HPV vaccine and use thereof

Abstract

Provided are an HPV vaccine containing a nucleic acid molecule encoding an HPV antigen and an immune costimulatory factor, and the use thereof.

Description

HPV vaccine and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the field of nucleic acid vaccine. Specifically, the present application provides an HPV vaccine and application thereof, wherein the HPV vaccine comprises a nucleic acid molecule encoding an HPV antigen and an immune co-stimulatory factor.

[0002] BACKGROUND

[0003] Human papillomavirus (HPV) is a group of highly infectious viruses, mainly transmitted through sexual contact. HPV infection can cause a variety of diseases, from benign lesions such as common warts and genital warts, to malignant lesions such as cervical cancer, anal cancer, oropharyngeal cancer, and genital cancer. High-risk HPV is closely related to the occurrence of cervical cancer, among which HPV16 and HPV18 are the most common high-risk types, accounting for about 70% of cervical cancer cases.

[0004] After HPV infection, the normal function of host cells is affected by encoding a variety of key proteins. E6 and E7 are two major oncogenic proteins that play an important role in the development of HPV-related cancer. E6 protein can bind to tumor suppressor protein p53 and promote its degradation and inactivation, leading to abnormal cell growth, promoting malignant transformation and tumor occurrence. E7 protein mainly binds to retinoblastoma protein (pRb), preventing its binding to E2F transcription factor, leading to uncontrolled cell proliferation, further promoting the carcinogenic process.

[0005] WO2022 / 008519 describes a therapeutic RNA containing HPV E6 or E7, wherein the vaccine antigen encoded by the therapeutic RNA has the structure sec-GS(1)-E6 / E7-GS(2)-P2P16-GS(3)-MITD, wherein sec is a signal peptide that guides the nascent peptide chain to the endoplasmic reticulum; P2P16 is an amino acid sequence derived from tetanus endotoxin, which destroys immune tolerance of the body; MITD is the transmembrane and cytoplasmic domain of MHC class I molecule, which enhances antigen processing and / or presentation. The elements of the vaccine antigen are connected by GS linker peptides to form a fusion polypeptide.

[0006] WO2024 / 140767 describes an HPV mRNA vaccine containing E6 and E7 antigen polypeptide coding sequences of types 16 and 18 HPV, comprising, from 5' to 3', in order: linked tPA-SP, Flt3L, coding sequence of HPV E2 (if any), E6 / E7 protein or variant thereof, which expresses a fusion polypeptide.

[0007] SUMMARY

[0008] The present application includes, but is not limited to, the following embodiments:

[0009] Embodiment 1. A nucleic acid molecule comprising an open reading frame (ORF), the ORF comprising:

[0010] a nucleotide sequence encoding an HPV antigen;

[0011] a nucleotide sequence encoding an immune costimulatory factor; and

[0012] a nucleotide sequence encoding a self-cleaving peptide located between the nucleotide sequence encoding an HPV antigen and the nucleotide sequence encoding an immune costimulatory factor.

[0013] Embodiment 2. The nucleic acid molecule of embodiment 1, wherein the HPV antigen comprises at least one HPV immunogenic polypeptide from at least one HPV serotype.

[0014] Embodiment 3. The nucleic acid molecule of embodiment 2, wherein the at least one HPV serotype is selected from the group consisting of serotypes 1, 2, 3, 4, 7, 10, 12, 15, 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, 5, 8, 14, 17, 20, 36, 38, 16, 18, 30, 31, 33, 35, 39, preferably the at least one HPV serotype is serotype 16 and / or serotype 18.

[0015] Embodiment 4. The nucleic acid molecule of embodiment 2 or 3, wherein the HPV immunogenic polypeptide is selected from the group consisting of an E1, E2, E3, E4, E5, E6, E7, L1, or L2 polypeptide of HPV or an immunogenic variant thereof, preferably the HPV immunogenic polypeptide is selected from the group consisting of an E6, E7, L2 polypeptide or an immunogenic variant thereof, more preferably the HPV immunogenic polypeptide is selected from the group consisting of an E6, E7 polypeptide or an immunogenic variant thereof.

[0016] Embodiment 5. The nucleic acid molecule of any one of embodiments 2-4, wherein the HPV antigen comprises two HPV immunogenic polypeptides from one HPV serotype, or the HPV antigen comprises two HPV immunogenic polypeptides from two HPV serotypes.

[0017] Embodiment 6. The nucleic acid molecule of any one of embodiments 2-5, wherein the HPV antigen comprises

[0018] i) an E6 polypeptide and an E7 polypeptide from HPV serotype 16;

[0019] ii) an E6 polypeptide and an E7 polypeptide from HPV serotype 18; or

[0020] iii) an E6 polypeptide and an E7 polypeptide from HPV serotype 16 and an E6 polypeptide and an E7 polypeptide from HPV serotype 18.

[0021] Embodiment 7. The nucleic acid molecule of embodiment 6, wherein

[0022] the E6 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 67 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 41 or SEQ ID NO: 67;

[0023] the E7 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 68 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 68;

[0024] the E6 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 69 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 43 or SEQ ID NO: 69;

[0025] the E7 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 70 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 70.

[0026] Embodiment 8. The nucleic acid molecule of any one of embodiments 2-7, wherein the HPV antigen further comprises a signal peptide, whereby secretion expression of the HPV immunogenic polypeptide is effected, for example, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0027] Embodiment 9. The nucleic acid molecule of any one of embodiments 2-8, wherein the different polypeptides comprised in the HPV antigen are directly linked or linked via a peptide linker, for example, the peptide linker comprises the sequence GnS, n being an integer from 1 to 5, for example n is 1 or 3.

[0028] Embodiment 10. The nucleic acid molecule of any one of embodiments 1-9, wherein the immune costimulatory factor is selected from Flt3Lecd, GM-CSF, OX40L, HMGB1, or HSP70p, preferably GM-CSF and OX40L, more preferably GM-CSF.

[0029] Embodiment 11. The nucleic acid molecule of embodiment 10, wherein the GM-CSF comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0030] Embodiment 12. The nucleic acid molecule of embodiment 11, wherein the self-cleaving peptide is a 2A polypeptide, preferably a T2A polypeptide or a P2A polypeptide, more preferably a T2A polypeptide.

[0031] Embodiment 13. The nucleic acid molecule of embodiment 12, wherein the T2A polypeptide amino acid sequence is set forth in SEQ ID NO: 64.

[0032] Embodiment 14. The nucleic acid molecule of any one of embodiments 1-13, wherein the ORF comprises, in the 5’ to 3’ direction, the nucleotide sequence encoding the HPV antigen, the nucleotide sequence encoding the self-cleaving peptide, and the nucleotide sequence encoding the immune costimulatory factor.

[0033] Embodiment 15. The nucleic acid molecule of any one of embodiments 1-14, wherein the ORF encodes an amino acid sequence comprising the structure: SP-HPV immunogenic polypeptide-2A-GMCSF, wherein SP is a signal peptide, 2A is a 2A polypeptide, and GMCSF is human or murine GM-CSF; preferably, the ORF encodes an amino acid sequence comprising the structure: SP-E6 16 -E7 16 -E6 18 -E7 18 -2A-GMCSF, wherein SP is a signal peptide, E6 16 and E7 16 are E6 and E7 polypeptides from HPV serotype 16, respectively, E6 18 and E7 18 are E6 and E7 polypeptides from HPV serotype 18, respectively, 2A is a 2A polypeptide, and GMCSF is human or murine GM-CSF.

[0034] Embodiment 16. The nucleic acid molecule of any one of embodiments 1-15, wherein the ORF encodes an amino acid sequence set forth in one of SEQ ID NOs: 1-18 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to one of SEQ ID NOs: 1-18,

[0035] Preferably, the ORF encodes the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 11.

[0036] Embodiment 17. The nucleic acid molecule of any one of embodiments 1-16, wherein the ORF comprises the nucleotide sequence of any one of SEQ ID NOs: 21-38 or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to any one of SEQ ID NOs: 21-38,

[0037] Preferably, the ORF comprises the nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 31.

[0038] Embodiment 18. The nucleic acid molecule of any one of embodiments 1-17, wherein the nucleic acid molecule is an mRNA molecule.

[0039] Embodiment 19. The nucleic acid molecule of embodiment 18, wherein the mRNA molecule further comprises a 5’ UTR and / or a 3’ UTR.

[0040] Embodiment 20. The nucleic acid molecule of embodiment 19, wherein the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 57 and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 58; or the 5’ UTR comprises the nucleotide sequence of SEQ ID NO: 59 and the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 60.

[0041] Embodiment 21. The nucleic acid molecule of any one of embodiments 18-20, wherein the mRNA molecule further comprises a poly(A) sequence.

[0042] Embodiment 22. The nucleic acid molecule of embodiment 21, wherein the poly(A) sequence comprises from about 20 to about 500 adenine nucleotides (A), preferably the poly(A) sequence comprises 120 adenine nucleotides (A).

[0043] Embodiment 23. The nucleic acid molecule of any one of embodiments 18-22, wherein the mRNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 66 or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 66.

[0044] Embodiment 24. The nucleic acid molecule of any one of embodiments 18-23, wherein the mRNA molecule further comprises a 5’ cap structure, for example, the 5’ cap structure is a Cap1 cap structure.

[0045] Embodiment 25. The nucleic acid molecule of any one of embodiments 18-24, wherein the mRNA molecule further comprises at least one modified nucleotide, preferably the modified nucleotide is selected from pseudouridine, N1-methyl-pseudouridine, 5-methylcytidine, or a combination thereof.

[0046] Embodiment 26. A lipid nanoparticle (LNP) comprising the nucleic acid molecule of any one of embodiments 1-25, the nucleic acid molecule is comprised in a lipid.

[0047] Embodiment 27. An immunogenic composition comprising the nucleic acid molecule of any one of embodiments 1-25 or the LNP of embodiment 26, and a pharmaceutically acceptable carrier.

[0048] Embodiment 28. An immunoprophylactic / therapeutic medicament comprising the nucleic acid molecule of any one of embodiments 1-25 or the LNP of embodiment 26, and a pharmaceutically acceptable carrier; preferably the immunoprophylactic / therapeutic medicament is a tumor immunotherapy medicament, more preferably the tumor immunotherapy medicament is a tumor vaccine.

[0049] Embodiment 29. Use of the nucleic acid molecule of any one of embodiments 1-25 or the LNP of embodiment 26 or the immunogenic composition of embodiment 27 in the manufacture of a vaccine for preventing and / or treating HPV infection and / or HPV infection related pathologies in a subject.

