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WO2025162105A1 - Human papilloma virus (hpv) mrna vaccines and uses thereof - Google Patents

Human papilloma virus (hpv) mrna vaccines and uses thereof

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Publication number
WO2025162105A1
WO2025162105A1 PCT/CN2025/073829 CN2025073829W WO2025162105A1 WO 2025162105 A1 WO2025162105 A1 WO 2025162105A1 CN 2025073829 W CN2025073829 W CN 2025073829W WO 2025162105 A1 WO2025162105 A1 WO 2025162105A1
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WO
WIPO (PCT)
Prior art keywords
dose
mrna
seq
vaccine
hpv
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/073829
Other languages
French (fr)
Inventor
Yijie DONG
Shan Cen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rinuagene Biotechnology Co Ltd
Rinuagene International Hk Ltd
Original Assignee
Rinuagene Biotechnology Co Ltd
Rinuagene International Hk Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rinuagene Biotechnology Co Ltd, Rinuagene International Hk Ltd filed Critical Rinuagene Biotechnology Co Ltd
Publication of WO2025162105A1 publication Critical patent/WO2025162105A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present application relates to a polynucleotide molecule encoding HPV antigen peptide, a delivery body comprising the polynucleotide molecule, a vaccine formulation, and a method for treating or preventing HPV infection related diseases.
  • HPV Human papillomavirus
  • the HPV genome has eight genes encoding viral proteins, which are classified into early genes (E1, E2, E4, E5 E6 and E7) and late genes (L1 and L2) according to the stage of their expression in viral life cycle. Early genes regulate viral replication and transformation of infected cells to cancer cells, while L1 and L2 are structural proteins which form a virus particle capsid (J Clin Virol 2005; 32 (Suppl. 1) : S7e15) .
  • Cervical cancer is one of the leading causes of death among women worldwide.
  • Major risk factors associated with the development of cervical cancer include high-risk human papillomavirus (hrHPV) infection, age, smoking, childbirth, use of oral contraceptives, and diet.
  • hrHPV human papillomavirus
  • HPV DNA has been identified in more than 99.7%of tumors biopsy specimens
  • HPV16 and HPV18 have been identified in all cervical biopsy specimens.
  • HPV16 and HPV18 are the most prevalent types, responsible for 50-60%and 10-20%of cervical cancer cases, respectively.
  • CIN cervical intraepithelial neoplasia
  • CIN2/3 pre-malignant cervical intraepithelial neoplasia 2 and 3
  • CIN3 has a higher risk of progressing to cancer, and the model estimates that 15%to 23%of untreated CIN3 will develop invasive cancer over an average of 13 years, with a lifetime risk of 40%.
  • prophylactic HPV vaccines (such as and ) have been shown to be effective in preventing health women from HPV infection, but do not treat pre-existing HPV infections, pre-malignant lesions, or malignant lesions.
  • the immunogen containing HPV16/18 E6 and E7 with N-terminus and C-terminus domains could induce strong specific cellular immune responses in vivo.
  • the addition of E2 conserved peptides could further enhance specific cellular immune responses and effectively induce humoral immune responses against E2.
  • the present application it is possible to induce effective cellular immune response, and potentially remedi patients with high-risk HPV related lesions. Accordingly, it is possible to prevent and/or treat infections with human papillomavirus. According to the present application, it is also possible to prevent and/or treat diseases caused by infections with HPV, such as cervical intraepithelial neoplasia (CIN) , cervical cancer.
  • CIN cervical intraepithelial neoplasia
  • Figs. 1A and 1B are the schematic diagram of the phase 1 and phase 2 study designs.
  • the present application provides a method for treating or preventing human papillomavirus (HPV) infection related diseases, comprising administering to a human subject at least one dose of a vaccine comprising a lipid nanoparticle (LNP) that comprises a messenger ribonucleic acid (mRNA) , wherein the mRNA comprises an open reading frame encoding a fusion protein of high-risk HPV antigen (s) , the one dose of the vaccine comprises 0.01 ⁇ g ⁇ 500 ⁇ g of the mRNA.
