompatible lipid bilayer shell to form the lipid nanoparticles of the present invention. In some embodiments, the compositions of the present invention comprise lipid nanoparticles (LNPs) or lipid polyplexes (LPPs). In some embodiments, the compositions of the present invention are lipid nanoparticles (LNPs) or lipid polyplexes (LPPs) comprising the nucleic acids or combinations of nucleic acids of the present invention.

[0229] In some embodiments, the lipids encapsulating the nucleic acids or combinations of nucleic acids of the present invention are selected from one or more of the following lipids: cationic lipids, phospholipids, steroids, and / or polyethylene glycol-modified lipids. In a preferred embodiment, the cationic lipid is an ionizable cationic lipid.

[0230] The compositions of the present invention comprise the nucleic acids or combinations of nucleic acids of the present invention and lipids encapsulating the nucleic acids or combinations of nucleic acids. The lipids encapsulating the nucleic acids or combinations of nucleic acids comprise cationic lipids, phospholipids, steroids, and polyethylene glycol-modified lipids.

[0231] In some embodiments, the composition comprises a cationic lipid. In some embodiments, the cationic lipid comprises a compound of formula (I) as described herein or a combination thereof. In some preferred embodiments, the cationic lipid comprises SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2, or SW-II-140-2. In a preferred embodiment, the cationic lipid comprises SW-II-140-2.

[0232] In some embodiments, the composition comprises a phospholipid and / or a steroid. In some embodiments, the composition comprises a phospholipid as described herein. In some embodiments, the phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a combination thereof. In some embodiments, the composition comprises a steroid as described herein. In some embodiments, the steroid comprises cholesterol. In some embodiments, the composition comprises a phospholipid and a steroid as described herein. In one embodiment, the composition comprises DOPE. In one embodiment, the composition comprises DSPC. In one embodiment, the composition comprises cholesterol. In one embodiment, the composition comprises DOPE and cholesterol. In one embodiment, the composition comprises DSPC and cholesterol.

[0233] In one embodiment, the composition comprises cationic lipids SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipids DOPE and cholesterol. In one embodiment, the composition comprises cationic lipids SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipids DSPC and cholesterol.

[0234] In some embodiments, the lipid encapsulating the nucleic acid or nucleic acid combination of the present invention further comprises a polyethylene glycol-modified lipid. In one embodiment, the polyethylene glycol-modified lipid comprises DMG-PEG (e.g., DMG-PEG 2000), DOG-PEG and DSPE-PEG or a combination thereof. In one embodiment, the polyethylene glycol-modified lipid is DSPE-PEG. In one embodiment, the polyethylene glycol-modified lipid is DMG-PEG (e.g., DMG-PEG 2000).

[0235] In one embodiment, the composition comprises a cationic lipid, DOPE, cholesterol and DSPE-PEG. In one embodiment, the composition comprises a cationic lipid, DSPC, cholesterol and DSPE-PEG. In one embodiment, the composition comprises a cationic lipid, DSPC, cholesterol and DMG-PEG. In a preferred embodiment, the composition comprises a cationic lipid, DOPE, cholesterol and DMG-PEG.

[0236] In a preferred embodiment, the composition comprises the cationic lipid SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, DOPE, cholesterol and DMG-PEG.

[0237] In some embodiments, the composition of the present invention further comprises a cationic polymer, which is associated with the polynucleic acid or nucleic acid combination as a complex and is co-encapsulated in the lipid. In one embodiment, the cationic polymer comprises poly-L-lysine, protamine, polyethyleneimine (PEI) or a combination thereof. In one embodiment, the cationic polymer is protamine.

[0238] In some embodiments, the amount of lipid in the composition is calculated as molar percentage (mol %), which is determined based on the total mole of lipid in the composition. Unless otherwise specified, the sum of the amount (mol %) of each lipid in the composition is 100 mol %, i.e., the sum of the amount (mol %) of cationic lipids, phospholipids, steroids and polyethylene glycol-modified lipids is 100 mol %.

[0239] In some embodiments, the amount of cationic lipid in the composition is about 10-about 70 mol%. In some embodiments, the amount of cationic lipid in the composition is about 20-about 60 mol%, about 30-about 50 mol%, about 35-about 50 mol%, about 35-about 45 mol%, about 38-about 45 mol%, about 40-about 45 mol%, about 40-about 50 mol% or about 45-about 50 mol%. For example, the amount of cationic lipid can be about 10, 15, 20, 25, 30, 32.5, 35, 37.5, 38, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 65, 70 mol%.

[0240] In some embodiments, the amount of phospholipids in the composition is about 10-about 70 mol%. In one embodiment, the amount of phospholipids in the composition is about 20-about 60 mol%, about 30-about 50 mol%, about 10-about 30 mol%, about 10-about 20 mol%, or about 10-about 15 mol%. For example, the amount of phospholipids can be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 mol%.

[0241] In some embodiments, the amount of cholesterol in the composition is about 10-about 70 mol%. In one embodiment, the amount of cholesterol in the composition is about 20-about 60 mol%, about 24-44 mol%, about 30-about 50 mol%, about 35-about 40 mol%, about 35-about 45 mol%, about 40-about 45 mol% or about 45-about 50 mol%. For example, the amount of cholesterol can be about 10, 15, 17.5, 18.75, 20, 22.5, 24, 25, 27.5, 28.75, 30, 32.5, 33.75, 35, 40, 42.5, 44, 45, 46.25, 47.5, 48.75, 50, 52.5, 53.75, 55, 60, 62.5, 63.75, 65 or 70 mol%.

[0242] In some embodiments, the amount of polyethylene glycol-modified lipids in the composition is about 0.05-about 20 mol%. In one embodiment, the amount of polyethylene glycol-modified lipids in the composition is about 0.5-about 15 mol%, about 1-about 10 mol%, about 5-about 15 mol%, about 1-about 5 mol%, about 1-about 1.5 mol%, about 1.5-about 3 mol%, or about 2-5 mol%. For example, the amount of polyethylene glycol-modified lipids can be about 0.05, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15 or 20 mol%.

[0243] In one embodiment, the composition comprises 10-70 mol % of cationic lipids, 10-70 mol % of phospholipids, 10-70 mol % of steroids and 0.05-20 mol % of polyethylene glycol-modified lipids. In one embodiment, the composition comprises 35-50 mol % of cationic lipids, 10-30 mol % of phospholipids, 24-44 mol % of steroids and 1-1.5 mol % of polyethylene glycol-modified lipids.

[0244] In one embodiment, LPP comprises a nucleic acid or a combination of nucleic acids of the present invention and a lipid encapsulating the nucleic acid or combination of nucleic acids, wherein the lipid encapsulating the nucleic acid or combination of nucleic acids comprises a cationic lipid, a phospholipid, a steroid and a lipid modified with polyethylene glycol; LPP further comprises a cationic polymer, wherein the cationic polymer is associated with the nucleic acid or combination of nucleic acids as a complex. In one embodiment, the composition of the present invention comprises a nucleic acid or a combination of nucleic acids of the present invention and a lipid encapsulating the nucleic acid or combination of nucleic acids, wherein the lipid encapsulating the nucleic acid or combination of nucleic acids comprises a cationic lipid, a phospholipid, a steroid and a lipid modified with polyethylene glycol; the composition further comprises a cationic polymer, wherein the cationic polymer is associated with the nucleic acid or combination of nucleic acids as a complex, and is encapsulated in the lipid to form a lipid polymer complex. In one embodiment, the phospholipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or a combination thereof. In one embodiment, the steroid is cholesterol. In one embodiment, the cationic polymer is protamine. In one embodiment, the polyethylene glycol-modified lipid is selected from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) or a combination thereof. In one embodiment, the cationic lipid is selected from SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In a preferred embodiment, the cationic lipid is SW-II-140-2.

[0245] In one embodiment, the lipid encapsulating the complex comprises 50 mol% of SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10 mol% of DOPE, 38.5 mol% of cholesterol and 1.5 mol% of DMG-PEG. In one embodiment, the lipid encapsulating the complex comprises 40 mol% of SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 15 mol% of DOPE, 43.5 mol% of cholesterol and 1.5 mol% of DMG-PEG. In a preferred embodiment, the lipid encapsulating complex comprises 40 mol% SW-II-140-2, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG.

[0246] In one embodiment, LNP comprises nucleic acid of the present invention or nucleic acid combination and the lipid of encapsulation nucleic acid or nucleic acid combination, wherein the lipid of said encapsulation nucleic acid or nucleic acid combination comprises cationic lipid, phospholipid, steroid and polyethylene glycol-modified lipid.In one embodiment, cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.In one embodiment, phospholipid is DSPC.In one embodiment, polyethylene glycol-modified lipid is DMG-PEG 2000. In one embodiment, the cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, the phospholipid is DSPC, and the polyethylene glycol-modified lipid is DMG-PEG 2000.

[0247] In one embodiment, the lipid encapsulating the nucleic acid or nucleic acid combination comprises 50 mol% SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG 2000.

[0248] In one aspect, the present invention provides a vaccine formulation comprising a polypeptide or a combination of polypeptides described herein or a composition comprising the polypeptide or a combination of polypeptides.

[0249] In yet another aspect, the present invention also provides a vaccine preparation comprising the nucleic acid or nucleic acid combination described herein or a composition comprising the nucleic acid or nucleic acid combination.

[0250] In some embodiments, vaccine formulations of the invention (also referred to as "vaccine agents") comprise a nucleic acid or combination of nucleic acids described herein.

[0251] In some embodiments, the vaccine formulation of the present invention (also referred to as "vaccine agent") comprises a composition described herein, wherein the lipid comprises 10-70 mol% SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10-70 mol% DOPE, 10-70 mol% cholesterol and 0.05-20 mol% DMG-PEG 2000; wherein the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a polypeptide or polypeptide combination described herein.