[0050] Embodiment 30. A method for preventing and / or treating HPV infection and / or HPV infection related pathologies in a subject in a subject, the method comprising administering to the subject an effective amount of the nucleic acid molecule of any one of embodiments 1-25 or the LNP of embodiment 26 or the immunogenic composition of embodiment 27.

[0051] Embodiment 31. The use of embodiment 29 or the method of embodiment 30, wherein the HPV infection is an HPV infection selected from the group consisting of serotypes 1, 2, 3, 4, 7, 10, 12, 15, 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, 5, 8, 14, 17, 20, 36, 38, 16, 18, 30, 31, 33, 35, 39.

[0052] Embodiment 32. The use or method of embodiment 31, wherein the HPV infection is an infection with a single HPV serotype or the HPV infection is a mixed infection with two or more different HPV serotypes.

[0053] Embodiment 33. The use or method of embodiment 31 or 32, wherein the HPV infection is an infection with HPV 16 and / or HPV 18.

[0054] Embodiment 34. The use of embodiment 29 or the method of embodiment 30, wherein the HPV infection related pathology is selected from the group consisting of cutaneous warts, such as common warts, plantar warts, flat warts, Bowen's disease, butcher's warts or condyloma acuminata; HPV-positive tumors such as HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, vaginal cancer, rectal cancer, bladder cancer, endometrial cancer, oropharyngeal cancer, head and neck squamous cell carcinoma, prostate cancer or bladder cancer.

[0055] Embodiment 35. A combination comprising:

[0056] (1) the nucleic acid molecule of any one of embodiments 1-25, the LNP of embodiment 26 or the immunogenic composition of embodiment 27, and

[0057] (2) an immune checkpoint inhibitor.

[0058] Embodiment 36. The combination of embodiment 35, wherein the immune checkpoint inhibitor is a PD1 signaling pathway inhibitor, for example, the PD1 signaling pathway inhibitor is selected from the group consisting of a PD1 antibody, a PDL1 antibody, a PDL2 antibody, or a combination thereof, preferably a PD1 antibody.

[0059] Embodiment 37. The combination of embodiment 35 or 36 for use in the prevention and / or treatment of a tumor and / or a cancer disease in a subject; preferably, the tumor and / or cancer disease is a HPV-positive tumor and / or cancer; preferably, the tumor and / or cancer disease is selected from the group consisting of HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, vaginal cancer, rectal cancer, bladder cancer, endometrial cancer, oropharyngeal cancer, head and neck squamous cell carcinoma, prostate cancer or bladder cancer.

[0060] Embodiment 38. A method for preventing and / or treating a tumor and / or a cancer disease in a subject, the method comprising administering to the subject an effective amount of the combination of Embodiment 35 or 36; preferably, the tumor and / or the cancer disease is a HPV-positive tumor and / or a cancer disease; preferably, the tumor and / or the cancer disease is selected from the group consisting of a HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, vaginal cancer, rectal cancer, bladder cancer, endometrial cancer, oropharyngeal cancer, head and neck squamous cell carcinoma, prostate cancer, or bladder cancer.

[0061] SUMMARY

[0062] Figure 1. shows that the designed mRNA is well expressed in cells.

[0063] Figure 2. shows HPV16 E7 polypeptide group ELISpot results.

[0064] Figure 3. shows the results of the pharmacodynamic screening of mRNA vaccines (TGI, tumor growth inhibition rate; CR, complete remission rate; n=5, mean±SD, n.s: no significance, *P<0.05, **P<0.01).

[0065] Figure 4. shows the results of Western blot detection of HPV vaccine mRNA expression in HEK293T cells.

[0066] Figure 5. Western blot experiment to detect HPV antigen content in DC2.4 cells.

[0067] Figure 6. ELISA detection of human or murine GM-CSF content in cell supernatant.

[0068] Figure 7. Results of the pharmacodynamic screening of mRNA vaccines (TGI, tumor growth inhibition rate; CR, complete remission rate; n=5, mean±SD, **P<0.01).

[0069] Figure 8. Results of the pharmacodynamic screening of M21-2 molecules (TGI, tumor growth inhibition rate; n=3, mean±SD, **P<0.01).

[0070] Figure 9. Results of ELISpot detection of immune responses of splenocytes to M21-2 (SPF, spot forming units; n=3, mean±SD, **P<0.01).

[0071] Figure 10. Results of HPV-specific CTL content detection in splenocytes and tumor tissue cells (n=3, mean±SD, **P<0.01).

[0072] Figure 11. The results of detecting the content of HPV-specific CTL in spleen cells and tumor tissue cells (n=3, mean±SD, **P<0.01).

[0073] Figure 12. The results of evaluating the pharmacodynamic activity of M21-2 molecules in an HPV16+ large tumor model (TGI, tumor growth inhibition rate; n=5, mean±SD, *P<0.05, **P<0.01).

[0074] Figure 13. The results of comparing the pharmacodynamic activity of M21-2, M21-2-Neg and BMK in an HPV16+ large tumor model (TGI, tumor growth inhibition rate; n=5, mean±SD, *P<0.05, **P<0.01).

[0075] Figure 14. The pharmacodynamic activity of M21-2 molecules in an MC38 (HPV18-E6 & E7)-100mm 3 transplanted tumor model (TGI, tumor growth inhibition rate; n=5, mean±SD, *P<0.05, **P<0.01).

[0076] Figure 15. The pharmacodynamic activity of M21-2 molecules in an MC38 (HPV18-E6 & E7)-200mm 3 transplanted tumor model (TGI, tumor growth inhibition rate; n=5, mean±SD, *P<0.05, **P<0.01).

[0077] Figure 16. The expression of PD-L1 in MC38 (HPV18-E6 & E7).

[0078] Figure 17. The synergistic pharmacodynamic effect of M21-2 molecules and aPD-1 in an MC38 (HPV18-E6 & E7) transplanted tumor model (TGI, tumor growth inhibition rate; n=6, mean±SD, **P<0.01).

[0079] DETAILED DESCRIPTION

[0080] I. DEFINITIONS

[0081] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are the terms and conventional procedures widely used in the corresponding fields.

[0082] As used herein, the term "and / or" encompasses all combinations of the items connected by the term, should be read as if each combination was individually listed. For example, "A and / or B" covers the cases where "A" is true, "B" is true, and "A and B" are true. For example, "A, B, and / or C" covers the cases where "A" is true, "B" is true, "C" is true, "A and B" are true, "A and C" are true, "B and C" are true, and "A and B and C" are true.

[0083] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single letter designation: "A" for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" for cytosine or deoxycytosine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. While nucleotide sequences herein can be represented in DNA sequence (containing T), the corresponding RNA sequences (i.e., with U in place of T) can be readily determined by one of skill in the art when RNA is referred to.

[0084] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of natural amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" can also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0085] The word "comprising" is used herein to mean that the protein or nucleic acid can be composed of the recited sequence, or can have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.

[0086] “Sequence identity” between two polypeptide sequences or between two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods to assess the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various sequence analysis software known. For example, sequence identity can be assessed by the online alignment tool of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm, using default parameters. Sequence identity can be over the full length of a given sequence.

[0087] “mRNA” or “messenger RNA” are used interchangeably herein to refer to a single-stranded ribonucleic acid molecule that carries genetic information capable of directing protein synthesis within a cell. An mRNA typically comprises a 5’ UTR, a protein-coding sequence (CDS), and a “3’ UTR”. An mRNA can also comprise a poly(A) sequence at the 3’ end and a “5’ cap”. An mRNA is generally linear, however the term can also encompass covalently closed circular RNA molecules.

[0088] As used herein, “5’ UTR” generally refers to the sequence of an mRNA molecule between the 5’ end and the translation initiation codon, which is capable of recruiting the ribosomal complex and initiating translation of the mRNA. The 5’ UTR regulates processes such as post-transcriptional modifications, formation and stability of the translation initiation complex, by interacting with transcription factors, ribosomes, and other transcriptional regulatory proteins. The term “5’ UTR” also encompasses ribosome entry site (IRES) sequences, in particular for circular RNA molecules.

[0089] As used herein, “3’ UTR” refers to the sequence of an mRNA between the stop codon of the polypeptide-coding sequence and the poly(A) sequence. The 3’ UTR can regulate translation of the mRNA by interacting with mRNA-binding proteins, miRNAs, and the like. Sequence and structural features of the 3’ UTR can influence the stability of the mRNA, scanning of the ribosome, and formation of the translation termination complex, thereby influencing the expression level of the protein.

[0090] A poly(A) sequence typically comprises a plurality of adenine nucleotides. The addition of a poly(A) sequence aids in the stability and transport of the mRNA, preventing its degradation, and plays an important role in post-transcriptional modification processes. The poly(A) sequence can be a continuous chain of pure adenine nucleotides, but can also be a variant comprising non-adenine nucleotides, as long as it is functionally equivalent to a conventional poly(A) sequence, i.e. is capable of providing similar biological functions as a natural poly(A) sequence, such as affecting the stability, translation efficiency or ribosome binding of the mRNA. Known poly(A) sequences are, for example, the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence. These variants can differ in the nucleotide composition, but are functionally identified as equivalent to a conventional poly(A) sequence.

[0091] As used herein, a "5' cap" for an RNA includes the 5' cap structure present on a natural mRNA as well as analogs thereof. The 5' cap structure on a natural mRNA refers to a methylated guanylate linked via a pyrophosphate to the 5' terminal nucleotide of the RNA, forming a 5',5'-triphosphate linkage. There are generally three types of 5' caps (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), referred to as Cap0, Cap1 and Cap2, respectively. Cap0 refers to the ribose of the terminal nucleotide being unmethylated, Cap1 refers to the ribose of the terminal one nucleotide being methylated, and Cap2 refers to the ribose of the terminal two nucleotides being methylated.

[0092] Methods for capping mRNA molecules are known in the art. The 5' cap structure of the mRNA molecule can be added using an enzymatic reaction after the mRNA molecule has been obtained by chemical synthesis or in vitro transcription (e.g. by using a commercial kit comprising a vaccinia capping enzyme and a mRNA cap structure 2'-0-methyltransferase). However, it is also possible to produce mRNA with a cap structure by directly incorporating a nucleotide analogue with a cap structure as the first nucleotide into the transcript during in vitro transcription.