  • a vaccine comprising a lipid nanoparticle (LNP) that comprises a messenger ribonucleic acid (mRNA)
  • mRNA messenger ribonucleic acid
  • s high-risk HPV antigen
  • the amount of the mRNA in the one dose is selected from the group consisting of: 0.01 ⁇ g ⁇ 500 ⁇ g, 0.05 ⁇ g ⁇ 500 ⁇ g, 0.1 ⁇ g ⁇ 500 ⁇ g, 1 ⁇ g ⁇ 500 ⁇ g, 2.5 ⁇ g ⁇ 500 ⁇ g, 5 ⁇ g ⁇ 500 ⁇ g, 10 ⁇ g ⁇ 500 ⁇ g, 10 ⁇ g ⁇ 480 ⁇ g, 10 ⁇ g ⁇ 450 ⁇ g, 12.5 ⁇ g ⁇ 450 ⁇ g, 25 ⁇ g ⁇ 450 ⁇ g, 25 ⁇ g ⁇ 400 ⁇ g, 25 ⁇ g ⁇ 350 ⁇ g, 25 ⁇ g ⁇ 250 ⁇ g, 25 ⁇ g ⁇ 200 ⁇ g, 25 ⁇ g ⁇ 150 ⁇ g, 50 ⁇ g ⁇ 350 ⁇ g, 75 ⁇ g ⁇ 350 ⁇ g, 75 ⁇ g ⁇ 300 ⁇ g, 75 ⁇ g ⁇ 200 ⁇ g, and 75 ⁇ g ⁇ 150 ⁇ g.
  • the amount of the mRNA in the one dose is selected from the group consisting of: 0.01 ⁇ g, 0.015 ⁇ g, 0.1 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, 2.5 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 6 ⁇ g, 7 ⁇ g, 8 ⁇ g, 9 ⁇ g, 10 ⁇ g, 11 ⁇ g, 12.5 ⁇ g, 14 ⁇ g, 15 ⁇ g, 17.5 ⁇ g, 18 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 105 ⁇ g, 110 ⁇ g, 120 ⁇ g, 125 ⁇ g, 130 ⁇ g, 135 ⁇ g, 140 ⁇ g, 145 ⁇ g, 150 ⁇ g, 155 ⁇ g, 160 ⁇ g, 165 ⁇ g, 170 ⁇ g, 175 ⁇ g, 180 ⁇ g, 185 ⁇
  • the method further comprises administering to the subject a second dose of the vaccine.
  • the second dose of the vaccine is administered at least 2 weeks after the first dose is administered.
  • the method further comprises administering to the subject a third dose of the vaccine.
  • the third dose of the vaccine is administered at least 2 weeks after the second dose is administered.
  • the amount of the mRNA in the second dose or the third dose is selected from the group consisting of: 0.01 ⁇ g ⁇ 500 ⁇ g, 0.05 ⁇ g ⁇ 500 ⁇ g, 0.1 ⁇ g ⁇ 500 ⁇ g, 1 ⁇ g ⁇ 500 ⁇ g, 2.5 ⁇ g ⁇ 500 ⁇ g, 5 ⁇ g ⁇ 500 ⁇ g, 10 ⁇ g ⁇ 500 ⁇ g, 10 ⁇ g ⁇ 480 ⁇ g, 10 ⁇ g ⁇ 450 ⁇ g, 12.5 ⁇ g ⁇ 450 ⁇ g, 25 ⁇ g ⁇ 450 ⁇ g, 25 ⁇ g ⁇ 400 ⁇ g, 25 ⁇ g ⁇ 350 ⁇ g, 25 ⁇ g ⁇ 250 ⁇ g, 25 ⁇ g ⁇ 200 ⁇ g, 25 ⁇ g ⁇ 150 ⁇ g, 50 ⁇ g ⁇ 350 ⁇ g, 75 ⁇ g ⁇ 350 ⁇ g, 75 ⁇ g ⁇ 300 ⁇ g, 75 ⁇ g ⁇ 200 ⁇ g, and 75 ⁇ g ⁇ 150 ⁇ g.