[0252] In some embodiments, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125, 126, 128, 129, 130, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122. In some embodiments, the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68, 69, 18, 42, 70, 71, 72, 73, 74, 75, 76, 1, 21, 22, 23, 24, 25, 26, 27, 28, 29, 2, 32, 33, 34, 35, 36, 37, 38, 39, 40, 3, 43, 44, 45, 46, 47, 48, 49, 50, 51, 4, 54, 55, 56, 57, 58, 59, 60, 61, and 62. In some embodiments, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151, 152, 154, 155, 156, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148. In some embodiments, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99, 100, 102, 103, 104, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, and 96. In a preferred embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 1, 2, 3, and 4.

[0253] In one embodiment, the vaccine formulation comprises a nucleic acid or a combination of nucleic acids as described herein, the nucleic acid or a combination of nucleic acids comprises a polynucleotide encoding a polypeptide or a combination of polypeptides as described herein, and the vaccine formulation comprises a lipid encapsulating the nucleic acid or a combination of nucleic acids, the lipid comprising 10 to 70 mol% of SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10 to 70 mol% of DOPE, 10 to 70 mol% of cholesterol and 0.05 to 20 mol% of DMG-PEG 2000. In a preferred embodiment, the lipid comprises 40 mol% of SW-II-140-2, 15 mol% of DOPE, 43.5 mol% of cholesterol and 1.5 mol% of DMG-PEG2000. In some embodiments, the polynucleotide comprises a nucleotide sequence as set forth in one of SEQ ID NOs: 124, 125, 126, 128, 129, 130, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68, 69, 18, 42, 70, 71, 72, 73, 74, 75, 76, 1, 21, 22, 23, 24, 25, 26, 27, 28, 29, 2, 32, 33, 34, 35, 36, 37, 38, 39, 40, 3, 43, 44, 45, 46, 47, 48, 49, 50, 51, 4, 54, 55, 56, 57, 58, 59, 60, 61, and 62. In some embodiments, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151, 152, 154, 155, 156, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148. In some embodiments, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 98, 99, 100, 102, 103, 104, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, and 96. In a preferred embodiment, the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 1, 2, 3, and 4. Optionally, the vaccine formulation further comprises a cationic polymer, wherein the cationic polymer is associated with the nucleic acid or a combination of nucleic acids as a complex and are co-encapsulated in lipids to form a lipid polymer complex.

[0254] Cationic lipids

[0255] A cationic lipid is a lipid that can carry a net positive charge at a specified pH. The lipid with a net positive charge can associate with nucleic acids through electrostatic interactions.

[0256] Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Didecyldimethylammonium bromide (DDAB), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), dioctadecyldimethyl ammonium chloride (DDAB), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium chloride (DDAB), dioctadecyldimethyl ammonium bromide ... bromide (DDAB), dioctadecyldimethyl ammonium bromide (DDAB), chloride, DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3-(N(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylammonium (2,3-di(tetradecoxy)propane yl-(2-hydroxyethyl)-dimethylazanium, DMRIE), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),N-dimethylaminopropane, DLenDMA), 3-dimethylamino-2-(cholest-5-eh-3-beta-oxybutan-4-oxy)-1-(cis, cis-9,12-octadecadienoxy)propane, CLinDMA), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide bromide, DMORIE), N, N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N, N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), dioctadecylamidolycyl spermine ( spermine, DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), a compound of formula (I) as described herein, or a combination thereof.

[0257] In some embodiments, the cationic lipid is preferably an ionizable cationic lipid. An ionizable cationic lipid carries a net positive charge at, for example, acidic pH, but is neutral at higher pH (eg, physiological pH). Examples of ionizable cationic lipids include, but are not limited to, dioctadecylamidoglycylspermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), a compound of formula (I) as described herein, or a combination thereof.

[0258] In one embodiment, the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0259]

[0260] in,

[0261] R 1 and R 2 Each independently selected from a bond, C 1 -C 12 Alkyl and C 2 -C 12 alkenyl;

[0262] R 3 and R 4 Each independently selected from C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 6 -C 10 aryl and 5-10 membered heteroaryl; and R 3 and R 4 Each independently selected by t R 6 Substitution, t is an integer selected from 1-5;

[0263] R 6 Each independently selected from C 1 -C 12 Alkyl and C 2 -C12 alkenyl;

[0264] M 1 and M 2 Each is independently selected from a bond, H, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, -C(S)S-, a 3-10 membered heterocycle, -NR 7 -,or

[0265] R 5 With M 1 and M 2 One of them together with the nitrogen atom to which it is connected forms a 3-10 membered heterocyclic ring, and the corresponding R 1 / R 3 or R 2 / R 4 No

[0266] exists, the heterocyclic ring is optionally replaced by R 7 replace;

[0267] R 5 Selected from C 3-8 Carbon ring, -C 1-12 Alkylene-Q, Q is selected from H, -OR 7 、-SR 7 、-OC(O)R 7 、-C(O)OR 7 、-N(R 7 )C(O)R 7 、-N(R 7 )S(O) 2 R 7 、-N(R 7 )C(S)R 7 、-N(R 7 ) 2 , cyano, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C 6 -C 10 Aryl, each of the above groups is optionally substituted with one or more C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 1 -C 12 Alkoxy, C 6 -C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O) substitution;

[0268] m and n are each independently an integer selected from 0-12;

[0269] The alkyl, alkenyl and alkylene groups are each optionally independently interrupted by one or more groups selected from: -O-, -S-, -NR 7 -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocyclic ring, and the alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R 7 replace;

[0270] R 7 Each independently selected from H, C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 1 -C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C 6 -C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclic ring, halogen, C 3-8 Carbocyclic ring, each of the above groups is optionally substituted by one or more C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 1 -C 12 Alkoxy, C 6 -C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O) substitution.

[0271] In one embodiment, the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0272]

[0273] R 1 and R 2 Each independently selected from C 1 -C 12 Alkyl and C 2 -C 12 alkenyl;

[0274] R 3 and R 4 Each independently selected from C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 6 -C 10 Aryl and 5-10 membered heteroaryl;

[0275] The condition is R3 and R 4 At least one of them is C 6 -C 10 aryl or 5-10 membered heteroaryl, and R 3 and R 4 Each independently selected by t R 6 substituted, t is an integer selected from 1-5; R 6 Each independently selected from C 1 -C 12 Alkyl and C 2 -C 12 alkenyl;

[0276] M 1 and M 2 each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, and -C(S)S-;

[0277] R 5 Selected from -C 1-12 Alkylene-Q, Q is selected from -OR 7 and-SR 7 , R 7 Independently selected from H, C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 1 -C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C 6 -C 10 Aryl and 5-10 membered heteroaryl;

[0278] m and n are each independently an integer selected from 1-12.

[0279] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:

[0280]

[0281]

[0282] In some embodiments, the cationic lipid comprises the following lipid compounds: SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In a preferred embodiment, the cationic lipid comprises SW-II-140-2.

[0283] Phospholipids

[0284] The composition of the present invention includes phospholipids, which can assist in cell penetration of the composition.

[0285] Examples of phospholipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC ... Undecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oil Acylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof.

[0286] Steroids

[0287] The compositions of the present invention contain steroids which may serve as a structural component of the composition.

[0288] Examples of steroids include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and derivatives thereof.

[0289] PEGylated lipids

[0290] As used herein, the term "polyethylene glycol-modified lipid" or "PEG-modified lipid" or "PEG lipid" refers to a molecule comprising a polyethylene glycol portion and a lipid portion, which is a lipid modified with polyethylene glycol. The PEG lipid can be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG-modified dialkylglycerol, or a combination thereof. For example, examples of polyethylene glycol-modified lipids include, but are not limited to, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-Dioleoyl-rac-glycerol, methoxypolyethylene Glycol (DOGPEG), and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG).

[0291] In one embodiment, the polyethylene glycol-modified lipid is DMG-PEG, such as DMG-PEG 2000. In one embodiment, DMG-PEG 2000 has the following structure:

[0292]

[0293] The average value of n is 44.

[0294] Cationic polymers

[0295] As used herein, the term "cationic polymer" refers to any ionic polymer that can carry a net positive charge at a specified pH, thereby electrostatically binding to nucleic acids. Examples of cationic polymers include, but are not limited to, poly-L-lysine, protamine, polyethyleneimine (PEI), or a combination thereof. The polyethyleneimine can be linear or branched polyethyleneimine.

[0296] The term "protamine" refers to a low molecular weight basic protein rich in arginine, which exists in sperm cells of various animals (especially fish) and binds to DNA instead of histones. In a preferred embodiment, the cationic polymer is protamine (eg, protamine sulfate).

[0297] Vectors and host cells

[0298] In another aspect, the present invention also provides an expression vector comprising a nucleic acid or a combination of nucleic acids of the present invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The nucleic acid or combination of nucleic acids of the present invention may be present in one or more expression vectors.

[0299] The present invention also provides a host cell comprising the nucleic acid or nucleic acid combination or expression vector of the present invention. The nucleic acid or nucleic acid combination or expression vector of the present invention can be introduced into a suitable host cell by various methods known in the art. Such methods include but are not limited to liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0300] In a preferred embodiment, host cells are used to express the human papillomavirus E6 protein and / or E7 protein of the present invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for expression include, but are not limited to, human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, such as HEK293 cells), Chinese hamster ovary (CHO) cells, DC2.4 cells (mouse bone marrow derived dendritic cells), CaSki cells (human cervical cancer metastatic cells) and other mammalian cells suitable for expression.