[0093] "Lipid nanoparticle" or "LNP" refers to a particle comprising a lipid component, having a nanoscale size.

[0094] As used herein, "treatment" of an individual having a disease or condition means that the individual's symptoms are partially or totally alleviated, or remain unchanged following the treatment. Thus, treatment includes prevention, therapy and / or cure. Prevention refers to preventing the underlying disease and / or preventing the symptoms from worsening or the disease from developing. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided herein and any composition provided herein.

[0095] As used herein, "therapeutic effect" means an effect resulting from the treatment of an individual that alters, usually improves or ameliorates symptoms of a disease or condition, or cures a disease or condition.

[0096] As used herein, "therapeutically effective amount" or "therapeutically effective dose" means the amount of a substance, compound, material, or composition comprising a compound that, after administration to a subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, retard or partially retard symptoms of a disease or condition.

[0097] As used herein, "prophylactically effective amount" or "prophylactically effective dose" means the amount of a substance, compound, material, or composition comprising a compound that, upon administration to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset of a disease or symptoms, reducing the likelihood of the onset of a disease or symptoms. A fully prophylactically effective dose does not necessarily occur with administration of one dose, and can only occur after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0098] As used herein, the term "subject" refers to a mammal, e.g., a human.

[0099] II. Nucleic acid molecules encoding at least one HPV antigen and at least one immune costimulatory factor

[0100] In one aspect, the present application provides a nucleic acid molecule comprising an open reading frame (ORF) comprising: a nucleotide sequence encoding an HPV antigen; and a nucleotide sequence encoding an immune costimulatory factor.

[0101] In some embodiments, the HPV antigen comprises at least one HPV immunogenic polypeptide from at least one HPV serotype. The HPV antigen can comprise any HPV immunogenic polypeptide from any HPV serotype or combinations thereof.

[0102] In the present application, the HPV can be any serotype HPV. In some embodiments, the HPV is a low-risk HPV, e.g., types 1, 2, 3, 4, 7, 10, 12, 15, 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, etc. In some embodiments, the HPV is a high-risk HPV, e.g., types 5, 8, 14, 17, 20, 36, 38, 16, 18, 30, 31, 33, 35, 39, etc. In some preferred embodiments, the HPV is a HPV of serotype 16 and / or serotype 18.

[0103] In the present application, the HPV immunogenic polypeptide can be selected from an E1, E2, E3, E4, E5, E6, E7, LI, or L2 polypeptide of HPV or an immunogenic variant thereof. In some preferred embodiments, the HPV immunogenic polypeptide is selected from an E6, E7, L2 polypeptide or an immunogenic variant thereof. In some more preferred embodiments, the HPV immunogenic polypeptide is selected from an E6, E7 polypeptide or an immunogenic variant thereof.

[0104] In some embodiments, the HPV antigen can comprise one, two, three, four or more HPV immunogenic polypeptides from one, two, three, four or more HPV serotypes. In some embodiments, the HPV antigen can comprise one HPV immunogenic polypeptide from one HPV serotype. In some embodiments, the HPV antigen can comprise two HPV immunogenic polypeptides from one HPV serotype. In some embodiments, the HPV antigen can comprise two HPV immunogenic polypeptides from two HPV serotypes.

[0105] In some embodiments, the HPV antigen comprises an E6 polypeptide and an E7 polypeptide from HPV serotype 16. In some embodiments, the HPV antigen comprises an E6 polypeptide and an E7 polypeptide from HPV serotype 18. In some embodiments, the HPV antigen comprises an E6 polypeptide and an E7 polypeptide from HPV serotype 16, and an E6 polypeptide and an E7 polypeptide from HPV serotype 18.

[0106] In some embodiments, the E6 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 41.

[0107] In some embodiments, the E6 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 67 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 67.

[0108] In some embodiments, the E7 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 42 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 42. In some embodiments, the E7 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 43.

[0109] In some embodiments, the E7 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 68 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 68.

[0110] In some embodiments, the E6 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 43.

[0111] In some embodiments, the E6 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 69 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 69.

[0112] In some embodiments, the E7 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 44 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 44.

[0113] In some embodiments, the E7 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 70 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 70.

[0114] In some embodiments, the HPV antigen can further comprise one or more epitope peptides derived from an L2 polypeptide. For example, an epitope peptide derived from an L2 polypeptide from HPV serotype 16 can comprise the amino acid sequence of SEQ ID NO: 45 or 46.

[0115] In some embodiments, the HPV antigen can further comprise a signal peptide, thereby resulting in secreted expression of the HPV immunogenic polypeptide. An exemplary signal peptide can comprise the amino acid sequence of SEQ ID NO: 48.

[0116] In some embodiments, the HPV antigen can further comprise an amino acid sequence that disrupts host immune tolerance and / or an amino acid sequence that enhances antigen processing and / or presentation. The amino acid sequence that disrupts host immune tolerance is, for example, P2P16 from tetanus endotoxin, the amino acid sequence of which is set forth in SEQ ID NO: 56. The amino acid sequence that enhances antigen processing and / or presentation is, for example, the transmembrane and cytoplasmic domain of MHC class I molecule (MITD), the amino acid sequence of which is set forth in SEQ ID NO: 55. However, in some preferred embodiments, the HPV antigen does not comprise an amino acid sequence that disrupts host immune tolerance and / or an amino acid sequence that enhances antigen processing and / or presentation.

[0117] In some embodiments, the different elements / polypeptides, amino acid sequences comprised in the HPV antigen are fused directly or via a peptide linker. The peptide linker can be a flexible peptide linker or a rigid peptide linker, preferably a flexible peptide linker. An exemplary peptide linker comprises the sequence GnS, n being an integer from 1 to 5, for example n being 1 or 3.

[0118] The "immune co-stimulatory factor" as used herein refers to a molecule that, when co-expressed with an antigen, enhances the immune response of the host to the antigen. Exemplary immune co-stimulatory factors include, but are not limited to, Flt3Lecd, GM-CSF, OX40L, HMGB1 or HSP70p.

[0119] In some embodiments, the Flt3Lecd comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the HMGB1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the HSP70p comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the OX40L comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the GM-CSF comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the GM-CSF comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 51.

[0120] In some preferred embodiments of the application, the immune co-stimulatory factor is OX40L or GM-CSF, most preferably GM-CSF.

[0121] In some embodiments, the immune co-stimulatory factor can further comprise a signal peptide, thereby resulting in secreted expression of the immune co-stimulatory factor. An exemplary signal peptide can comprise the amino acid sequence of SEQ ID NO: 48.

[0122] In some preferred embodiments, the ORF further comprises a nucleotide sequence encoding a self-cleaving peptide located between the nucleotide sequence encoding the HPV antigen and the nucleotide sequence encoding the immune co-stimulatory factor.

[0123] As used herein, "self-cleaving peptide" means a peptide that can achieve self-cleavage within a cell. The presence of the self-cleaving peptide can allow the HPV antigen and the immune co-stimulatory molecule to be translated to form separate polypeptides rather than a fusion protein.

[0124] For example, the self-cleaving peptide can comprise a protease recognition site, so as to be recognized and specifically cleaved by a protease within the cell. Alternatively and preferably, the self-cleaving peptide can be a 2A polypeptide. 2A polypeptides are a class of short peptides from viruses, whose self-cleavage occurs during translation. Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea asigna virus, E2A from equine rhinitis A virus, and F2A from foot-and-mouth disease virus. A variety of functional variants of these 2A polypeptides are also known in the art, which can also be used in the present application. In some preferred embodiments, the 2A polypeptide is a T2A polypeptide. An exemplary T2A polypeptide amino acid sequence is set forth in SEQ ID NO: 64. In some preferred embodiments, the 2A polypeptide is a P2A polypeptide. An exemplary P2A polypeptide amino acid sequence is set forth in SEQ ID NO: 65.

[0125] In some embodiments, the ORF comprises, in the 5' to 3' direction:

[0126] i) the nucleotide sequence encoding the HPV antigen and the nucleotide sequence encoding the immune co-stimulatory factor; or

[0127] ii) the nucleotide sequence encoding the HPV antigen, the nucleotide sequence encoding the self-cleaving peptide, and the nucleotide sequence encoding the immune co-stimulatory factor.

[0128] In some embodiments, the ORF encodes an amino acid sequence comprising one of SEQ ID NOs: 3-6 and 8-18 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to one of SEQ ID NOs: 3-6 and 8-18.

[0129] In some embodiments, the ORF encodes an amino acid sequence comprising the following structure: SP-E6 16 -E7 16 -E6 18 -E7 18 -2A-GMCSF, wherein SP is a signal peptide, E6 16 and E716 These are E6 and E7 peptides from HPV serotype 16, respectively. 18 and E7 18 The E6 and E7 peptides are derived from HPV serotype 18, respectively; 2A is a 2A peptide such as T2A; and GMCSF is human or mouse GM-CSF. In some preferred embodiments, the ORF encodes the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:11.

[0130] In some embodiments, the ORF encodes an amino acid sequence comprising one of SEQ ID NO:23-26 and 28-38, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with one of SEQ ID NO:23-26 and 28-38. In some preferred embodiments, the ORF comprises the nucleotide sequence shown in SEQ ID NO:31, or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:31.

[0131] In some embodiments, the nucleic acid molecule is an mRNA molecule. In some embodiments, the mRNA molecule also contains a 5' UTR and / or a 3' UTR.

[0132] Various available 5'UTRs and / or 3'UTRs are known in the art. Those skilled in the art can determine the 5'UTR and / or 3'UTRs suitable for this invention.

[0133] In some embodiments, the 5'UTR comprises the nucleotide sequence shown in SEQ ID NO:57 or 59. In some embodiments, the 3'UTR comprises the nucleotide sequence shown in SEQ ID NO:58 or 60. In some embodiments, the 5'UTR comprises the nucleotide sequence shown in SEQ ID NO:57, and the 3'UTR comprises the nucleotide sequence shown in SEQ ID NO:58. In some embodiments, the 5'UTR comprises the nucleotide sequence shown in SEQ ID NO:59, and the 3'UTR comprises the nucleotide sequence shown in SEQ ID NO:60.

[0134] In some implementations, the mRNA molecule also includes a poly(A) sequence.