  • the amount of the mRNA in the second dose or the third dose is selected from the group consisting of: 0.01 ⁇ g, 0.015 ⁇ g, 0.1 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, 2.5 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 6 ⁇ g, 7 ⁇ g, 8 ⁇ g, 9 ⁇ g, 10 ⁇ g, 11 ⁇ g, 12.5 ⁇ g, 14 ⁇ g, 15 ⁇ g, 17.5 ⁇ g, 18 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 105 ⁇ g, 110 ⁇ g, 120 ⁇ g, 125 ⁇ g, 130 ⁇ g, 135 ⁇ g, 140 ⁇ g, 145 ⁇ g, 150 ⁇ g, 155 ⁇ g, 160 ⁇ g, 165 ⁇ g, 170 ⁇ g, 175 ⁇ g, 180 ⁇ g
  • the amount of the mRNA in the first dose , second dose or the third dose is selected from the group consisting of: 25 ⁇ g, 75 ⁇ g or 150 ⁇ g.
  • the subject is a 9-70 years old subject.
  • the subject is an 18-70 years old subject.
  • the subject is an 18-55 years old subject.
  • the subject is an 18-45 years old subject.
  • the high-risk HPV antigen encoded by the ORF of the mRNA has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the amino acid sequence of SEQ ID NO: 3; in some embodiments, the high-risk HPV antigen encoded by the ORF of the mRNA comprises or consists of the amino acid sequence of SEQ ID NO: 3, or a conserved substitution variant of SEQ ID NO: 3. In some embodiments, the encoding sequence of the high-risk HPV antigen comprises or consists of a polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the encoding sequence of the high-risk HPV antigen comprises or consists of a synonymous mutant of SEQ ID NO: 10.
  • the polynucleotide molecules further include coding sequences of immunostimulatory factors or their functional domains.
  • the coding sequence of the immunostimulating factor or its functional domain is located at the 3' or 5' end of the coding sequence of the HPV antigen peptide.
  • the immunostimulatory factor is Flt3L.
  • the polypeptide sequence of the immunostimulatory factor comprises at least an amino acid sequence of SEQ ID NO: 1, a conserved substituent variant of SEQ ID NO: 1, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 1.
  • the polypeptide sequence of the immunostimulating factor consists of the amino acid sequence of SEQ ID NO: 1, or a conserved substitution variant of SEQ ID NO: 1, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 1.
  • the encoding sequence of the immunostimulatory factor comprises or consists of a polynucleotide sequence of SEQ ID NO: 9.
  • the encoding sequence of the immunostimulating factor comprises or consists of a synonymous mutant of SEQ ID NO:9.
  • the polynucleotide molecule further comprises a signal peptide encoding sequence.
  • the signal peptide encoding sequence is located at the 5' end of the coding sequence of the HPV antigen.
  • the signal peptide is a human secretory signal peptide.
  • the secretory signal peptide is tPA-SP.
  • the polypeptide sequence of the secretory signal peptide comprises an amino acid sequence of SEQ ID NO: 2, a conserved substituent variant of SEQ ID NO: 2, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 2.
  • the polypeptide sequence of the secretory signal peptide consists of an amino acid sequence of SEQ ID NO: 2, or a conserved substituent variant of SEQ ID NO: 2, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 2.
  • the encoding sequence of the secretory signal peptide comprises or consists of a polynucleotide sequence of SEQ ID NO: 8.
  • the encoding sequence of the secretory signal peptide comprises or consists of a synonymous mutant of SEQ ID NO: 8.
  • the high-risk HPV antigen encoded by the ORF of the mRNA has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the amino acid sequence of SEQ ID NO: 4.
  • the high-risk HPV antigen encoded by the ORF of the mRNA comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a conserved substituent variant of SEQ ID NO: 4.
  • the mRNA or polynucleotide molecule encoding the high-risk HPV antigen has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the polynucleotide sequence of SEQ ID NO: 11.
  • the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of the amino acid sequence of SEQ ID NO: 11.
  • the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of a synonymous mutant of SEQ ID NO: 11.
  • the mRNA or polynucleotide molecule further comprises a 5'UTR structure. In some embodiments, the mRNA or polynucleotide molecule comprises a 3'UTR structure. In some embodiments, the mRNA or polynucleotide molecule further comprises a 5'UTR structure and a 3'UTR structure. In some embodiments, the 5’ UTR structure comprises at least a polynucleotide sequence of SEQ ID NO: 5, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 5.