[0301] Therapeutic applications

[0302] The present invention provides a polypeptide or polypeptide combination, a nucleic acid or a nucleic acid combination (especially RNA), a composition or a vaccine preparation of the present invention, which is used to treat human papillomavirus type 16 infection, human papillomavirus type 16-positive cancer or precancerous lesions, human papillomavirus type 18 infection, human papillomavirus type 18-positive cancer or precancerous lesions in a subject.

[0303] The present invention provides use of the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (especially RNA), composition or vaccine preparation of the present invention in the preparation of a medicament for treating human papillomavirus type 16 infection, human papillomavirus type 16-positive cancer or precancerous lesion, human papillomavirus type 18 infection, human papillomavirus type 18-positive cancer or precancerous lesion in a subject.

[0304] The present invention provides a method for treating human papillomavirus type 16 infection, human papillomavirus type 16 positive cancer or precancerous lesions, human papillomavirus type 18 infection, human papillomavirus type 18 positive cancer or precancerous lesions in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide or polypeptide combination, a nucleic acid or a nucleic acid combination (particularly RNA), a composition or a vaccine formulation of the present invention. In one embodiment, the method comprises administering a therapeutically effective amount of a composition comprising a polypeptide or polypeptide combination, or a nucleic acid or a nucleic acid combination (particularly RNA) of the present invention, in particular a composition comprising an LNP or LPP as described herein.

[0305] In some embodiments, HPV-16 positive cancers include, but are not limited to, cervical cancer, anogenital cancer, penile cancer, and head and neck cancers, such as genital cancer, head and neck squamous cell carcinoma.

[0306] In some embodiments, HPV-18 positive cancers include, but are not limited to, cervical cancer, anogenital cancer, penile cancer, and head and neck cancers, such as genital cancer, head and neck squamous cell carcinoma.

[0307] The term "therapeutically effective amount" refers to an amount sufficient to inhibit the occurrence of a disease or symptom and / or slow down, alleviate, or delay the development or severity of a disease or symptom. The therapeutically effective amount is affected by factors including, but not limited to, the rate of development and severity of the disease or symptom, the age, sex, weight, and physiological condition of the subject, the duration of treatment, and the specific route of administration. The therapeutically effective amount can be administered in one or more doses. The therapeutically effective amount can be achieved by continuous or intermittent administration.

[0308] In some embodiments, the therapeutically effective amount is provided in one or more administrations. In some embodiments, the therapeutically effective amount is provided in two administrations. In some embodiments, the therapeutically effective amount is provided in three administrations. In some embodiments, the therapeutically effective amount is provided in four administrations. In some embodiments, the therapeutically effective amount is provided in five administrations. In some embodiments, the therapeutically effective amount is provided in six administrations. In some embodiments, the therapeutically effective amount is provided in seven administrations. In some embodiments, the therapeutically effective amount is provided in eight administrations. In some embodiments, the therapeutically effective amount is provided in greater than eight administrations.

[0309] In some embodiments, the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition or vaccine formulation of the present invention can be administered to a subject by any method known to those skilled in the art, such as parenteral, oral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous or intraperitoneal. Preferably, the composition or vaccine formulation of the present invention is administered by subcutaneous injection.

[0310] As used herein, the term "subject" describes an organism, such as a mammal, to which a polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition or vaccine formulation of the present invention can be provided for therapeutic immunity. In some embodiments, the subject is a mouse. In a preferred embodiment, the subject is a human.

[0311] In some embodiments, the uses and methods of the present invention further comprise administering an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0312] Combination therapy

[0313] For the treatment of human papillomavirus type 16-positive cancer or human papillomavirus type 18-positive cancer, the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (especially RNA), composition or vaccine preparation of the present invention can be used in combination with other treatment methods, including but not limited to: surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, angiogenesis inhibition and palliative treatment.

[0314] The polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition or vaccine formulation of the present invention can also be co-administered with at least one or more therapeutic agents described herein. The mode of co-administration is not limited. For example, the following therapeutic agents can be all administered at one time or separately. When administered separately (using different administration schemes), they can be administered continuously without interruption or at predetermined intervals.

[0315] In certain embodiments, the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition or vaccine preparation of the present invention is further co-administered with one or more therapeutic agents selected from the following: chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors and tumor antigen targeting drugs. In a preferred embodiment, the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition or vaccine preparation of the present invention is co-administered with immune checkpoint inhibitors.

[0316] In one embodiment, the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (especially RNA), composition or vaccine preparation of the present invention is administered in combination with an anti-PD-L1 antibody or an antigen-binding fragment thereof, thereby mediating a more excellent tumor inhibition effect.

[0317] Reagent test kit

[0318] The present invention also provides a kit comprising a polypeptide or polypeptide combination of the present invention, a nucleic acid or a nucleic acid combination (particularly RNA), a composition or a vaccine formulation, and instructions for use. The kit may also include a suitable container. In certain embodiments, the kit also includes a device for administration. The kit generally includes a label indicating the intended use and / or method of use of the contents of the kit. The term "label" includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0319] In one embodiment, the kit comprises a polypeptide or a combination of polypeptides, a nucleic acid or a combination of nucleic acids (particularly RNA), a composition or a vaccine preparation, and one or more therapeutic agents selected from the following: chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen targeting drugs.

[0320] In one embodiment, the kit comprises a polypeptide or a combination of polypeptides, a nucleic acid or a combination of nucleic acids (particularly RNA), a composition or a vaccine preparation, and an immune checkpoint inhibitor.

[0321] In one embodiment, the kit comprises a polypeptide or a combination of polypeptides, a nucleic acid or a combination of nucleic acids (particularly RNA), a composition or a vaccine formulation, and an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0322] The present invention provides a kit of the present invention for treating human papillomavirus type 16 infection, human papillomavirus type 16 positive cancer or precancerous lesion, human papillomavirus type 18 infection, human papillomavirus type 18 positive cancer or precancerous lesion in a subject in need thereof.

[0323] The present invention provides use of the kit of the present invention in preparing a medicament for treating human papillomavirus type 16 infection, human papillomavirus type 16-positive cancer or precancerous lesions, human papillomavirus type 18 infection, human papillomavirus type 18-positive cancer or precancerous lesions in a subject in need thereof.

[0324] The present invention provides a method for treating human papillomavirus type 16 infection, human papillomavirus type 16-positive cancer or precancerous lesions, human papillomavirus type 18 infection, human papillomavirus type 18-positive cancer or precancerous lesions in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent in the kit of the present invention (e.g., a polypeptide or polypeptide combination, a nucleic acid or a nucleic acid combination (particularly RNA), a composition or a vaccine preparation of the present invention).

[0325] In some embodiments, HPV-16-positive cancers or HPV-18-positive cancers include, but are not limited to, cervical cancer, anogenital cancer, penile cancer, and head and neck cancers, such as genital cancer, head and neck squamous cell carcinoma.

[0326] Beneficial Effects

[0327] The polypeptides or polypeptide combinations, nucleic acids or nucleic acid combinations, compositions, vaccine preparations, and kits for HPV16 of the present invention can exhibit excellent effects, such as but not limited to: (1) inducing a potent cellular immune response, showing excellent immunogenicity even at low doses; (2) the immune efficacy can be maintained for 3 months or even longer; (3) inducing protective memory T cells to help eliminate tumor cells positive for the corresponding type of HPV-related antigens encountered again in the body, thereby preventing tumor recurrence and helping the body maintain a long-term tumor-free state; (4) slowing down the growth rate of HPV-16 positive tumors; (5) mediating the complete regression of HPV-16 positive tumors; (6) improving the survival rate; (7) having good safety; (8) the combined administration of the polypeptides or polypeptide combinations, nucleic acids or nucleic acid combinations, compositions, vaccine preparations, and immune checkpoint inhibitors of the present invention can further improve the tumor inhibition effect and survival rate; (9) can be used in combination with chemotherapy, radiotherapy, targeted therapy, and immunotherapy to enhance the therapeutic effect.

[0328] The polypeptides, polynucleotides, compositions, vaccine preparations, and kits for HPV18 of the present invention can exhibit excellent effects, such as but not limited to: (1) high expression in cells; (2) inducing a strong cellular immune response; (3) inducing protective memory T cells to help eliminate tumor cells positive for the corresponding type of HPV-related antigens encountered again in the body, thereby preventing tumor recurrence and helping the body maintain a long-term tumor-free state; (4) slowing down the growth rate of HPV-18 positive tumors; (5) mediating the complete regression of HPV-18 positive tumors; (6) improving the survival rate; (7) having good safety; (8) the combined administration of the polypeptides, polynucleotides, compositions, vaccine preparations, and immune checkpoint inhibitors of the present invention can further improve the tumor inhibition effect and survival rate; (9) can be used in combination with chemotherapy, radiotherapy, targeted therapy, and immunotherapy to enhance the therapeutic effect.

[0329] Exemplary embodiments

[0330] Embodiment 1. A polypeptide or a combination of polypeptides, wherein the N-terminus of the polypeptide or the polypeptide in the combination of polypeptides comprises a signal peptide sequence, the C-terminus comprises a transmembrane-intracellular domain sequence of MHC-I, and the polypeptide or the combination of polypeptides comprises an amino acid sequence of human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, and / or an amino acid sequence of human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof; or

[0331] The polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV18 E6 protein or its immunogenic fragment, and / or the amino acid sequence of human papillomavirus HPV18 E7 protein or its immunogenic fragment; wherein,

[0332] The human papillomavirus HPV16 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 105 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 105; the human papillomavirus HPV16 E7 protein comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 6; the human papillomavirus HPV18 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 131 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 131; the human papillomavirus HPV18 E7 protein comprises the amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 79.