[0135] In some embodiments of the application, the poly(A) sequence comprises about 20 to about 500 (e.g., contiguous) adenine nucleotides (A), e.g., about 25, about 50, about 100, about 150, about 175, about 200, about 300, about 400, about 500 (e.g., contiguous) adenine nucleotides (A). In some preferred embodiments, the poly(A) sequence comprises 120 (e.g., contiguous) adenine nucleotides (A). The addition of a poly(A) sequence aids in the stability and transport of the mRNA, preventing its degradation, and plays an important role in the post-transcriptional modification process. The poly(A) sequence can be a continuous chain of pure adenine nucleotides, but can also be a variant comprising nucleotides other than adenine, as long as it is functionally equivalent to a conventional poly(A) sequence, i.e., is capable of providing similar biological functions as a natural poly(A) sequence, such as affecting the stability, translation efficiency or ribosome binding of the mRNA. Known poly(A) sequences are, e.g., the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence. These variants can differ in the nucleotide composition, but are functionally recognized as equivalent to a conventional poly(A) sequence. In some particular embodiments, the poly(A) sequence comprises the nucleotide sequence set forth in SEQ ID NO: 61.

[0136] In some particularly preferred embodiments, the mRNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 66 or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 66.

[0137] In some embodiments, the mRNA molecule further comprises a 5’ cap structure. In some embodiments of the application, the 5’ cap structure is a Cap 1 cap structure.

[0138] In some embodiments, the mRNA molecule of the application can further comprise at least one nucleotide modification. The at least one nucleotide modification includes, but is not limited to, a cytidine modification, a uridine modification, or an adenosine modification. In some embodiments, the at least one nucleotide modification includes, but is not limited to, 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methoxyuridine (5moU).

[0139] In some embodiments, the mRNA is chemically synthesized. In some embodiments, the mRNA molecule is obtained by in vitro transcription.

[0140] In addition, the mRNA molecules of the present application have a certain stability, and can tolerate the insertion of certain additional sequences (such as microRNA binding sites) without affecting their ability to be translated, nor affecting the stability of the mRNA molecule. In some embodiments, additional sequences (such as microRNA binding sites) can be inserted into the 3' UTR. The microRNA binding sites include the full-length reverse complement of a microRNA (exemplary length can be 19-25 nt) or the reverse complement of the seed sequence thereof (exemplary length can be 7-8 nt).

[0141] In one aspect, the present application provides a nucleic acid vector comprising a coding sequence of a nucleic acid molecule, such as an mRNA molecule, of the present application. In some embodiments, the nucleic acid vector is used to generate a nucleic acid molecule, such as an mRNA molecule, of the present application.

[0142] As used herein, "vector" refers to a piece of DNA extracted from a virus, plasmid, or cell of a higher organism into which a foreign DNA segment can be or has been inserted for cloning and / or expression purposes. In certain embodiments, a vector can be stably maintained in an organism. A vector can comprise, for example, an origin of replication, a selectable marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.

[0143] In some embodiments, the nucleic acid vector further comprises an RNA polymerase promoter sequence operably linked to the coding sequence of the nucleic acid molecule, such as an mRNA molecule. The operably linked promoter allows for the in vivo and / or in vitro transcription of the RNA molecule. The promoter is, for example, a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, a SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0144] In some embodiments, the nucleic acid vector is a plasmid vector. In some embodiments, the nucleic acid vector comprises a restriction endonuclease site, such as a type IIS restriction endonuclease site, flanking the 3' of the coding sequence of the nucleic acid molecule, such as an mRNA molecule. Suitable restriction endonucleases include, but are not limited to, BsmBI, BsaI, SapI, and the like. The restriction endonuclease site can be used to linearize the nucleic acid vector for in vitro transcription.

[0145] Methods for in vitro transcription of mRNA molecules from nucleic acid vectors are known in the art, for example, in vitro transcription can be performed using commercial kits.

[0146] III. LNPs, immunogenic compositions, disease prevention and / or treatment

[0147] The present application also provides a lipid nanoparticle (LNP) comprising a nucleic acid molecule of the present application, said nucleic acid molecule being comprised in a lipid. In some embodiments, said nucleic acid molecule is encapsulated in a lipid. The lipid can for example be MTS002, which comprises the following formula:

[0148] Various methods and materials for constructing LNPs comprising a particular nucleic acid molecule are known in the art and can be applied to the present application.

[0149] The present application also provides an immunogenic composition comprising a nucleic acid molecule of the present application or an LNP of the present application, and a pharmaceutically acceptable carrier. The immunogenic composition is for use in preventing and / or treating an HPV infection and / or an HPV infection related pathology in a subject.

[0150] The present application also provides the use of a nucleic acid molecule of the present application or an LNP of the present application or an immunogenic composition of the present application for the manufacture of a vaccine for preventing and / or treating an HPV infection and / or an HPV infection related pathology in a subject.

[0151] The vaccine described herein can be a prophylactic vaccine or a therapeutic vaccine.

[0152] In the present application, the HPV infection can be an infection with any HPV serotype. In some embodiments, the HPV is a low-risk HPV, such as type 1, 2, 3, 4, 7, 10, 12, 15, 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, etc. In some embodiments, the HPV is a high-risk HPV, such as type 5, 8, 14, 17, 20, 36, 38, 16, 18, 30, 31, 33, 35, 39, etc. In some embodiments, the HPV infection is an infection with a single HPV serotype. In some embodiments, the HPV infection is a mixed infection with two or more different HPV serotypes. In some preferred embodiments, the HPV infection is an infection with HPV 16 and / or HPV 18.

[0153] HPV infection related pathologies include, but are not limited to, cutaneous warts, such as common warts, plantar warts, flat warts, butcher’s warts or condyloma acuminata; HPV-positive tumors such as HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, head and neck squamous cell carcinoma, prostate cancer or bladder cancer, etc. In some preferred embodiments, the HPV infection related pathology is a HPV-positive tumor such as HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, head and neck squamous cell carcinoma, prostate cancer or bladder cancer, etc.

[0154] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical (e.g., by injection or infusion) administration.

[0155] Actual dosage levels of the active ingredients in the immunogenic compositions of the present application can be varied to obtain an amount effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level depends upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present application employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

[0156] An "effective amount" of a nucleic acid molecule of the present application or an LNP of the present application preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability because of the disease affliction. For example, for the treatment of HPV-positive tumors, an "effective amount" of a nucleic acid molecule of the present application or an LNP of the present application preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to a subject who has not received treatment. The ability to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, the ability to inhibit cell growth can also be evaluated, such inhibition can be determined in vitro by assays known to the skilled artisan. An effective amount of a nucleic acid molecule of the present application or an LNP of the present application is capable of reducing tumor size, or otherwise ameliorating the symptoms of the subject, such as preventing and / or treating metastasis or recurrence. Such amounts can be determined by one of skill in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

[0157] The nucleic acid molecule of the application or the LNP of the application or the immunogenic composition of the application can be administered by one or more routes of administration using one or more methods known in the art. It will be appreciated by one skilled in the art that the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration of the nucleic acid molecule of the application or the LNP of the application include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., injection or infusion. As used herein, the phrase“parenteral administration” means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, infracapsular, subarachnoid, intraspinal, epidural, and intrastemal injection and infusion.

[0158] The present application also provides a method of preventing and / or treating an HPV infection and / or an HPV infection related pathology in a subject, said method comprising administering to said subject an effective amount of the nucleic acid molecule of the application or the LNP of the application or the immunogenic composition of the application.

[0159] In some embodiments, in particular for the prevention and / or treatment of HPV positive tumors, the nucleic acid molecule of the application, the LNP of the application, the immunogenic composition of the application, or the method of the application can be used in combination with a chemotherapeutic agent, an immune checkpoint inhibitor, an antibody targeting a tumor specific antigen or radiotherapy.

[0160] The chemotherapeutic agent, immune checkpoint inhibitor, or antibody targeting a tumor antigen that can be used in combination with the nucleic acid molecule of the present application, the LNP of the present application, or the immunogenic composition of the present application is not particularly limited. Examples of the chemotherapeutic agent, immune checkpoint inhibitor, and antibody targeting a tumor antigen include, but are not limited to, ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enocitabine, capecitabine, carmofur, cladrabine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, doxifluridine, nelarabine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate, irinotecan, etoposide, eribulin, sobuzoxane, docetaxel, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine, actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, zinostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, mitoxantrone, oxaliplatin, carboplatin, cisplatin, nedaplatin, anastrozole, exemestane, ethinyl estradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, bicalutamide, toremifene, flutamide, prednisolone, phosphocin, mitotane, methyltestosterone, leuprolide, letrozole, megestrol, ibritumomab tiuxetan, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, tretinoin, panitumumab, bevacizumab, bortezomib, lapatinib, atezolizumab, pembrolizumab, tislelizumab, camrelizumab, sugemalimab, and nivolumab, and the like.

[0161] The nucleic acid molecule of the present application, the LNP of the present application, or the immunogenic composition of the present application and the chemotherapeutic agent, immune checkpoint inhibitor, or antibody targeting a tumor antigen can be all administered at once or administered separately. When administered separately (in the case of different administration schedules from each other), they can be administered consecutively without interruption or at predetermined intervals.

[0162] The combined dose of the nucleic acid molecule of the present application, the LNP of the present application, or the immunogenic composition of the present application and the chemotherapeutic agent, immune checkpoint inhibitor, or antibody targeting a tumor antigen is not particularly limited. As described above, the dose of the nucleic acid molecule of the present application can be determined by referring to the dose when the nucleic acid molecule is used alone. The chemotherapeutic agent, immune checkpoint inhibitor, and antibody targeting a tumor antigen can be used according to the dose indicated for each drug or can be reduced (in consideration of the combined effect with the nucleic acid molecule of the present application).

[0163] The immune checkpoint inhibitor described herein in the context of the present application can be a PD1 signaling pathway inhibitor. For example, the PD1 signaling pathway inhibitor can be selected from the group consisting of a PD1 antibody, a PDL1 antibody, a PDL2 antibody, or a combination thereof; preferably a PD1 antibody.

[0164] The nucleic acid molecule of the present application, the LNP of the present application, or the immunogenic composition of the present application can also be combined with radiotherapy, for example comprising the administration of ionizing radiation to the subject, earlier in the course of, during and / or later than the administration of the nucleic acid molecule, LNP or immunogenic composition of the present application.

[0165] The present application also provides a combination comprising 1) the nucleic acid molecule of the present application, the LNP of the present application, or the immunogenic composition of the present application; and 2) a chemotherapeutic agent, an immune checkpoint inhibitor or an antibody targeting a tumor antigen.