  • the 5’UTR structure consists of a polynucleotide sequence of SEQ ID NO: 5, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 5.
  • the 3'UTR structure comprises at least a polynucleotide sequence of SEQ ID NO: 6, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 6.
  • the 3'UTR structure consists of a polynucleotide sequence of SEQ ID NO: 6, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 6.
  • the mRNA or polynucleotide molecule further comprises a 5'cap structure, preferably the 5'cap structure is m7G (5') ppp (5') (2'-OMeA) pG.
  • the mRNA or polynucleotide molecule further comprises a poly (A) tail; preferably, the poly (A) tail sequence comprises at least 50, at least 60, or at least 100 A nucleotides; preferably, the poly (A) tail comprises or consists of a polynucleotide sequence as shown in SEQ ID NO: 7, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 7.
  • the mRNA molecule of the present application sequentially comprises 5’ cap, 5'UTR, ORF, 3'UTR, and poly A tail, wherein the5’ cap, 5'UTR, ORF, 3'UTR, and poly A tail are operably connected to each other.
  • the mRNA or polynucleotide molecule encoding the high-risk HPV antigen has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of the amino acid sequence of SEQ ID NO: 12.
  • the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of a synonymous mutant of SEQ ID NO: 12.
  • part or all of the uridines in the mRNA molecule are chemically modified uridines, preferably 1-methyl-pseuduridine modification.
  • the lipid nanoparticle comprises ionizable lipid (s) , phospholipids, cholesterol, and polyethylene glycol (PEG) -lipids.
  • the lipid nanoparticles comprise ionizable lipid (s) , phospholipids, cholesterol, and polyethylene glycol (PEG) –lipids; wherein the content of the ionizable lipid (s) is 35mol%-65mol%, the content of the sum of the phospholipids and cholesterol is 35mol%-65mol%, the content of the PEG–lipids is 0.5mol%-5mol%.
  • the lipid nanoparticles comprise 40mol%-50mol%ionizable lipid (s) , 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 1.5mol%-2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 48mol%-49mol%ionizable lipid (s) , 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 1.5mol%-2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 15mol%phospholipids, 42.5mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 15mol%phospholipids, 43.5mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 48.5mol%ionizable lipid (s) , 5mol%phospholipids, 45mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 44.3mol%ionizable lipid (s) , 9.3mol%phospholipids, 45mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 5mol%phospholipids, 43mol%cholesterol, and 2.1mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 12.5mol%phospholipids, 45mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 46mol%ionizable lipid (s) , 11mol%phospholipids, 41mol%cholesterol, and 2mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 47.5mol%ionizable lipid (s) , 5mol%phospholipids, 45mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 13mol%phospholipids, 45mol%cholesterol, and 2mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 12.5mol%phospholipids, 35mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 8mol%phospholipids, 39.5mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 42.3mol%ionizable lipid (s) , 15mol%phospholipids, 40.3mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 46mol%ionizable lipid (s) , 11mol%phospholipids, 41mol%cholesterol, and 2mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 47.5mol%ionizable lipid (s) , 15mol%phospholipids, 35mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 48.5mol%ionizable lipid (s) , 15mol%phospholipids, 35mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 5mol%phospholipids, 43.5mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 15mol%phospholipids, 43.5mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
  • the lipid nanoparticles comprise 48mol%ionizable lipid (s) , 10.3mol%phospholipids, 39.6mol%cholesterol, and 2.1mol%polyethylene glycol (PEG) –lipids.
  • the molar ratio of the ionizable lipids, the sum of the phospholipids and the structural lipids, and the PEG-lipids is (35-65) : (35-65) : (0.5-5) ; wherein the 35-65 can be any value between 35-65, for example, it can be 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; the 0.5-5
  • the above mol % is calculated based on the total moles of the lipids of lipid nanoparticle composition.
  • the lipid nanoparticle (LNP) futher comprises Ca 2+ , Mg 2+ , Zn 2+ and /or Mn 2+ .