[0333] Embodiment 2. The polypeptide or polypeptide combination of embodiment 1, wherein the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO: 7 or 77; and / or wherein the transmembrane-intracellular domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO: 8.

[0334] Embodiment 3. The polypeptide or polypeptide combination of embodiment 1 or 2 further comprises one or more linkers, preferably a GS linker; more preferably, the linker comprises the amino acid sequence shown in SEQ ID NO: 9 or 10.

[0335] Embodiment 4. The polypeptide or polypeptide combination of embodiment 1, which comprises the amino acid sequence shown in SEQ ID NO:106 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:106; and / or the amino acid sequence shown in SEQ ID NO:5 or 78 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:5 or 78; or the amino acid sequence shown in SEQ ID NO:132 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:132; and / or the amino acid sequence shown in SEQ ID NO:80 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:80.

[0336] Embodiment 5. A nucleic acid or a combination of nucleic acids, comprising a polynucleotide encoding a human papillomavirus (HPV16) E6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125 and 126, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125 and 126; and / or the nucleic acid or a combination of nucleic acids comprises a polynucleotide encoding a human papillomavirus (HPV16) E7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69; or

[0337] The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus (HPV18) E6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is RNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs: 150, 151 and 152, or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs: 150, 151 and 152; and / or the nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus (HPV18) E7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is RNA, and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs: 98, 99 and 100, or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs: 98, 99 and 100.

[0338] Embodiment 6. A nucleic acid or a combination of nucleic acids, comprising a polynucleotide encoding the human papillomavirus (HPV16) E6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 128, 129 and 130, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 128, 129 and 130; and / or the nucleic acid or a combination of nucleic acids comprises a polynucleotide encoding the human papillomavirus (HPV16) E7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76; or

[0339] The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156, or a nucleotide sequence having at least 85% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156; and / or the nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1-4, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 102, 103 and 104, or a nucleotide sequence having at least 85% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 102, 103 and 104.

[0340] Embodiment 7. A nucleic acid or a combination of nucleic acids comprising a polynucleotide encoding the polypeptide or the combination of polypeptides of any one of embodiments 1-4.

[0341] Embodiment 8. The nucleic acid or nucleic acid combination of embodiment 7, wherein the polynucleotide is RNA, the polynucleotide comprising a nucleotide sequence as shown in one of SEQ ID NOs: 107, 108, 109 and 110, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 107, 108, 109 and 110; and / or the polynucleotide comprising a nucleotide sequence as shown in one of SEQ ID NOs: 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29; or

[0342] The polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 133, 134, 135 and 136, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 133, 134, 135 and 136; and / or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 81, 82, 83 and 84, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 81, 82, 83 and 84.

[0343] Embodiment 9. The nucleic acid or nucleic acid combination of Embodiment 8, wherein the polynucleotide further comprises a 5'-UTR sequence; preferably, the 5'-UTR sequence comprises the nucleotide sequence of SEQ ID NO:11; the polynucleotide further comprises a 3'-UTR sequence; preferably, the 3'-UTR sequence comprises the nucleotide sequence of SEQ ID NO:12; and / or the polynucleotide further comprises a poly(A) sequence; preferably, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:15.

[0344] Embodiment 10. The nucleic acid or nucleic acid combination of Embodiment 7, wherein the polynucleotide is RNA, the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:111, 112, 113, and 114 or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NO:111, 112, 113, and 114; and / or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:2, 32, 33, 34, 35, 36, 37, 38, 39, and 40 or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NO:2, 32, 33, 34, 35, 36, 37, 38, 39, and 40; or

[0345] the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:137, 138, 139, and 140 or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NO:137, 138, 139, and 140; and / or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:85, 86, 87, and 88 or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NO:85, 86, 87, and 88.

[0346] Embodiment 11. The nucleic acid or nucleic acid combination of Embodiment 7, wherein the polynucleotide is DNA, the polynucleotide comprises the nucleotide sequences shown in SEQ ID NO:115, 116, 117, and 118 or a nucleotide sequence having at least 85% identity with the nucleotide sequences shown in SEQ ID NO:115, 116, 117, and 118; and / or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:3, 43, 44, 45, 46, 47, 48, 49, 50, and 51 or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NO:3, 43, 44, 45, 46, 47, 48, 49, 50, and 51; or

[0347] The polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 141, 142, 143 and 144, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 141, 142, 143 and 144; and / or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 89, 90, 91 and 92, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 89, 90, 91 and 92.

[0348] Embodiment 12. The nucleic acid or nucleic acid combination of embodiment 11, wherein the polynucleotide further comprises a DNA sequence corresponding to a 5'-UTR sequence; preferably, the DNA sequence corresponding to the 5'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 13; the polynucleotide further comprises a DNA sequence corresponding to a 3'-UTR sequence; preferably, the DNA sequence corresponding to the 3'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 14; and / or the polynucleotide further comprises a DNA sequence corresponding to a poly(A) sequence; preferably, the DNA sequence corresponding to the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 16.

[0349] Embodiment 13. The polynucleotide of embodiment 7, wherein the polynucleotide is a DNA, comprising a nucleotide sequence as shown in one of SEQ ID NOs: 119, 120, 121 and 122, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 119, 120, 121 and 122; and / or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62, or a nucleotide sequence having at least 85% identity to a nucleotide sequence as shown in one of SEQ ID NOs: 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62; or

[0350] The polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 145, 146, 147 and 148, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 145, 146, 147 and 148; and / or the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 93, 94, 95 and 96, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 93, 94, 95 and 96.

[0351] Embodiment 14. A composition comprising the polypeptide or polypeptide combination of any one of embodiments 1-4.

[0352] Embodiment 15. A composition comprising the nucleic acid or combination of nucleic acids according to any one of embodiments 5-13.

[0353] Embodiment 16. The composition of embodiment 15, comprising lipids encapsulating the nucleic acid or combination of nucleic acids.

[0354] Embodiment 17. The composition of embodiment 15 or 16, comprising lipid nanoparticles or lipid multiplexes.

[0355] Embodiment 18. The composition of embodiment 17, wherein the lipids encapsulating the nucleic acid or the combination of nucleic acids comprise cationic lipids, phospholipids, steroids and polyethylene glycol-modified lipids; optionally, the composition further comprises a cationic polymer, wherein the cationic polymer is associated with the nucleic acid or the combination of nucleic acids into a complex and are co-encapsulated in the lipid to form a lipid polymer complex; preferably, the cationic polymer is protamine.

[0356] Embodiment 19. The composition of embodiment 18, wherein the cationic lipid comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof

[0357]

[0358] in,

[0359] R 1 and R 2 Each independently selected from a bond, C 1 -C 12 Alkyl and C 2 -C 12 alkenyl;

[0360] R 3 and R 4 Each independently selected from C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 6 -C 10 aryl and 5-10 membered heteroaryl; and R 3 and R 4 Each independently selected by t R 6 Substitution, t is an integer selected from 1-5;

[0361] R 6 Each independently selected from C 1 -C 12 Alkyl and C 2 -C 12alkenyl;

[0362] M 1 and M 2 Each is independently selected from a bond, H, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, -C(S)S-, a 3-10 membered heterocycle, -NR 7 -,or

[0363] R 5 With M 1 and M 2 One of them together with the nitrogen atom to which it is connected forms a 3-10 membered heterocyclic ring, and the corresponding R 1 / R 3 or R 2 / R 4 No

[0364] exists, the heterocyclic ring is optionally replaced by R 7 replace;

[0365] R 5 Selected from C 3-8 Carbon ring, -C 1-12 Alkylene-Q, Q is selected from H, -OR 7 、-SR 7 、-OC(O)R 7 、-C(O)OR 7 、-N(R 7 )C(O)R 7 、-N(R 7 )S(O) 2 R 7 、-N(R 7 )C(S)R 7 、-N(R 7 ) 2 , cyano, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C 6 -C 10 Aryl, each of the above groups is optionally substituted with one or more C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 1 -C 12 Alkoxy, C 6 -C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O) substitution;

[0366] m and n are each independently an integer selected from 0-12;

[0367] The alkyl, alkenyl and alkylene groups are each optionally independently interrupted by one or more groups selected from: -O-, -S-, -NR 7 -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocyclic ring, and the alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R 7 replace;

[0368] R 7 Each independently selected from H, C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 1 -C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C 6 -C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclic ring, halogen, C 3-8 Carbocyclic ring, each of the above groups is optionally substituted by one or more C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 1 -C 12 Alkoxy, C 6 -C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O) substitution.

[0369] Embodiment 20. The composition of embodiment 19, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:

[0370]

[0371]

[0372] Preferably, the cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2; more preferably, the cationic lipid is SW-II-140-2.

[0373] Embodiment 21. The composition of any one of Embodiments 15-20, comprising 40 mol% SW-II-140-2, 15 mol% DOPE, 43.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0374] Embodiment 22. A vaccine formulation comprising the polypeptide or polypeptide combination of any one of embodiments 1-4 or the composition of embodiment 14.

[0375] Embodiment 23. A vaccine formulation comprising a nucleic acid or a combination of nucleic acids according to any one of embodiments 5-13 or a composition according to any one of embodiments 15-21.

[0376] Embodiment 24. An expression vector comprising the nucleic acid or nucleic acid combination of any one of embodiments 5-13.

[0377] Embodiment 25. A host cell comprising the nucleic acid or nucleic acid combination of any one of embodiments 5-13 or the expression vector of embodiment 24.