[0166] Thus, in the method of the present application for preventing and / or treating an HPV infection and / or an HPV infection related pathology in a subject, it can also comprise administering to said subject an effective amount of a chemotherapeutic agent, an immune checkpoint inhibitor or an antibody targeting a tumor antigen or radiotherapy.

[0167] The present application also provides a kit for use in the method of the present application for preventing and / or treating an HPV infection and / or an HPV infection related pathology, said kit comprising the nucleic acid molecule of the present application, the LNP of the present application, or the immunogenic composition of the present application, and optionally a chemotherapeutic agent, an immune checkpoint inhibitor or an antibody targeting a tumor antigen. Examples

[0168] The present application can be further understood by reference to the specific examples described herein, which are intended to be purely exemplary of the application and are not intended to limit the scope of the application. Obviously, many modifications and variations of the present application are possible in light of the above teachings, the scope of the application is not limited to the examples described herein. It is therefore to be understood that within the scope of the appended claims, various modifications can be made and equivalents can be substituted for elements thereof.

[0169] Synthesis of lipid molecules

[0170] Synthesis of compound MTS002:

[0171] To a stirred solution of methyl isobutyrate (4.4 g) in tetrahydrofuran (100.0 mL) was added lithium diisopropylamide (43.0 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 30 min, to the above solution was added 1,5-dibromo-pentane (20.0 g) and the resulting mixture was stirred at room temperature for 5 h. The reaction was quenched with saturated ammonium chloride solution (1.0 mL), diluted with water and extracted with dichloromethane. The combined organic layers were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 10 g (46%) of 1-4 as a light yellow oil;

[0172] To a stirred solution of 1-4 (10.0 g) in tetrahydrofuran (20.0 mL) was added borane-tetrahydrofuran complex solution (100.0 mL) at 0 °C. The resulting solution was stirred at 75 °C for 3 h. The mixture was cooled to room temperature, diluted with water and extracted with dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 8 g (90%) of 1-5 as a colorless oil;

[0173] To a stirred solution of 1-4 (10.0 g) in tetrahydrofuran (20.0 mL) was added borane-tetrahydrofuran complex solution (100.0 mL) at 0 °C. The resulting solution was stirred at 75 °C for 3 h. The mixture was cooled to room temperature, diluted with water and extracted with dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 8 g (90%) of 1-5 as a colorless oil;

[0174] To a stirred solution of 1-4 (10.0 g) in tetrahydrofuran (20.0 mL) was added borane-tetrahydrofuran complex solution (100.0 mL) at 0 °C. The resulting solution was stirred at 75 °C for 3 h. The mixture was cooled to room temperature, diluted with water and extracted with dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 8 g (90%) of 1-5 as a colorless oil;

[0175] To a solution of 2-2 (1 g) and ethanolamine (1.56 g) in acetonitrile (15 mL), potassium carbonate (1.06 g) was added under nitrogen protection. The reaction was stirred at 50 °C, after the reaction was completed, it was cooled to room temperature, diluted with water, then extracted with dichloromethane, the organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, the organic phase was collected by filtration, the organic solvent was removed to obtain a crude product, which was purified by silica gel column to obtain compound 2-3 (400 mg, 42%) as yellow oil;

[0176] To a solution of 2-3 (150 mg) and 2-1 (234.2 mg) in N,N-dimethylformamide (2 mL), potassium carbonate (165.6 mg) and sodium iodide (150.0 mg) were added under nitrogen protection. The reaction was stirred at 70 °C, after the reaction was completed, it was cooled to room temperature, diluted with water, then extracted with dichloromethane, the organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, the organic phase was collected by filtration, the organic solvent was removed to obtain a crude product, which was purified by high performance liquid chromatography (column: Xselect CSH F-Phenyl OBD column 19x250 mm, 5 μm; A phase: acetonitrile / water (10 mmol / L ammonium bicarbonate + 0.05% ammonia water), B phase: isopropanol / acetonitrile; flow rate: 20 mL / min; gradient: 75% B to 95% B, 9 min) to obtain compound MTS002 (99 mg, 31%) as yellow oil; 1H NMR (300 MHz, CD3OD) δ: 0.80-0.90 (m, 15H), 1.29-1.34 (m, 54H), 1.52-1.64 (m, 12H), 2.28-2.35 (m, 4H), 2.52-2.57 (m, 4H), 2.63-2.68 (m, 2H), 3.60-3.64 (m, 2H), 3.80 (s, 2H), 4.87-4.89 (m, 1H); MS m / z [M+H]+(ESI): 780.80.

[0177] Pharmacological Examples

[0178] Example 1, Preparation and expression detection of HPV mRNA vaccine

[0179] Flt3Lecd, GMCSF, OX40L, HMGB1 and HSP70p were selected as co-stimulatory factors for HPV mRNA vaccines. Using the artificial intelligence driven RNA design platform (AiRNA) built by the applicant, mRNA sequences were designed for different structures of HPV mRNA vaccines. The specific structure information of the vaccine mRNA sequence is shown in Tables 1-3. The nucleic acid fragments containing T7 promoter, 5'UTR, vaccine ORF coding sequence, 3'UTR, polyA (SEQ ID NO: 61) and IIS type restriction endonuclease cleavage site were synthesized in vitro and cloned into the in vitro transcription vector (pIVTRup, Addgene). The 5'UTR and 3'UTR of M20 1-4 and M21 1-4 were selected from SEQ ID NO. 57 and SEQ ID NO. 58, respectively; the 5'UTR and 3'UTR of P007, P008, P014, M20, M21, P019, P022, P025, P029 and P030 were selected from SEQ ID NO. 59 and SEQ ID NO. 60, respectively. The obtained vectors were linearized and used for in vitro transcription to produce mRNA molecules using T7-RNA polymerase, and 5'-cap structure was added by co-transcription capping to produce mRNA molecules with Cap1 structure. The obtained mRNA molecules were purified and resuspended in water, and stored at -80°C after determination of the concentration.

[0180] The mouse dendritic cell DC2.4 cells were plated at 2.5x10 5 The 6-well plates were plated at a density of 6x105 cells / well, and after overnight culture, each group of vaccine mRNA (2 μg) was transfected into cells per well using Lipofectamine MessengerMAX (Thermofisher). After 24 h of continuous culture, the cells were lysed and Western blotting was performed to detect the expression of different HPV mRNA sequences using HPV16 E7 antibody (Cell Signaling Technology). BNT113E7 was the positive control group (Benchmark, BMK, the sequence was from WO2022 / 008519, the antigen structure was HPV16 SP-E7-Tetanus P2P16-MITD, and the nucleotide sequence of the mRNA is shown in SEQ ID NO: 40).

[0181] The results showed that the mRNA of each vaccine group in Table 1 and the mRNA of the positive control group were well expressed in the cells (Figure 1).

[0182] Table 1

[0183] Example 2, in vivo immunogenicity detection of HPV mRNA vaccine

[0184] HPV mRNA vaccine in the form of LNP is delivered into the animal body by intramuscular injection, which can significantly increase the number of HPV antigen-specific CD4+ and / or CD8+ T lymphocytes in the animal body. The in vivo immunogenicity of mRNA drugs is evaluated by detecting the number of T cells that respond to HPV mRNA vaccine and specifically secrete IFNγ.

[0185] In this experiment, LNP-mRNA preparation was prepared using ionizable lipid SM102 (Xiamen Senobon) classic formula (SM102:DSPC:cholesterol:DMG-PEG2000=50:10:38.5:1.5). Six to eight-week-old C57BL / 6N female mice were selected for grouping, and different HPV mRNA vaccine groups were set up, with empty LNP as the negative control group. The mice were immunized by intramuscular injection (i.m.) of 1 μg / mouse of mRNA drugs at QWx2 intervals and frequencies. Seven days after the second immunization, the mouse spleen was taken, ground, red blood cells were lysed, and the spleen single cell suspension was obtained after filtration.

[0186] The mouse spleen single cell suspension was diluted to 0.5x10 5 The ELISpot 96-well plate was plated at a density of 0.5x10

[0187] The results show (Figure 2) that almost no IFNy+T cells were detected in the empty LNP group, a strong T cell immune response was detected in the PMA+Ionomycin positive group, and the P014 (GMCSF) and P022 (OX40L) groups had a stronger T cell immune response, indicating that GMCSF and OX40L as costimulatory factors can significantly improve the immunogenicity of the HPV mRNA vaccine.

[0188] Example 3, Evaluation of Tumor Inhibition Synergistic Activity of Costimulatory Factors

[0189] This example uses a subcutaneous transplanted tumor mouse model of HPV16 positive mouse lung cancer cell line TC-1 to evaluate mRNA vaccines corresponding to different protein structures. The dosing regimen is: the dose is set at 0.05, 0.15, 0.5 pg / mouse; the administration method is i.m.; the administration window is ~6mm 3 The drug was administered on the day of small tumor implantation; the administration interval and frequency were Q5Dx2 (once every 5 days for a total of 2 times). The specific experimental operation is as follows:

[0190] 3.1 Experimental materials

[0191] 3.1.1 TC-1 mouse tumor model

[0192] The subcutaneous transplanted tumor model was modeled by tumor transmission, and the large tumor mass taken from the passaged mice was cut into small round pieces with a particle size of about 2mm using a scalpel, and the small tumor mass was buried in the right dorsal axillary of the mice using a push needle.

[0193] 3.1.2 Drug

[0194] LNP formulation: SM102:DSPC:cholesterol:DMG-PEG2000=50:10:38.5:1.5

[0195] mRNA: The specific structure information of the experimental groups is shown in Table 2; the mRNA nucleotide sequence is shown in SEQ ID NO:40; Benchmark is the positive control.