  • the PEG –lipid is DMG-PEG2000, and the phospholipid is selected from DSPC or DOPE; preferably, the phospholipid is DSPC.
  • the administration is intratumoral, perilymphatic, intradermal (ID) , or intramuscular injection; preferably intramuscular injection.
  • the vaccine is administered to the subject once, twice, thrice, 4 times, 5 times, 6 times, 7 times, 8 times 9 times, 10 times or until the disease is alleviated or cured.
  • the vaccine is administered to the subject for a total of 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses or 10 doses.
  • the vaccine is administered to the subject every 7 days, every 10 days, every 14 days, every 21 days, every 28 days, every 35 days, every 60 days, every 90 days, or every 120 days.
  • the vaccine is administered to the subject every 7 days, every 14 days, every 21 days or every 28 days.
  • the vaccine is administered to the subject every 1 week or every 2 weeks, every 3 weeks or every 4 weeks.
  • the vaccine is administered intramuscularly at assigned dose level, with administration frequency of every 2 weeks (D1, D15, and D29) for a total of 3 doses.
  • the vaccine is administered intramuscularly at assigned dose level of 25 ⁇ g, 75 ⁇ g or 150 ⁇ g mRNA respectively, with administration frequency of every 2 weeks (D1, D15, and D29) for a total of 3 doses.
  • the method further comprises administering immune stimulating factors, chemotherapy, radiation therapy, and/or targeted therapy to an individual.
  • the present invention provides a method of expressing the E6 and E7 antigens of human papillomavirus in vitro, comprising introducing into cells a composition containing the above-described lipid particle. Further, the present invention also provides a method of expressing the E6 and E7 antigens of human papillomavirus in vivo, comprising administering to a mammal a composition containing the above-described lipid particle.
  • the present application provides a use of any one of the vaccines described above in the manufacture of a medicament for inducing an immune response in a subject.
  • the present invention also provides a method of inducing immune response to human papillomavirus, comprising administering to a mammal a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) .
  • a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) comprising administering to a mammal a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) .
  • the present application provides a use of any one of the vaccines described above in treating or preventing HPV type 16 and/or 18HPV infection related disease, comprising administering to a mammal a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) .
  • a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) comprising administering to a mammal a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) .
  • the HPV type 16 and/or 18 HPV infection related disease is selected from the group consisting of: cervical cancer, cervical precancerous lesions, high-grade intraepithelial lesions of the cervix, high-grade squamous intraepithelial lesions (HSIL) , cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN2/3) , cervical intraepithelial neoplasia (CIN) grade 1 (CIN1) .
  • the HPV type 16 and/or 18HPV infection related disease is cervical cancer.
  • the HPV type 16 and/or 18 HPV infection related disease is Human Papillomavirus (HPV) 16 or 18 Associated Cervical Intraepithelial Neoplasia Grade 2 or 3 (CIN2/3) .
  • the present application provides a composition for use in the method described above.
  • the lipid particle encapsulating a nucleic acid molecule according to the present invention may be prepared by various methods, such as a thin film method, a reverse phase evaporation method, an ethanol injection method, an ether injection method, a dehydration-rehydration method, a detergent dialysis method, a hydration method, a freezing-thawing method, and so forth.
  • the lipid particle encapsulating a nucleic acid molecule may be prepared by the methods described in WO2015/005253.
  • the lipid particle encapsulating a nucleic acid molecule according to the present invention can also be prepared by mixing a nucleic acid solution and a solution of lipids in a micro flow channel.
  • the lipid particle may be prepared with NanoAssemblrTM from Precision NanoSystems, according to the method described in the attached protocol.
  • the mean particle size of the particle of the present invention may be 30 nm to 300 nm, preferably 30 nm to 200 nm, and more preferably 30 nm to 100 nm.
  • the particle of the present invention may be used as a pharmaceutical drug or an experimental reagent.
  • the particle of the present invention is usually added to a carrier (such as water, buffer, saline, etc. ) , and the resultant formulation (composition) may be introduced into a cell (in vitro) or administered to a mammal (in vivo) .
  • the carrier may be a pharmacologically acceptable carrier (e.g., saline) .