[0378] Embodiment 26. A kit comprising a polypeptide or polypeptide combination of any one of embodiments 1-4, a nucleic acid or nucleic acid combination of any one of embodiments 5-13, a composition of any one of embodiments 14-21, or a vaccine preparation of embodiment 22 or 23, and one or more therapeutic agents selected from the following: a chemotherapeutic agent, a radioisotope, an immune checkpoint inhibitor, and a tumor antigen targeted drug; preferably, the therapeutic agent is an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0379] Embodiment 27. Use of the polypeptide or polypeptide combination of any one of embodiments 1-4, the nucleic acid or nucleic acid combination of any one of embodiments 5-13, the composition of any one of embodiments 14-21, the vaccine preparation of embodiment 22 or 23, or the kit of embodiment 26 in the preparation of a medicament for treating human papillomavirus type 16 infection, human papillomavirus type 16-positive cancer or precancerous lesions, human papillomavirus type 18 infection, human papillomavirus type 18-positive cancer or precancerous lesions in a subject.

[0380] Embodiment 28. The use of embodiment 27, wherein the human papillomavirus type 16-positive cancer or the human papillomavirus type 18-positive cancer comprises cervical cancer, anogenital cancer, penile cancer, head and neck cancer, such as genital cancer, head and neck squamous cell carcinoma.

[0381] Example

[0382] The present invention is further described by reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the present invention. The following materials and instruments are commercially available or prepared according to methods known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0383] Example 1 Preparation of mRNA

[0384] 1.1. Design and synthesis of DNA template

[0385] The applicant designed and synthesized a codon-optimized DNA open reading frame (ORF) sequence encoding the E7 protein or the E6 protein of the human papillomavirus HPV16, which contains a signal peptide sequence sec1.0 (SEQ ID NO: 7) or sec2.0 (SEQ ID NO: 77) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a transmembrane-intracellular domain (MITD) sequence of MHC-I (SEQ ID NO: 8) at the C-terminus. The nucleotide sequences of nucleic acids encoding HPV16 E7 protein (SEQ ID NO: 5 or 78) containing a signal peptide sequence and MITD at both ends (named 22#, 29#, 31#, 33#, 24#, 47#, 50#, 51#, respectively) and nucleic acids encoding the same form of HPV16 E6 protein (SEQ ID NO: 106) (named E6-2, E6-3, E6-4, respectively) are shown in Table 1.

[0386] At the same time, the applicant designed and synthesized an original unoptimized wild-type DNA ORF sequence encoding only HPV16E7 protein (SEQ ID NO: 6) (named 8#), a codon-optimized DNA ORF sequence encoding only HPV16 E7 protein (named 10#), an original unoptimized DNA ORF sequence encoding HPV 16E7 protein containing a signal peptide sequence sec2.0 (SEQ ID NO: 77) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a transmembrane-intracellular domain (MITD) sequence of MHC-I at the C-terminus (named 18#), and an original unoptimized DNA ORF sequence encoding an HPV 16E7 protein containing a signal peptide sequence sec1.0 (SEQ ID NO: 7) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a transmembrane-intracellular domain (MITD) sequence of MHC-I at the C-terminus. The DNA ORF sequence of 16E7 protein (named 19#) and the original unoptimized DNA ORF sequence encoding HPV 16E6 protein (named E6-1) containing a signal peptide sequence see1.0 (SEQ ID NO: 7) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a MHC-I transmembrane-intracellular domain (MITD) sequence (SEQ ID NO: 8) at the C-terminus were used as controls, and their corresponding nucleotide sequences are also shown in Table 1.

[0387] The applicant also designed and synthesized a codon-optimized DNA open reading frame (ORF) sequence encoding the E7 protein of human papillomavirus HPV 18, which contains a signal peptide sequence sec 1.0 (SEQ ID NO: 7) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a transmembrane-intracellular domain (MITD) sequence of MHC-I at the C-terminus. The nucleotide sequences of the nucleic acids encoding the HPV18 E7 protein (SEQ ID NO: 80) containing SP and MITD at both ends (named 18-2, 18-3 and 18-4, respectively) are shown in Table 2. At the same time, the applicant designed and synthesized an original non-optimized DNA ORF sequence encoding the HPV 18E7 protein containing a signal peptide sequence sec1.0 (SEQ ID NO: 7) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a MHC-I transmembrane-intracellular domain (MITD) sequence (SEQ ID NO: 8) at the C-terminus (named 18-1) as a control, and its corresponding nucleotide sequence is also shown in Table 2.

[0388] In addition, the applicant designed and synthesized a codon-optimized DNA ORF sequence encoding the HPV 18E6 protein containing a signal peptide sequence sec1.0 (SEQ ID NO: 7) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a transmembrane-intracellular domain (MITD) sequence of MHC-I (SEQ ID NO: 8) at the C-terminus. The nucleotide sequences of the nucleic acids encoding the HPV18 E6 protein (SEQ ID NO: 132) containing a signal peptide sequence and MITD at both ends (named 18E6-2, 18E6-3, and 18E6-4, respectively) are shown in Table 2. At the same time, the applicant designed and synthesized an original non-optimized DNA ORF sequence encoding the HPV 18E6 protein containing a signal peptide sequence sec1.0 (SEQ ID NO: 7) and a GS linker (SEQ ID NO: 9) at the N-terminus and a GS linker (SEQ ID NO: 10) and a MHC-I transmembrane-intracellular domain (MITD) sequence (SEQ ID NO: 8) at the C-terminus (named 18E6-1) as a control, and its corresponding nucleotide sequence is also shown in Table 2.

[0389] The T7 promoter sequence (TAATACGACTCACTATA, SEQ ID NO: 157), 5'-UTR sequence (SEQ ID NO: 13) and 3'-UTR sequence (SEQ ID NO: 14) were also designed. The Kozak sequence "GCCACC" was included in the 5'-UTR sequence (SEQ ID NO: 13).

[0390] Then, the T7 promoter sequence, 5'-UTR, DNA ORF, 3'-UTR and PolyA were connected in order, and whole gene synthesis was performed using pUC57 as a vector (Nanjing GenScript Biotechnology Co., Ltd.) to obtain a plasmid DNA template.

[0391] The plasmid was linearized using restriction endonucleases and purified (Takara purification kit) to obtain the linearized plasmid, which is the DNA template required for in vitro transcription and synthesis of mRNA.

[0392] 1.2. In vitro transcription of mRNA from DNA template

[0393] Cap1 mRNA was produced by co-transcriptional capping reaction with T7 RNA polymerase and in vitro transcription of RNA. 1-Methyl-pseudouridine-triphosphate was used instead of uridine triphosphate (UTP) in in vitro transcription, so the modification ratio of 1-methyl-pseudouridine in in vitro transcribed Cap1 mRNA was 100%. After transcription, DNaseI (Thermo Fisher Scientific) was used to digest the DNA template to reduce the risk of residual DNA template.

[0394] The mRNA was purified using Dynabeads Myone (Thermo Fisher Scientific Inc.). The purified mRNA was dissolved in 1 mM sodium citrate buffer (pH 6.5 + / - 0.1), sterile filtered, and frozen at -80°C until use. The obtained mRNA sequences are shown in Tables 1 and 2.

[0395] Table 1. Nucleic acids encoding E6 and E7 proteins of human papillomavirus HPV 16

[0396]

[0397]

[0398] Table 2. Nucleic acids encoding E6 and E7 proteins of human papillomavirus HPV 18

[0399]

[0400] Example 2 Expression of candidate HPV16 mRNA in cells

[0401] To further detect the expression of the designed and synthesized candidate mRNA in cells, the applicant verified the expression of the mRNA for HPV16 type prepared in Example 1.2 in DC2.4 cells. The specific method is as follows: mouse bone marrow-derived dendritic cells DC2.4 cells (Shanghai Cell Bank) were cultured at 1.2×10 6 Cells / well were seeded in a 6-well plate. 18 hours after seeding, the corresponding mRNA stock solution was transfected into DC2.4 cells using the transfection reagent Lipofectamine Messenger Max (Invitrogen, LMRNA015), with 2 μg mRNA transferred into each well. The transfected cells were placed in a cell culture incubator at 37°C and 5% CO 2 Continue to culture for 24 hours. Then collect the cells, treat the collected cells with 6x loading buffer (full gold, DL101-02), heat at 100°C for 10 minutes, perform polyacrylamide gel electrophoresis on the treated cell samples and transfer them to PVDF membranes, and block them with 5% skim milk powder at room temperature for 1 hour. Use HPV16 E7 antibody (HPV Type 16E7Antibody, Invitrogen#28-0006) diluted 1:200 as the primary antibody, incubate overnight at 4°C with shaking, wash 5 times with PBST (PBS solution containing 0.5% Tween 20), use 1:5000 as the secondary antibody (Goat anti-Mouse IgG1Cross-AdsorbedSecondary Antibody, HRP Invitrogen#A 10551), incubate at room temperature for 1 hour, wash 5 times with PBST, add ECL luminescent solution (Pierce TM ECL Plus Western Blotting Substrate, Thermo Scientific TM #32132X3) for color development and imaging. DC CTRL is DC2.4 cells that were not transfected with mRNA (as a negative control).

[0402] The results are as follows Figure 1 As shown, compared with wild-type mRNA 8# encoding HPV16 E7 protein, the expression of sequence-optimized mRNAs (10#, 22#, 24#, 29#, 31#, 33#, 47#, 50# and 51# mRNA) increased significantly. Among them, under the same conditions, the sequence-optimized 29# mRNA expressed the most E7 protein and had the highest expression efficiency, so it was selected as the preferred solution and further tested.