[0196] Table 2

[0197] Solvent: Tris + 10% sucrose

[0198] 3.1.3 Experimental animals

[0199] Animal species: C57BL / 6N mice

[0200] Animal gender: female

[0201] Animal source: Vivotecnia

[0202] Weeks of age: 6 weeks of age

[0203] 3.2 Experimental method

[0204] 3.2.1 Administration

[0205] Administration time point: administration on the day of tumor implantation, tumor volume ~ 6mm 3

[0206] Administration method: i.m., right hind limb rectus femoris

[0207] Administration dose: 0.05, 0.15, 0.5 μg / 50 μL / mouse / time

[0208] Administration frequency: Q5Dx2

[0209] 3.2.2 Experimental grouping

[0210] Blank control group: solvent group (Neg)

[0211] Negative control group: SM102-Empty (lipid molecule content is the same as the high dose group)

[0212] Positive control group: Benchmark drug group (BMK)

[0213] Experimental group: 6 kinds of mRNA are set to high, medium and low doses (0.05, 0.15 and 0.5 μg / 50 μl / mouse / time)

[0214] 5 mice in each group

[0215] 3.2.3 Evaluation index

[0216] Body weight (2-3 times a week after initial administration); tumor volume (2-3 times a week after initial administration); experimental endpoint: significant difference can be seen between groups or the experiment reaches the humane endpoint (mouse weight reduction of more than 20% or / and transplanted tumor volume more than 2000mm 3 )

[0217] 3.3 Experimental results

[0218] There was no difference in mouse weight between groups. Tumor volume data showed that the high dose of positive control drug significantly inhibited tumor growth, while the medium and low dose groups did not show obvious efficacy; the high and medium doses of the five METiS-mRNAs showed significant pharmacodynamic activity. In the low dose group, all mice models CR after P014 administration, P022 did not show pharmacodynamic activity, P019, P007 and P025 showed moderate antitumor activity (Figure 3).

[0219] 3.4 Experimental summary

[0220] The 6 mRNAs were ranked according to the efficacy data in this round of experiments as follows: P014 > P019 > P025 ≈ P022 ≈ P007 > positive drug. This shows that the GM-CSF element has the optimal efficacy enhancement activity for the HPV mRNA vaccine.

[0221] Example 4, Preparation and Expression Detection of HPV mRNA Vaccine Containing Co-stimulatory Factor GM-CSF

[0222] The HPV mRNA vaccine containing the co-stimulatory factor GM-CSF was prepared according to the method of Example 1, and the specific structural information of the vaccine mRNA sequence is shown in Table 3, and the ORF coding sequence is SEQ ID NO: 29-38.

[0223] Table 3

[0224] HPV antigen expression detection: HEK293T cells and DC2.4 cells were plated at a density of 8x10 5 and 5x10 5 in 6-well plates, respectively, and cultured overnight. Then 2 μg mRNA was transfected into each well of cells using Lipofectamine MessengerMAX (Thermofisher), and the cells were cultured for another 24 h. After that, the cells were lysed, and Western blotting was performed to detect the expression of different HPV mRNA sequences using HPV18E7 antibody (NOVUS). Solvent blank and GFP were used as negative control groups.

[0225] Co-stimulatory factor (GMCSF) expression detection: HEK293T cells were plated at a density of 8x10 5 in 6-well plates, and cultured overnight. Then 2 μg mRNA was transfected into each well of cells using Lipofectamine MessengerMAX (Thermofisher), and the cells were cultured for another 24 h. After that, the cell culture supernatant was collected, and the content of GM-CSF in the supernatant was detected using ELISA (Abeam).

[0226] Results:

[0227] 1. Western blotting results showed that HPV18 E7 expression was detected in HEK293T and DC2.4 cells transfected with different HPV mRNA vaccines, and the expression level of HPV antigen located upstream of T2A was higher (Figures 4 and 5).

[0228] 2、ELISA results showed that the hGM-CSF content in the supernatant of MTS-M20, MTS-M20-1 and MTS-M20-3 groups was significantly higher than that of MTS-M20-2 and MTS-M20-4 groups; the mGM-CSF content in the supernatant of transfected MTS-M21, MTS-M21-1 and MTS-M21-3 cells was significantly higher than that of MTS-M21-2 and MTS-M21-4 groups (Figure 6), which indicated that the expression level of GM-CSF located upstream of T2A was higher.

[0229] Example 5, Evaluation of the Anti-tumor Activity of HPV mRNA Vaccines Containing the Costimulatory Factor GM-CSF

[0230] This example uses a subcutaneous transplanted tumor mouse model of HPV16 positive mouse lung cancer cell line TC-1 to evaluate HPV mRNA vaccines with different structures containing the costimulatory factor GM-CSF. The administration scheme is: the administration dose is set to 0.05, 0.15, 0.5 μg / 50 μl / mouse / time; the administration mode is i.m.; the administration window is ~ 6 mm 3 The small tumor pieces were administered on the day of implantation; the administration interval and frequency were Q5Dx2.

[0231] 5.1 Experimental materials

[0232] 5.1.1 TC-1 mouse tumor model

[0233] The subcutaneous transplanted tumor model was modeled by tumor transmission, and the large tumor pieces taken from the passaged mice were cut into nearly circular small pieces with a particle size of about 2 mm using a scalpel, and the small tumor pieces were implanted in the right dorsal axillary of the mice using a push needle.

[0234] 5.1.2 Drug

[0235] LNP formula: MTS002-23 (MTS002: DOPE: cholesterol: DMG-PEG2000 = 40: 15: 43.5: 1.5)

[0236] mRNA: See Table 4 for specific structural information

[0237] Table 4

[0238] Solvent: Tris + 10% sucrose

[0239] 5.1.3 Experimental animals

[0240] Animal species: C57BL / 6N mice

[0241] Animal gender: female

[0242] Animal source: Vivotecno

[0243] Weeks of age: 6 weeks of age

[0244] 5.2 Experimental method

[0245] 5.2.1 Administration

[0246] Administration time point: administration on the day of tumor implantation, volume of transplanted tumor ~ 6 mm3

[0247] Administration mode: i.m., right hind limb rectus femoris

[0248] Administration dose: 0.02, 0.1, 0.5 μg / 50 μL / mouse / time

[0249] Administration frequency: Q5D x 2

[0250] 5.2.2 Experimental grouping

[0251] Blank control group: solvent group

[0252] Negative control group (Empty): MTS002-23-Empty (lipid molecule content is the same as the high-dose group)

[0253] Positive control: Benchmark (BMK)

[0254] Experimental group: 5 kinds of mRNA are set to high, medium and low 3 doses

[0255] 5 mice in each group

[0256] 5.2.3 Evaluation index

[0257] Body weight (2-3 times a week after initial administration); tumor volume (2-3 times a week after initial administration)

[0258] 5.3 Experimental results

[0259] There was no difference in the body weight of mice among groups. M21-2 performed best at a high dose, and all mice achieved complete remission (CR) (Figure 7, each line in the figure represents one mouse).

[0260] 5.4 Experimental summary

[0261] M21-2 and M21-4 have better pharmacodynamic activity than M21-1 and M21-3, indicating that HPV mRNA vaccines perform better activity when the GM-CSF coding sequence is at the 3' end; M21-2 and M21-1 have better pharmacodynamic activity than M21-4 and M21-3, indicating that HPV mRNA vaccines without MITD coding sequences have better pharmacodynamic activity.

[0262] Example 6, Vaccine molecules produce pharmacodynamic activity by activating HPV-specific CTLs

[0263] The present embodiment uses a subcutaneous transplanted tumor mouse model of HPV16 positive mouse lung cancer cell line TC-1 to analyze the pharmacological mechanism of the mRNA vaccine molecule, and the administration scheme is: the administration dose is 0.05, 0.15, 0.5 μg / mouse; the administration mode is i.m.; the administration window is ~ 6 mm 3 The small tumor pieces were implanted on the same day, and the administration interval and frequency were Q5Dx2. The specific experimental operation is as follows:

[0264] 6.1 Experimental materials

[0265] 6.1.1 Homologous mouse transplantation model

[0266] The subcutaneous transplanted tumor model was modeled by tumor transmission, and the large tumor pieces taken from the passaged mice were cut into nearly circular small pieces with a particle size of about 2 mm using a scalpel, and the small tumor pieces were implanted in the right dorsal axillary of the mice using a push needle.

[0267] 6.1.2 Drug

[0268] Lipid molecules: MTS002

[0269] LNP formula: MTS002-23 (MTS002: DOPE: cholesterol: DMG-PEG2000 = 40: 15: 43.5: 1.5)

[0270] mRNA: M21-2 (HPV 16-18SP-E6-E7-E6-E7-2A-mGMCSF)

[0271] Solvent: Tris + 10% sucrose

[0272] 6.1.3 Experimental animals

[0273] Animal species: C57BL / 6N mice

[0274] Animal gender: female

[0275] Animal source: Vivotecno

[0276] Weeks of age: 6 weeks of age

[0277] 6.2 Experimental method

[0278] 6.2.1 Administration

[0279] Administration time point: administration on the day of tumor piece implantation, transplanted tumor volume ~ 6 mm 3

[0280] Administration mode: i.m., right hind limb rectus femoris

[0281] Dose: 0.05, 0.15, 0.5 pg / 50 pL / mouse / injection

[0282] Dosing frequency: Q5Dx2

[0283] 6.2.2 Experimental grouping

[0284] Blank control group: solvent group Tris

[0285] Negative control group (Empty): MTS002-23-Empty (same lipid molecule content as high dose group)

[0286] Experimental group: high, medium and low three doses

[0287] Three mice were set in each group

[0288] 6.2.3 Evaluation index

[0289] Body weight (2-3 times per week after the first administration); tumor volume (2-3 times per week after the first administration); experimental endpoint: one week after the second injection, i.e. D12.

[0290] 6.3 Experimental results

[0291] There was no difference in the body weight of mice between groups. The tumor volume data showed that M21-2 molecules exhibited significant dose-dependent pharmacodynamic activity at D12 (Figure 8).

[0292] The immune response to HPV16 E7 was detected by ELISpot method for the spleen cells of each group, and the results showed that with the increase of the dose of M21-2, the number of IFN-γ secreting positive points in response to HPV16 E7 also showed a dose-dependent increase, and there was a significant difference (Figure 9).

[0293] Further staining of spleen cells and tumor tissue cells of each group with Tetramer of HPV16 E7, and identifying specific killer T cells (CTL) by flow cytometry. The results showed that after administration of M21-2, the content of HPV E7 specific CTL was significantly up-regulated, and had a dose-dependent effect (Figure 10).

[0294] 6.4 Experimental summary

[0295] The results of ex vivo ELISpot and flow cytometry detection were consistent, which proved that mRNA vaccine M21-2 successfully activated HPV specific CTL, and the specific CTL had a significant positive correlation with pharmacodynamics (Figure 11). Therefore, the pharmacological mechanism of M21-2 is to activate the production of HPV specific CTL, and then exert specific killing activity.