  • the particle of the present invention may also be prepared into such formulations as cream, paste, ointment, gel, lotion or the like that comprise fat, fatty oil, lanolin, vaseline, paraffin, wax, resin, plastic, glycols, higher alcohol, glycerol, water, emulsifier, suspending agent, and the like as base materials.
  • the particle of the present invention may be administered to a mammal such as human, mouse, rat, hamster, guinea pig, rabbit, pig, monkey, cat, dog, goat, sheep, cattle, etc. orally or parenterally through various routes such as intramuscular, intravenous, rectal, transdermal, transmucosal, subcutaneous or intradermal administration.
  • a mammal such as human, mouse, rat, hamster, guinea pig, rabbit, pig, monkey, cat, dog, goat, sheep, cattle, etc. orally or parenterally through various routes such as intramuscular, intravenous, rectal, transdermal, transmucosal, subcutaneous or intradermal administration.
  • the mRNA in the particle may be administered, for example, at an approximate dose of 0.01 ⁇ g ⁇ 500 ⁇ g per adult per administration either once or several times by intramuscular injection, subcutaneous injection, intradermal injection, intravenous infusion or intravenous injection.
  • the dose and the number of times of administration may be changed appropriately depending on the type and symptoms of the disease, the age of the patient, administration route, etc.
  • the particle of the present invention When the particle of the present invention is used as an experimental reagent, it is possible to express the E6 and E7 antigens of human papillomavirus in vitro by introducing the particle into a cell in which expression of the E6 and E7 antigens of human papillomavirus is desired [e.g., HEK293 cells and cells derived therefrom (HEK293T cells, FreeStyle 293 cells, Expi293 cells, etc. ) , CHO cells, C2Cl2 mouse myoblast cells, immortalized mouse dendritic cells (MutuDC1940) , or the like] .
  • HEK293 cells and cells derived therefrom HEK293T cells, FreeStyle 293 cells, Expi293 cells, etc.
  • CHO cells C2Cl2 mouse myoblast cells
  • immortalized mouse dendritic cells MotuDC1940
  • the expression of the E6 and E7 antigens of human papillomavirus may be analyzed by detecting the E6 and E7 antigen proteins of human papillomavirus in samples based on Western blotting or by detecting peptide fragments specific to the E6 and E7 antigens of human papillomavirus based on mass spectrometry.
  • treat refers to recovery, amelioration, relaxation and/or delaying the progression of clinical symptoms of diseases in patients who are developing infections with viruses or bacteria or diseases caused by such infections (e.g., precancerous lesion or cancer) .
  • the term “prevent” refers to reducing the incidence rate of diseases caused by infections with viruses or bacteria. “Prevent” encompasses lowering the risk of progression of diseases caused by infections with viruses or bacteria, or reducing exacerbation of such diseases. Since the particle of the present invention induces protective immune response, the particle of the present invention shows effectiveness on prevention and/or treatment of the above-described diseases.
  • RG002 is the active ingredient as a non-self-amplifying mRNA, which is formulated in LNPs to ensure that RG002 is stabilized and encapsulated, and to facilitate its uptake into cells and release into the cytosol.
  • the mRNA is designed to express the E2, E6, and E7 antigens of high-risk HPV16 and HPV18 types as a therapeutic vaccine when administrated by intramuscular injection.
  • Dose regimen RG002 injection is administered intramuscularly as a series of 3 doses with a two-week interval;
  • Solubility soluble in water
  • RG002 is a non-self-amplifying mRNA.
  • the theoretical length of the mRNA sequence is 3518nt, the nucleotide sequence is shown as SEQ ID NO: 12; the fusion protein sequence of RG002 contained a total of 1071 amino acid residues, the amino acid sequence is shown as SEQ ID NO: 4.
  • RG002 mRNA is encapsulated in an LNP composed of four lipids: ionizable lipid, DMG-PEG 2000, cholesterol, and DSPC.
  • the lipid nanoparticles comprise 40mol%-55mol%ionizable lipid, 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 0.5mol%-2.5mol%PEG–lipids.
  • cytokines in serum i.e., IL-1 ⁇ , IL-1ra, IL-2, IL-6, IL-10, GM-CSF, TNF- ⁇ and IFN- ⁇ measured by Assays.