[0403] Example 3 Preparation of mRNA vaccine preparation

[0404] This embodiment uses a lipid polymer Lipopolyplex (LPP) delivery system to deliver mRNA. LPP is a nano delivery system with a "core-shell" structure. First, negatively charged mRNA is microfluidically mixed with a positively charged polymer to form a core structure with uniform particle size. Then, the lipid molecule mixture dissolved in the organic phase (core ionizable lipid molecule SW-II-140-2, auxiliary phospholipid DOPE, PEGylated lipid molecule PEG-DMG2K and cholesterol) is microfluidically mixed with the core structure in the second step to form uniform LPP-mRNA nanoparticles.

[0405] 3.1 Experimental Materials

[0406] Cationic lipid SW-II-140-2 was synthesized by Simicroorganisms (for the specific synthesis method, see WO2022 / 233291); auxiliary phospholipid (DOPE) was purchased from CordenPharma; cholesterol was purchased from Sigma-Aldrich; mPEG2000-DMG (ie, DMG-PEG 2000 or PEG-DMG2K) was purchased from AvantiPolarLipids, Inc.; PBS was purchased from Invitrogen; protamine sulfate was purchased from Beijing Silian Pharmaceutical Co., Ltd.

[0407] 3.2. Preparation of mRNA lipid polyplex (LPP)

[0408] Preparation of mRNA aqueous solution: The mRNA prepared as in Example 1.2 was diluted with 10 mM citric acid-sodium citrate buffer (pH 4.0) to a 0.2 mg / mL mRNA solution.

[0409] Preparation of lipid solution: cationic lipid (SW-II-140-2): DOPE: cholesterol: mPEG2000-DMG were dissolved in anhydrous ethanol at a molar ratio of 40:15:43.5:1.5 to prepare a 10 mg / mL lipid solution.

[0410] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.25 mg / mL.

[0411] Preparation of core nanoparticle solution: Using microfluidic technology (Mianna (Shanghai) Technology Co., Ltd., model: InanoD), the protamine sulfate solution and the mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution formed by protamine and mRNA: volume = 4.0 mL; flow rate ratio = 5 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, start waste = 0.35 mL, end waste = 0.1 mL, room temperature.

[0412] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: volume = 4.0 mL, flow rate ratio = 1 (lipid solution): 3 (core nanoparticle solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature, diluted with PBS to obtain LPP solution.

[0413] Centrifugal ultrafiltration: The LPP solution was centrifuged by ultrafiltration to remove ethanol (speed 3000g, centrifugation time 60min, temperature 4°C) to obtain LPP preparations containing the mRNA prepared in Example 1.2. Among them, the LPP preparation containing 29#mRNA was named SW0128, and the LPP preparations containing mRNA 18-1, 18-2, 18-3, and 18-4 were named 1801, 1802, 1803, and 1804, respectively.

[0414] Example 4 SW0128 HPV16 LPP vaccine formulation induces cellular immune response

[0415] 4.1 Experimental mice

[0416] Female, 6-week-old Taconic C57BL / 6NTac mice and Vital River C57BL / 6J mice were used. C57BL / 6NTac and C57BL / 6J mice were bred and cared for by Shanghai Model Organisms Center Co., Ltd. Animal studies were performed in strict accordance with the recommendations in the Shanghai Guide for the Care and Use of Laboratory Animals.

[0417] 4.2 Experimental steps and results

[0418] In order to evaluate the immunogenicity of the LPP preparation SW0128 prepared in Example 3.2, this example used SW0128 to immunize C57BL / 6NTac mice and evaluate its ability to activate specific CD8 + Levels of T cell immune response.

[0419] Mice were randomly divided into 4 groups (n = 10 mice per group). On day 0 (D0), primary immunization (one immunization) was performed by subcutaneous two-point immunization. PBS, SW0128 mRNA LPP vaccine containing 3μg, 10μg, and 30μg mRNA were administered to each group of mice, respectively. On day 6 (D6), 5 mice in each group were killed, and the spleen lymphocytes of the mice were collected to determine the IFN-γ ELISpot effect after the first immunization; on day 7 (D7), the remaining mice were immunized by two subcutaneous injections for the second immunization. On day 13 (D13), the remaining mice were killed, and the spleen cells of the mice were collected to determine the IFN-γ ELISpot effect after the second immunization.

[0420] ELISpot assay

[0421] Mouse IFN-γ ELISpot assay was performed using the IFN-γ ELISpotPLUS kit (Mabtech, 3321-4APT-10) according to the manufacturer's instructions. Briefly, the plates were blocked and incubated in RPMI 1640 medium (supplemented with 10% FBS) for 30 minutes. Splenocytes were plated at 3×10 5 Cells / well were plated and stimulated in vitro with 2 μg / ml HPV16E7 peptide (RAHYNIVTF, Gill Biochemical), irrelevant 2 μg / ml vesicular stomatitis virus VSV peptide (RGYVYQGL, Gill Biochemical), positive stimulation 500 ng / mL phorbol ester (PMA) + 10 μg / mL ionomycin (Ionomycin) (Dako, 2030421) and PBS alone (negative control) and incubated for 20 hours at 37°C, 5% CO2. Afterwards, biotinylated IFN-γ-detection antibody and streptavidin-alkaline phosphatase (ALP) detection were used, BCIP / NBT-plus (5-bromo-4-chloro-3-indole-phosphate / nitro blue tetrazolium-plus) substrate was added for color development and counted with an ELISpot plate reader (ImmunoSpot S6 Core Analyzer (CTL)).

[0422] The experimental results are as follows Figure 2B and 2C As shown in Figure 2, SW0128 can effectively activate E7 peptide-specific CD8 + T cells, and this cellular immune response is dose-dependent. As the SW0128 immune dose increases, the number of IFN-γ-spots increases, that is, the cellular immune response is enhanced. Figure 2D As shown, this specific CD8 + The activation of T cell immunity was further enhanced 6 days (D13) after the second immunization (D7).

[0423] To further investigate whether immunization with a lower dose of SW0128 could also activate specific CD8 + For T cell immunization, the applicant immunized mice with SW0128 containing 0.3 μg or 1 μg mRNA and followed them for a long time.

[0424] In this experiment, mice were randomly divided into 5 groups, each with 20 mice. On day 0 (D0), primary immunization (one immunization) was performed by subcutaneous two-point immunization, and each group of mice was injected with PBS, SW0128mRNA LPP vaccine containing 0.3μg, 1μg, 3μgmRNA, or LPP preparation containing 3μgEGFPmRNA as a control; on day 6 (D6), 5 mice in each group were killed, and the spleen lymphocytes of the mice were collected to test the IFN-γELISpot effect after one immunization; on day 7 (D7), the remaining mice were immunized by two subcutaneous injections. On day 13 (D13), 5 mice in each group were killed, and the spleen lymphocytes of the second batch of mice were collected to determine the IFN-γELISpot effect after the second immunization. ot effect; on the 14th day (D14), the remaining mice were immunized three times by two-point subcutaneous injection; on the 20th day (D20), 5 mice from each group were killed, and the spleen lymphocytes of the third batch of mice were collected to measure the IFN-γ ELISpot effect after the three immunizations; on the 90th day (D90), 5 mice from each group were killed, and the spleen lymphocytes of the fourth batch of mice were collected, and the persistence of the mouse T cell immune response at three months was measured by IFN-γ ELISpot.

[0425] The experimental results are shown in 3B. At 6 days after the initial immunization (D6), even a low dose of 0.3 μg of SW0128 can effectively activate specific CD8 + T cell immunity, and the induced T cell immune response is further enhanced after the second and third immunizations. This induced immune response can be maintained for a long time, and activated specific CD8 + T cell immunity.

[0426] Taken together, these data indicate that SW0128 can induce sensitive, potent, and sustained specific T cell immune responses against HPV in mice.

[0427] Example 5 Tumor inhibition effect of SW0128 LPP vaccine preparation

[0428] 5.1SW0128 LPP vaccine formulation induces regression of HPV-16 positive TC-1 tumors in C57BL / 6NTac mice

[0429] The applicant further tested the tumor-suppressing effect of SW0128 in the syngeneic transplanted mouse TC-1 tumor model. The TC-1 tumor model was constructed with TC-1 cells, which are primary lung cells of C57BL / 6 mice containing HPV16 E6 / E7 genes obtained by HPV16 E6 / E7 retroviral transduction and immortalization. The specific modeling and testing process is described as follows.

[0430] C57BL / 6NTac mice were injected subcutaneously with 2x10 s TC-1 tumor cells were cultured and the tumor growth of mice was monitored regularly. 3 When cutting, cut it to about 30mm 3 , and implanted into the subcutaneous space of the flank of C57BL / 6NTac mice (n=15 per group).

[0431] Immunization procedures such as Figure 4A As shown, 4 days after tumor implantation (D4), SW0128 containing 3 μg, 10 μg, and 30 μg mRNA was administered to the tumor-implanted mice at a frequency of immunization once a week for 3 weeks (QW, immunization was performed on the 4th, 11th, and 18th days, respectively) or immunization twice a week for 3 weeks (BIW, immunization was performed on the 4th, 7th, 11th, 14th, 18th, and 21st days, respectively; and PBS and EGFP were administered to the tumor-implanted mice at the same time point as controls. The tumor volume was measured in a non-blind manner every 3 or 4 days, and the tumor volume was calculated as (a 2 xb) / 2 (a represents tumor width; b represents tumor length). The experimental endpoint is when the tumor volume reaches 2000mm 3 The mice were killed when the tumor ulcerated or had severe tissue necrosis, or when the weight loss of mice was greater than 20%.

[0432] Tumor volume detection results Figure 4B As shown, within 18-30 days after tumor implantation, the tumors of most control mice grew rapidly, leading to the experimental endpoint (each broken line represents one mouse). In contrast, most mice immunized with SW0128 containing 3μg, 10μg or 30μg mRNA showed complete tumor regression, and no significant difference was found in the complete regression (complete remission, CR) rate between the QW administration group and the BIW administration group.