[0296] Example 7, pharmacodynamic evaluation of HPV16+ large tumor model

[0297] This embodiment uses a subcutaneous xenograft mouse model of HPV16-positive mouse lung cancer cell line TC-1 to evaluate the pharmacological activity of mRNA molecules. The dosing regimen was as follows: dosage was 0.1, 1, and 10 μg / mouse; administration was intramuscular (IM); and the administration window was defined as xenografts with a tumor volume of 100 mm². 3 and 500mm 3 Dosing began at [time]; the dosing interval and frequency were QW×3 (one dose per week, for a total of three times). Specific experimental procedures are as follows:

[0298] 7.1 Experimental Materials

[0299] 7.1.1 Homologous mouse transplantation model

[0300] The subcutaneous xenograft model was created using a tumor-to-tumor transmission method. A large tumor mass taken from a passaged mouse was cut into nearly circular pieces with a diameter of about 2 mm using a scalpel. The small tumor pieces were then implanted into the right back of the mouse near the armpit using a push needle.

[0301] 7.1.2 Drugs

[0302] Lipid molecule: MTS002

[0303] LNP formulation: MTS002-23 (MTS002:DOPE:cholesterol:DMG-PEG2000 = 40:15:43.5:1.5)

[0304] mRNA: BMK (Benchmark), M21-2-Neg (without GM-CSF, encoding amino acid sequence see SEQ ID NO:19), M21-2

[0305] Solvent: Tris + 10% sucrose

[0306] 7.1.3 Laboratory Animals

[0307] Animal species: C57BL / 6N mouse

[0308] Animal sex: Female

[0309] Animal source: Vitalliwa

[0310] Age in weeks: 6 weeks

[0311] 7.2 Experimental Methods

[0312] 7.2.1 Administration

[0313] Administration time point: tumor volume ~100mm 3 and ~500mm 3

[0314] Dosing method: i.m., right hind limb rectus femoris

[0315] Dosing dose: 0.1, 1, 10 μg / 50 μL / mouse / time

[0316] Dosing frequency: QWx3

[0317] 7.2.2 Experimental grouping

[0318] Negative control group (Empty): MTS002-23-Empty (lipid content is the same as the high-dose group)

[0319] Experimental group: three kinds of mRNA are set to high, medium and low three doses

[0320] 5 mice in each group

[0321] 7.2.3 Evaluation index

[0322] Body weight (2-3 times a week after the first administration); tumor volume (2-3 times a week after the first administration)

[0323] 7.3 Experimental results

[0324] There is no difference in the body weight of mice among groups. The tumor volume data shows that M21-2 has significant tumor inhibition activity at high, medium and low doses when the first dose is given when the tumor volume is ~100 mm 3 and 3 doses are accumulated. When the first dose is given when the tumor volume is ~500 mm 3 and 2 doses are accumulated, M21-2 shows significant dose-dependent tumor inhibition efficacy (Figure 12).

[0325] At the same time, in the large tumor efficacy model with a tumor volume of ~500 mm 3 , two control mRNA M21-2-Neg and BMK were given for 2 doses. Compared with the efficacy of M21-2, the results show that the efficacy of M21-2 at high, medium and low doses is better than that of the other two mRNA vaccines, and the superior efficacy of M21-2 at high dose has a significant difference from M21-2-Neg and BMK (Figure 13).

[0326] 7.4 Experimental summary

[0327] In this round of experiments, M21-2 showed significant dose-dependent tumor inhibition activity in the large tumor model; at the same time, compared with M21-2-Neg (without mGM-CSF) and BMK (BioNTech product), M21-2 showed better efficacy. This indicates that M21-2 has good tumor inhibition activity in HPV16 + large tumors, and GM-CSF has a drug efficacy holding effect.

[0328] Example 8, Pharmacodynamic evaluation of MC38 (HPV18-E6&E7) tumor xenograft model

[0329] This example uses a subcutaneous tumor xenograft mouse model of colon cancer cells MC38 (HPV18-E6&E7) stably expressing HPV18 E6 and E7 (Cat No. KC-3790, purchased from KYINNO) to evaluate the pharmacodynamic activity of mRNA molecules. The drug administration was started when the tumor volume was ~100mm 3 and ~200mm 3 , respectively, i.e. the inhibitory effect of mRNA molecules on HPV18+ large tumors was evaluated simultaneously. The drug administration scheme was as follows: the drug dose was set to 0.05, 0.5, 5 μg / mouse; the drug administration mode was i.m.; the drug administration window was ~100mm 3 and ~200mm 3 ; the drug administration interval and frequency were QWx3 (once a week for a total of three times) or Q5Dx3 (once every five days for a total of three times). The specific experimental operation was as follows:

[0330] 8.1 Experimental materials

[0331] 8.1.1 Homologous mouse transplantation model

[0332] The subcutaneous tumor xenograft model was established by subcutaneously inoculating MC38 (HPV18-E6&E7) tumor cells, and 0.1 mL of cell suspension with a concentration of 2x10 7 cells / mL was inoculated at the right dorsal axillary of the mouse.

[0333] 8.1.2 Drug

[0334] Lipid molecules: MTS002

[0335] LNP formulation: MTS002-23 (MTS002:DOPE:cholesterol:DMG-PEG2000=40:15:43.5:1.5)

[0336] mRNA:

[0337] Solvent: Tris + 10% sucrose

[0338] 8.1.3 Experimental animals

[0339] Animal species: C57BL / 6J mice

[0340] Animal gender: female

[0341] Animal source: Beijing Yaoke Biological

[0342] Weeks of age: 6 weeks

[0343] 8.2 Experimental methods

[0344] 8.2.1 Dosing

[0345] Dosing time point: tumor volume ~100mm 3 or ~200mm 3

[0346] Dosing method: i.m., right hind limb rectus femoris

[0347] Dosing dose: 0.05, 0.5, 5 μg / 50 μL / mouse / time

[0348] Dosing frequency: Q5Dx3 or QWx3

[0349] 8.2.2 Experimental grouping

[0350] Negative control group (Empty): MTS002-23-Empty (lipid molecule content same as high dose group)

[0351] Experimental group: M21-2 set high, medium and low three doses, M20-2 set high and medium two doses

[0352] 5 mice per group

[0353] 8.2.3 Evaluation index

[0354] Body weight (2-3 times per week after initial administration); tumor volume (2-3 times per week after initial administration)

[0355] 8.3 Experimental results

[0356] 8.3.1 mRNA molecule in tumor volume ~100mm 3 time dosing efficacy activity

[0357] There was no difference in mice weight between groups. Tumor volume data results showed that M21-2 presented dose-dependent efficacy activity in this 100mm 3 transplanted tumor model, and the efficacy activity of medium and high doses showed significant difference compared with the control group. In high dose, M21-2 and M20-2 both showed good tumor inhibition rate (Figure 14).

[0358] 8.3.2 mRNA molecule in tumor volume ~200mm 3 time dosing efficacy activity

[0359] There was no difference in mice weight between groups. Tumor volume data results showed that M21-2 presented dose-dependent efficacy activity in this 200mm 3 transplanted tumor model, and the efficacy activity of medium and high doses showed significant difference compared with the control group (Figure 15).

[0360] 8.4 Experimental summary

[0361] M21-2 and M20-2 showed good pharmacodynamic activity in 100mm 3 and 200mm 3 Good pharmacodynamic activity in the xenograft model, indicating that it has a good tumor inhibition effect on HPV18+ large tumors.

[0362] Example 9, pharmacodynamic evaluation of combination of immune checkpoint inhibitors (ICIs)

[0363] This example uses a subcutaneous xenograft mouse model of MC38 (HPV18-E6 & E7) to test the synergistic effect of the combination of the vaccine drug and aPD-1. The specific experimental operation is as follows:

[0364] 9.1 Experimental materials

[0365] 9.1.1 Homologous mouse transplantation model

[0366] The subcutaneous xenograft model uses subcutaneous inoculation of MC38 (HPV18-E6 & E7) tumor cells to form tumors, and 0.1 mL of cell suspension with a concentration of 2 x 10 7 cells / mL is inoculated on the right side of the mouse back near the armpit.

[0367] 9.1.2 Drugs

[0368] Lipid molecules: MTS002

[0369] LNP formula: MTS002-23 (MTS002: DOPE: cholesterol: DMG-PEG2000 = 40: 15: 43.5: 1.5)

[0370] mRNA:

[0371] Solvent: Tris + 10% sucrose

[0372] ICIs: aPD-1 (BP0146, BioXcell)

[0373] Solvent: vaccine: Tris + 10% sucrose; ICIs: PBS

[0374] Preparation concentration: vaccine: 0.01 mg / mL; ICIs: 2 mg / mL

[0375] 9.1.3 Experimental animals

[0376] Animal species: C57BL / 6N mice

[0377] Animal gender: female

[0378] Animal source: Vivotecnia

[0379] Weeks of age: 6-8 weeks

[0380] 9.2 Experimental method

[0381] 9.2.1 Administration

[0382] Administration time point: when the tumor volume is 70-100 mm 3

[0383] Administration method: vaccine: i.m., right hind limb rectus femoris; ICIs: i.p., intraperitoneal injection

[0384] Administration dose: vaccine: 0.5 μg / 50 μL / mouse / time; ICIs: 200 μg / 100 μL / mouse / time

[0385] Administration frequency: vaccine: Q5Dx3; ICIs: Q3Dx6

[0386] 9.2.2 Experimental grouping

[0387] Negative control group (Empty)

[0388] Vaccine single-drug group: M21-2 0.5 μg

[0389] ICIs single-drug group: aPD-1 10 mpk

[0390] Combination group: M21-2 0.5 μg + aPD-1 10 mpk

[0391] 6 mice were set in each group

[0392] 9.2.3 Evaluation index

[0393] Body weight (2-3 times per week after initial administration); tumor volume (2-3 times per week after initial administration)

[0394] 9.3 Experimental results and analysis

[0395] 9.3.1 Detection of tool cell PD-L1 expression

[0396] Flow cytometry was used to detect the PD-L1 expression of tool cell MC38 (HPV18-E6 & E7), and the results showed that the PD-L1 expression was positive (Figure 16).

[0397] 9.3.2 Combination efficacy experiment

[0398] There was no significant difference in body weight data among groups. The single-drug administration groups had significant pharmacological activity relative to the blank control group, and the combination group had significantly enhanced efficacy relative to the single-drug administration groups (Figure 17).

[0399] 9.4 Research summary​

[0400] The vaccine drug of the present application has synergistic pharmacodynamic activity when combined with aPD-1.

[0401] Sequence information involved in the present application

Claims

1. A nucleic acid molecule comprising an open reading frame (ORF), the ORF comprising: a nucleotide sequence encoding an HPV antigen; a nucleotide sequence encoding an immune costimulatory factor; and a nucleotide sequence encoding a self-cleaving peptide located between the nucleotide sequence encoding the HPV antigen and the nucleotide sequence encoding the immune costimulatory factor.

2. The nucleic acid molecule of claim 1, wherein the HPV antigen comprises at least one HPV immunogenic polypeptide from at least one HPV serotype.

3. The nucleic acid molecule of claim 2, wherein the at least one HPV serotype is selected from the group consisting of serotypes 1, 2, 3, 4, 7, 10, 12, 15, 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, 5, 8, 14, 17, 20, 36, 38, 16, 18, 30, 31, 33, 35, 39, preferably, the at least one HPV serotype is serotype 16 and / or serotype 18.

4. The nucleic acid molecule of claim 2 or 3, wherein the HPV immunogenic polypeptide is selected from the group consisting of an El, E2, E3, E4, E5, E6, E7, LI, or L2 polypeptide of HPV or an immunogenic variant thereof, preferably, the HPV immunogenic polypeptide is selected from the group consisting of an E6, E7, L2 polypeptide or an immunogenic variant thereof, more preferably, the HPV immunogenic polypeptide is selected from the group consisting of an E6, E7 polypeptide or an immunogenic variant thereof.

5. The nucleic acid molecule of any one of claims 2-4, wherein the HPV antigen comprises two HPV immunogenic polypeptides from one HPV serotype, or, the HPV antigen comprises two HPV immunogenic polypeptides from two HPV serotypes.

6. The nucleic acid molecule of any one of claims 2-5, wherein the HPV antigen comprises i) an E6 polypeptide and an E7 polypeptide from HPV serotype 16; ii) an E6 polypeptide and an E7 polypeptide from HPV serotype 18; or iii) an E6 polypeptide and an E7 polypeptide from HPV serotype 16 and an E6 polypeptide and an E7 polypeptide from HPV serotype 18.

7. The nucleic acid molecule of claim 6, wherein the E6 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 67 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 41 or SEQ ID NO: 67; the E7 polypeptide from HPV serotype 16 comprises the amino acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 68 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 68; the E6 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 69 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity with SEQ ID NO: 43 or SEQ ID NO: 69; the E7 polypeptide from HPV serotype 18 comprises the amino acid sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 70 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity with SEQ ID NO: 44 or SEQ ID NO:

70.

8. The nucleic acid molecule of any one of claims 2-7, wherein the HPV antigen further comprises a signal peptide, whereby secretion expression of the HPV immunogenic polypeptide is achieved, for example, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

48.

9. The nucleic acid molecule of any one of claims 2-8, wherein the different polypeptides comprised in the HPV antigen are directly linked or linked via a peptide linker, for example, the peptide linker comprises the sequence GnS, n being an integer from 1 to 5, for example, n is 1 or 3.

10. The nucleic acid molecule of any one of claims 1-9, wherein the immune co-stimulatory factor is selected from Flt3Lecd, GM-CSF, OX40L, HMGB1 or HSP70p, preferably GM-CSF and OX40L, more preferably GM-CSF.

11. The nucleic acid molecule of claim 10, wherein the GM-CSF comprises the amino acid sequence set forth in SEQ ID NO:

50.

12. The nucleic acid molecule of claim 11, wherein the self-cleaving peptide is a 2A polypeptide, preferably a T2A polypeptide or a P2A polypeptide, more preferably a T2A polypeptide.

13. The nucleic acid molecule of claim 12, wherein the T2A polypeptide amino acid sequence is set forth in SEQ ID NO:

64.

14. The nucleic acid molecule of any one of claims 1-13, wherein the ORF comprises, in the 5’ to 3’ direction, the nucleotide sequence encoding the HPV antigen, the nucleotide sequence encoding the self-cleaving peptide and the nucleotide sequence encoding the immune co-stimulatory factor.

15. The nucleic acid molecule of any one of claims 1-14, wherein the ORF encodes an amino acid sequence comprising the structure: SP-HPV immunogenic polypeptide-2A-GMCSF, wherein SP is a signal peptide, 2A is a 2A polypeptide, and GMCSF is human or murine GM-CSF; preferably, the ORF encodes an amino acid sequence comprising the structure: SP-E6 16 -E7 16 -E6 18 -E7 18 -2A-GMCSF, wherein SP is a signal peptide, E6 16 and E7 16 are E6 and E7 polypeptides, respectively, from HPV serotype 16, E6 18 and E7 18 are E6 and E7 polypeptides, respectively, from HPV serotype 18, 2A is a 2A polypeptide, and GMCSF is human or murine GM-CSF.

16. The nucleic acid molecule of any one of claims 1-15, wherein the ORF encodes the amino acid sequence set forth in one of SEQ ID NOs: 3-6 and 8-18 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity with one of SEQ ID NOs: 3-6 and 8-18. Preferably, the ORF encodes an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO:

11.

17. The nucleic acid molecule of any one of claims 1-16, wherein the ORF comprises a nucleotide sequence as set forth in one of SEQ ID NOs: 23-26 and 28-38 or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to one of SEQ ID NOs: 23-26 and 28-38, Preferably, the ORF comprises a nucleotide sequence as set forth in SEQ ID NO: 31 or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO:

31.

18. The nucleic acid molecule of any one of claims 1-17, wherein the nucleic acid molecule is an mRNA molecule.

19. The nucleic acid molecule of claim 18, wherein the mRNA molecule further comprises a 5’ UTR and / or a 3’ UTR.

20. The nucleic acid molecule of claim 19, wherein the 5’ UTR comprises a nucleotide sequence as set forth in SEQ ID NO: 57 and the 3’ UTR comprises a nucleotide sequence as set forth in SEQ ID NO: 58; or the 5’ UTR comprises a nucleotide sequence as set forth in SEQ ID NO: 59 and the 3’ UTR comprises a nucleotide sequence as set forth in SEQ ID NO:

60.

21. The nucleic acid molecule of any one of claims 18-20, wherein the mRNA molecule further comprises a poly(A) sequence.

22. The nucleic acid molecule of claim 21, wherein the poly(A) sequence comprises about 20 to about 500 adenine nucleotides (A), preferably the poly(A) sequence comprises 120 adenine nucleotides (A).

23. The nucleic acid molecule of any one of claims 18-22, wherein the mRNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 66 or a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO:

66.

24. The nucleic acid molecule of any one of claims 18-23, wherein the mRNA molecule further comprises a 5’ cap structure, for example, the 5’ cap structure is a Cap 1 cap structure.

25. The nucleic acid molecule of any one of claims 18-24, wherein the mRNA molecule further comprises at least one modified nucleotide, preferably the modified nucleotide is selected from pseudouridine, N1-methyl-pseudouridine, 5-methylcytidine, or a combination thereof.

26. A lipid nanoparticle (LNP) comprising the nucleic acid molecule of any one of claims 1-25, said nucleic acid molecule being comprised in a lipid.

27. An immunogenic composition comprising the nucleic acid molecule of any one of claims 1-25 or the LNP of claim 26, and a pharmaceutically acceptable carrier.

28. An immunoprophylactic / therapeutic medicament comprising the nucleic acid molecule of any one of claims 1-25 or the LNP of claim 26, and a pharmaceutically acceptable carrier; preferably, the immunoprophylactic / therapeutic medicament is a tumor immunotherapy medicament, more preferably, the tumor immunotherapy medicament is a tumor vaccine.

29. Use of the nucleic acid molecule of any one of claims 1-25 or the LNP of claim 26 or the immunogenic composition of claim 27 for the manufacture of a vaccine for the prevention and / or treatment of HPV infection and / or HPV infection related pathologies in a subject.

30. A method for the prevention and / or treatment of HPV infection and / or HPV infection related pathologies in a subject, said method comprising administering to said subject an effective amount of the nucleic acid molecule of any one of claims 1-25 or the LNP of claim 26 or the immunogenic composition of claim 27.

31. The use of claim 29 or the method of claim 30, wherein the HPV infection is an HPV infection of a serotype selected from the group consisting of: 1, 2, 3, 4, 7, 10, 12, 15, 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, 5, 8, 14, 17, 20, 36, 38, 16, 18, 30, 31, 33, 35, 39.

32. The use or method of claim 31, wherein the HPV infection is an infection of a single HPV serotype or the HPV infection is a mixed infection of two or more different HPV serotypes.

33. The use or method of claim 31 or 32, wherein the HPV infection is an infection of HPV 16 and / or HPV 18.

34. The use of claim 29 or the method of claim 30, wherein the HPV infection related pathology is selected from the group consisting of cutaneous warts, such as common warts, plantar warts, flat warts, Bowen's disease, butcher's warts or condyloma acuminata; HPV-positive tumors such as HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, vaginal cancer, rectal cancer, bladder cancer, endometrial cancer, oropharyngeal cancer, head and neck squamous cell carcinoma, prostate cancer or bladder cancer.

35. A combination comprising: (1) the nucleic acid molecule of any one of claims 1-25, the LNP of claim 26 or the immunogenic composition of claim 27, and (2) an immune checkpoint inhibitor.

36. The combination of claim 35, wherein the immune checkpoint inhibitor is a PD1 signaling pathway inhibitor, for example, the PD1 signaling pathway inhibitor is selected from the group consisting of a PD1 antibody, a PDL1 antibody, a PDL2 antibody, or a combination thereof, preferably a PD1 antibody.

37. The combination of claim 35 or 36 for use in the prevention and / or treatment of a tumor and / or cancer disease in a subject; preferably, the tumor and / or cancer disease is a HPV-positive tumor and / or cancer; preferably, the tumor and / or cancer disease is selected from the group consisting of a HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, vaginal cancer, rectal cancer, bladder cancer, endometrial cancer, oropharyngeal cancer, head and neck squamous cell carcinoma, prostate cancer or bladder cancer.

38. A method for the prevention and / or treatment of a tumor and / or cancer disease in a subject, the method comprising administering to the subject an effective amount of the combination of claim 35 or 36; preferably, the tumor and / or cancer disease is a HPV-positive tumor and / or cancer disease; preferably, the tumor and / or cancer disease is selected from the group consisting of a HPV-positive cervical cancer, vulvar cancer, penile cancer, anal cancer, vaginal cancer, rectal cancer, bladder cancer, endometrial cancer, oropharyngeal cancer, head and neck squamous cell carcinoma, prostate cancer or bladder cancer.

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