[0433] The above results show that SW0128 under different immunization programs (QW and BIW) can induce complete regression of HPV-16 positive tumors. Figure 4CAs shown, there was no significant change or abnormality in the body weight of mice administered SW0128 (each broken line represents one mouse), further illustrating the excellent tumor inhibition effect and safety of SW0128.

[0434] 5.2SW0128 LPP vaccine formulation induces slower tumor growth rate in C57BL / 6J mice

[0435] In addition, the applicant also tested the tumor-suppressing effect of SW0128 in the C57BL / 6J mouse tumor model.

[0436] C57BL / 6J mice (female, 6 weeks old, Taconic Biosciences) were injected subcutaneously with 2×10 5 TC-1 tumor cells were cultured and the tumor growth of mice was monitored regularly. 3 When cutting, cut it to about 30mm 3 , and implanted into the subcutaneous space of the flank of C57BL / 6J mice (n=15 per group).

[0437] Immunization procedures such as Figure 5 As shown, 7 days after tumor implantation (D7), SW0128 containing 0.3 μg, 1 μg, and 3 μg mRNA was administered to mice implanted with tumors at a frequency of 1 immunization per week for 3 weeks (immunization was performed on the 7th, 14th, and 21st days, respectively) or 1 immunization per week for 2 weeks (immunization was performed on the 7th and 14th days, respectively); and PBS and EGFP were administered to mice implanted with tumors at the same time point as controls. Tumor volume was measured in a non-blind manner every 3 or 4 days. The tumor volume calculation formula and experimental endpoints are as described above.

[0438] Tumor volume detection results Figure 5 As shown, C57BL / 6J mice immunized with SW0128 containing 0.3μg, 1μg or 3μg mRNA did not show complete regression of tumors, but showed a significant slowing of tumor growth rate. This slowing of tumor growth rate was dose-dependent, and C57BL / 6J mice immunized with SW0128 containing 3μg mRNA showed the smallest tumor growth rate.

[0439] The applicant also noted that if Figure 4BAs shown, C57BL / 6NTac mice immunized with SW0128 QW containing 3 μg mRNA showed a significant reduction in tumor volume, and 10 of 15 mice showed complete regression, while C57BL / 6J mice immunized with SW0128 QW containing 3 μg mRNA only showed a significant slowing of tumor growth rate, and did not induce tumor regression. The difference in these data may be due to the difference in mice. It is generally believed that C57BL / 6NTac mice are a more suitable mouse strain for tumor syngeneic transplantation mouse experiments.

[0440] Example 6 SW0128 LPP vaccine formulation promotes the proliferation of memory T cells

[0441] In order to evaluate the protective efficacy of SW0128, the applicant continued to use the C57BL / 6NTac mice that had achieved complete tumor regression by treatment with 3 μg SW0128 in Example 5 in this example. These mice were kept in a tumor-free physiological state for at least 4 weeks and then randomly divided into new research groups (5 mice per group). 5 C57BL / 6NTac mice were subcutaneously inoculated with TC-1 tumor cells, and the mouse tumor model was prepared again as described in Example 5. At the same time, mice subcutaneously inoculated with the same amount of TC-1 cells and not treated with SW0128 were used as a control group.

[0442] The results are as follows Fig. 6A As shown in the figure, all mice treated with 3 μg SW0128 before were able to maintain a long-term tumor-free state after being challenged with tumor cells again, with a survival rate of 100%. The control group was killed 21-25 days after TC-1 implantation because the rapid growth of the tumor reached the experimental endpoint. These results illustrate the sustainability of the protective effect of SW0128, which can effectively inhibit tumor growth and cause tumor regression after being challenged with tumor cells again 4 months after immunization.

[0443] Furthermore, the applicant analyzed the reasons why the protective effect in mice with complete tumor regression after SW0128 treatment was persistent. Blood lymphocytes from C57BL / 6NTac mice with complete tumor regression after 3 μg SW0128 treatment in Example 5 (n=5 per group) or C57BL / 6NTac mice without SW0128 treatment (n=5 per group) were taken for E7 49-57 Dextramer + CD8 + Flow cytometry analysis of T cells, the reagent used is MHC I H-2Db(RAHYNIVTF), IMMUDEX, #JA2195.

[0444] The results are as follows Figure 6B As shown, up to 20% of E7 was expressed in the blood of SW0128-treated mice on day 59 (D59) after initial subcutaneous inoculation of TC-1 tumor cells. 49-57 CD8 + It can be seen that SW0128 immunization can induce potent antigen-specific CD8 T cells in mice. + The above experimental results show that SW0128 immunization can effectively induce and promote E7 49-57 Epitope-specific protective memory CD8 + The proliferation of T cells enables them to kill tumor cells when they encounter HPV-16 E7 antigen-positive tumor cells again.

[0445] In addition, antigen-specific-T cell responses in C57BL / 6NTac mice (6 mice) with complete tumor regression after treatment with 3 μg SW0128 and C57BL / 6NTac mice (3 mice) without SW0128 treatment were also detected by ELISpot at 93 days (D93) after the initial TC-1 tumor cell inoculation. The specific experimental method is described in Example 4.

[0446] The results are as follows Figure 6C As shown in Figure 2, IFN-γ spots in spleen lymphocytes of mice treated with SW0128 were still clearly detected 3 months after vaccination, which again demonstrated the durability of the protective efficacy of SW0128 and the ability to induce antigen-specific CD8 + T cell response.

[0447] Together, these data suggest that the HPV-16 mRNA vaccine SW0128 can induce regression of TC-1 tumors and induce robust protective cellular immune memory to HPV antigens that persists for at least 3 months, and possibly longer, to protect against viral rechallenge and recurrence.

[0448] Example 7 Tumor inhibition effect of combined administration of SW0128 LPP vaccine preparation and anti-PD-L1 antibody

[0449] It is reported that HPV mRNA vaccines upregulate the immune checkpoint molecules PD-1 and PD-L1 in the tumor microenvironment (TME) (Grunwitz C et al., HPV 16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishes protective T cell memory. Oncoimmunology. 201 9 Jul 11; 8 (9): e 1629259.). In order to further improve the immune efficacy of the SW0128 LPP vaccine preparation, the applicant tested the synergistic therapeutic effect of SW0128 and the checkpoint inhibitor anti-PD-L1 antibody in the TC-1 mouse tumor model. The specific detection method is as follows.

[0450] On day 0 (D0), C57BL / 6J mice (female, 6 weeks old, Taconic Biosciences) were injected subcutaneously with 5×10 5 TC-1 tumor cells were cultured and the tumor growth of mice was monitored regularly. 3 When cutting, cut it to about 30mm 3 , and implanted into the subcutaneous space of the flank of C57BL / 6J mice.

[0451] Dosage regimen Fig. 7AAs shown, mice were randomly divided into 7 groups (n=15). Among them, SW0128 containing 3 μg mRNA was administered to mice in Group 1 and Group 2 on Day 6 (D6), Day 13 (D13) and Day 20 (D20), SW0128 containing 10 μg mRNA was administered to mice in Group 3 and Group 4 at the same time points, LPP preparation containing 10 μg GFP mRNA was administered to mice in Group 5 and Group 6, and PBS was administered to mice in Group 7 as a negative control group. In addition, starting from day 9, 200 μg of PD-L1 antibody (InVivoMAb anti-mouse PD-L1 (B7-H1), BioX Cell, #BE0101) or IgG2b isotype control antibody (InVivoMAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin, Bio X Cell, #BE0090) was intraperitoneally injected every 3-4 days, that is, 200 μg of PD-L1 antibody was administered to Group 1, Group 3 and Group 5 mice on Day 9 (D9), Day 13 (D13), Day 16 (D16), Day 20 (D20) and Day 23 (D23), respectively, and IgG2b isotype control antibody was administered to Group 2, Group 4 and Group 6 mice at the same time points.

[0452] Tumor volume was measured in a non-blind manner every 3 or 4 days, and the tumor volume was calculated as follows: 2 xb) / 2 (a represents tumor width; b represents tumor length). The experimental endpoint is when the tumor volume reaches 2000mm 3 The mice were killed when the tumor ulcerated or had severe tissue necrosis, or when the weight loss of mice was greater than 20%.

[0453] Tumor volume detection results Figure 7B As shown, compared with the single administration of SW0128 or the single administration of PD-L1 antibody, the combined administration of SW0128 and PD-L1 antibody can enhance the inhibitory effect on tumor growth. Among them, the tumor growth inhibition rate of the second group of mice administered with 3μg SW0128 and IgG2b reached 45%; the tumor growth inhibition rate of the first group of mice administered with 3μg SW0128 and PD-L1 antibody reached 78%; the tumor growth inhibition rate of the fourth group of mice administered with 10μg SW0128 and IgG2b reached 71%; the tumor growth inhibition rate of the third group of mice administered with 10μg SW0128 and PD-L1 antibody reached 87%. It can be seen that the application of the immune checkpoint inhibitor PD-L1 antibody can further help reduce the TC-1 tumor burden.

[0454] The survival rate of mice Figure 7CAs shown, the PBS group, the mice in group 5 administered with LPP preparations containing GFP mRNA and PD-L1 antibodies, and the mice in group 6 administered with LPP preparations containing GFP mRNA and IgG2b were sacrificed within 33 days after TC-1 implantation because the tumors grew rapidly and reached the experimental endpoint. The survival rate of mice administered with 3 doses of SW0128 and 5 doses of PD-L1 antibodies increased significantly, and the mice in group 3 administered with 10 μg of SW0128 and PD-L1 antibodies had the highest survival rate.

[0455] The results of tumor regression analysis were as follows Fig.7D As shown, 4 mice in the 4th group of 15 mice that were administered with 10 μg SW0128 alone had complete tumor regression, and no mice in the 2nd group of 15 mice that were administered with 3 μg SW0128 alone had complete tumor regression. In contrast, 8 mice in the 3rd group of 15 mice that were administered with 10 μg SW0128 and PD-L1 antibody had complete tumor regression, and 5 mice in the 1st group of 15 mice that were administered with 3 μg SW0128 and PD-L1 antibody had complete tumor regression. It can be seen that the combined administration of SW0128 and PD-L1 antibody significantly increased the complete tumor regression rate.

[0456] Example 8 Expression of HPV18 E7 mRNA-LPP preparation in cells

[0457] In this example, the expression of four mRNA-LPP preparations against HPV18 prepared in Example 3 was verified. The specific detection method is as follows: human renal epithelial cells 293 (Shanghai Cell Bank) were cultured at 1.2×10 6 Cells / well were seeded in 6-well plates. 18 hours after seeding, 250 μL Opti-MEM reduced serum medium (Gibco, #31985070) was used to dilute HPV 18 mRNA LPP preparations 1801, 1802, 1803 and 1804 containing 2 μg mRNA and added dropwise to the 293 cells to mix well. The transfected cells were placed in a cell culture incubator at 37°C and 5% CO 2The cells were cultured for 24 hours. The cells were then collected and treated with 6× loading buffer (full gold, DL101-02) and heated at 100°C for 10 min. The treated cell samples were subjected to polyacrylamide gel electrophoresis and transferred to PVDF membranes, which were blocked with 5% skim milk powder for 1 h at room temperature. HPV18 E7 antibody (Anti-HPV18 E7 antibody [8E2] (ab100953)) diluted 1:1000 was used as the primary antibody, incubated overnight at 4°C with shaking, washed 5 times with PBST (PBS solution containing 0.5% Tween 20), and a secondary antibody (Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, HRP Invitrogen #A10551) diluted 1:5000 was used, incubated at room temperature for 1 h, washed 5 times with PBST, and ECL luminescent solution (Pierce TM ECLPlus Western Blotting Substrate, Thermo Scientific TM #32132X3) for color development and imaging.

[0458] The results are as follows Figure 8 As shown, compared with 1801 containing unoptimized mRNA, the expression of LPP preparations 1802 and 1804 containing sequence-optimized mRNA in cells was significantly increased.

[0459] Example 9 HPV18 E7 mRNA-LPP vaccine preparation induces cellular immune response

[0460] 9.1 Experimental mice

[0461] Female, 6-week-old C57BL / 6J mice from Vital River were used. C57BL / 6J mice were bred and cared for by Shanghai Model Organisms Center Co., Ltd. Animal studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Shanghai.

[0462] 9.2 Experimental steps and results

[0463] In order to evaluate the immunogenicity of LPP preparations 1801, 1802, 1803 and 1804 prepared in Example 3.2, this example used HPV18E7 mRNA-LPP vaccine preparations 1801, 1802, 1803 and 1804 to immunize C57BL / 6J mice and evaluated the level of activation of specific CD8+T cell immune responses in mice.

[0464] The mice were randomly divided into 5 groups (n = 5 mice per group). On day 0 (D0), primary immunization (one immunization) was performed by subcutaneous two-point immunization, and PBS, LPP preparations 1801, 1802, 1803, and 1804 containing 10 μg of mRNA 18-1, 18-2, 18-3, and 18-4 were administered to each group of mice, respectively. On the 7th day (D7) after the first immunization, the mice were killed, and the spleen lymphocytes of the mice were collected to test the IFN-γ ELISPOT effect after the first immunization.

[0465] ELISpot assay

[0466] Mouse IFN-γ ELISpot assay was performed using the IFN-γ ELISpotPLUS kit (Mabtech, 3321-4APT-10) according to the manufacturer's instructions. Briefly, the plates were blocked and incubated in RPMI 1640 medium (supplemented with 10% FBS) for 30 minutes. Splenocytes were plated at 3×10 5 Cells / well were plated and stimulated in vitro with 2 μg / ml HPV18 E7 peptide (Gill Biochemical, sequence as shown in Table 3 below), irrelevant 2 μg / ml vesicular stomatitis virus VSV peptide (RGYVYQGL, Gill Biochemical), positive stimulation 500 ng / mL phorbol ester (PMA) + 10 μg / mL ionomycin (Ionomycin) (Dako, 2030421) and PBS alone (negative control), and incubated for 20 hours at 37°C, 5% CO2. Afterwards, biotinylated IFN-γ-detection antibody and streptavidin-alkaline phosphatase (ALP) detection were used, BCIP / NBT-plus (5-bromo-4-chloro-3-indole-phosphate / nitro blue tetrazolium-plus) substrate was added for color development and counted using an ELISpot plate reader (ImmunoSpot S6 Core Analyzer (CTL)).

[0467] Table 3. HPV 18 E7 and E6 peptide libraries

[0468]

[0469]

[0470]

[0471] Test results such as Fig. 9B and 9C As shown, 1801, 1802, 1803 and 1804 all induced HPV18-specific T cell immune responses in non-tumor-bearing mice. Among them, 1802 induced the highest level of HPV18-specific T cell immune responses in non-tumor-bearing mice.

[0472] In summary, the HPV18-2 vaccine molecule has the best performance in both in vitro protein expression results and HPV18-specific T cell immune response results in non-tumor-bearing mice.

[0473] It will be clear to those skilled in the art that many modifications and variations of the present invention may be made without departing from its spirit and scope. The specific embodiments described herein are provided only by way of example and are not intended to be limited in any way. The true scope and spirit of the present invention are shown by the appended claims, and the description and examples are merely exemplary.

Claims

1. A polypeptide or a combination of polypeptides, wherein the N-terminus of the polypeptide or the polypeptide combination comprises a signal peptide sequence, and the C-terminus comprises a transmembrane-intracellular domain sequence of MHC-I, and The polypeptide or polypeptide combination comprises The amino acid sequence of human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof, and / or The amino acid sequence of human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof; or The polypeptide or polypeptide combination comprises The amino acid sequence of human papillomavirus HPV18 E6 protein or its immunogenic fragment, and / or The amino acid sequence of human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof; in, The human papillomavirus HPV16 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 105 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 105; The human papillomavirus HPV16 E7 protein comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:6; The human papillomavirus HPV18 E6 protein comprises the amino acid sequence shown in SEQ ID NO: 131 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 131; The human papillomavirus HPV18 E7 protein comprises the amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

79.

2. A nucleic acid or a combination of nucleic acids, The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125 and 126, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 124, 125 and 126; and / or The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 17, 20, 63, 64, 65, 66, 67, 68 and 69; or The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 150, 151 and 152, or a nucleotide sequence having at least 85% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 150, 151 and 152; and / or The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is RNA, and the polynucleotide comprises a nucleotide sequence shown in one of SEQ ID NOs: 98, 99 and 100 or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs: 98, 99 and 100.

3. A nucleic acid or a combination of nucleic acids, The nucleic acid or nucleic acid combination comprises a nucleic acid encoding the human papillomavirus HPV16 E6 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 128, 129 and 130 or a nucleotide sequence having at least 85% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 128, 129 and 130; and / or The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV16 E7 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76, or a nucleotide sequence having at least 85% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 18, 42, 70, 71, 72, 73, 74, 75 and 76; or The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV18 E6 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is a DNA, and the polynucleotide comprises a nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156 or a nucleotide sequence having at least 85% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 154, 155 and 156; and / or The nucleic acid or nucleic acid combination comprises a polynucleotide encoding the human papillomavirus HPV18 E7 protein or an immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is DNA, and the polynucleotide comprises a nucleotide sequence shown in one of SEQ ID NOs: 102, 103 and 104, or a nucleotide sequence having at least 85% identity with the nucleotide sequence shown in one of SEQ ID NOs: 102, 103 and 104.

4. A nucleic acid or a combination of nucleic acids, comprising a polynucleotide encoding the polypeptide or the combination of polypeptides according to claim 1.

5. A composition comprising the polypeptide or polypeptide combination of claim 1, or the nucleic acid or nucleic acid combination of any one of claims 2 to 4.

6. A vaccine preparation comprising the polypeptide or polypeptide combination of claim 1, the nucleic acid or nucleic acid combination of any one of claims 2 to 4, or the composition of claim 5.

7. An expression vector comprising the nucleic acid or nucleic acid combination according to any one of claims 2 to 4.

8. A host cell comprising the nucleic acid or nucleic acid combination according to any one of claims 2 to 4 or the expression vector according to claim 7.

9. A kit comprising the polypeptide or polypeptide combination of claim 1, the nucleic acid or nucleic acid combination of any one of claims 2-4, the composition of claim 5 or the vaccine preparation of claim 6, and one or more therapeutic agents selected from the following: chemotherapeutic agents, radioactive isotopes, immune checkpoint inhibitors and tumor antigen targeted drugs; preferably, the therapeutic agent is an anti-PD-L1 antibody or an antigen-binding fragment thereof.

10. Use of the polypeptide or polypeptide combination of claim 1, the nucleic acid or nucleic acid combination of any one of claims 2 to 4, the composition of claim 5, the vaccine preparation of claim 6 or the kit of claim 9 in the preparation of a medicament for treating human papillomavirus type 16 infection, human papillomavirus type 16-positive cancer or precancerous lesions, human papillomavirus type 18 infection, human papillomavirus type 18-positive cancer or precancerous lesions in a subject.

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