  • Exposure endpoints the plasma exposure level of RG002 Injection including mRNA as measured by RT-qPCR and the plasma exposure level of cationic lipids as measured by LC-MS/MS.
  • PEG-ADA endpoints the serum titer of anti-PEG IgG and anti-PEG IgM as measured by ELISA.
  • Primary efficacy endpoint the proportion of subjects with histopathological regression to either CIN1 or normal in 36 weeks.
  • cytokines in serum, i.e., IL-1 ⁇ , IL-1ra, IL-2, IL-6, IL-10, GM-CSF, TNF- ⁇ and IFN- ⁇ , measured by Assays.
  • Exposure endpoints the plasma exposure level of RG002 Injection including mRNA as measured by RT-qPCR and the plasma exposure level of cationic lipids as measured by LC-MS/MS.
  • PEG-ADA endpoints the serum titer of anti-PEG IgG and anti-PEG IgM as measured by ELISA.
  • phase 1/2 open-label clinical study to evaluate the safety, tolerability, immunogenicity, and efficacy of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
  • the study has two parts (see Figures 1A-1B) .
  • Part A is a phase 1 dose escalation study.
  • Part B is a phase 2 proof-of-concept (PoC) study.
  • the available data of Part A including safety, tolerability, immunogenicity, exposure levels and preliminary efficacy of RG002 will be reviewed to determine the RP2D.
  • Part B one or two dose levels including RP2D will be evaluated to optimize the efficacy of RG002 Injection in the subjects with HPV16/18 associated CIN2/3.
  • the schematic diagram of the phase 1/2 study design is shown in Figures 1A-1B.
  • dose escalation will start from 25 ⁇ g and the present 3 dose cohorts are 25 ⁇ g (Cohort A1) , 75 ⁇ g (Cohort A2) and 150 ⁇ g (Cohort A3) .
  • Part A will include 3 cohorts of 3 to 6 subjects each based on a “3+3” dose-escalation study design. A total of 9 to 18 subjects are planned to be enrolled. All subjects will receive three RG002 Injections at assigned dose level intramuscularly every 2 weeks (D1, D15, and D29) . Subjects will be evaluated for a minimum of 30 min for injection site reaction (such as pain, tenderness, erythema, and induration) after each vaccination, and they will be instructed to report any local or systemic AEs on diary cards after each dose. The DLT observation period of each cohort will be from the first dose administration to 4 weeks after the last dose administration (Week 1 ⁇ Week 9) . Safety and efficacy will be evaluated up to 36 weeks from the first dose of vaccination according to the Schedule of Activities.
  • injection site reaction such as pain, tenderness, erythema, and induration
  • Part A After all subjects in Part A completed DLT observations, the investigator and the sponsor will review all available safety data, immunogenicity data, and efficacy data from Part A, to determine the recommended dose level and immunization schedule for Part B.
  • Part B is to further evaluate the efficacy and safety of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
  • Part B is planned to enroll 15 or 30 subjects with 1 or 2 dose levels according to the results of Part A.
  • Part B comprises the vaccination period (Week 1 ⁇ Week 5) and follow-up period (Week 6 ⁇ Week 37) .
  • the vaccination period eligible subjects will receive RG002 Injection at assigned dose level intramuscularly every 2 weeks, and complete safety and immunogenicity evaluation. From Week 13 to Week 37, all subjects will return to the research site regularly for follow-up evaluation of safety, immunogenicity and efficacy.
  • dose escalation of Phase 1 three subjects will be treated in each dose group, and the occurrence of DLT will be evaluated during the observation period, from the first vaccination to 4 weeks after the last vaccination. If ⁇ 2/3 subjects in any group experience DLT, dose escalation will be terminated, and the lower next dose level will be determined as MTD; if 1/3 subjects experience DLT, 3 more subjects will be enrolled in the same dose group; if ⁇ 1/3 of the 3 newly enrolled subjects experience DLT (i.e. ⁇ 2/6 in total) , dose escalation will be terminated, and the lower next dose level will be determined as MTD; if none of the additional 3 subjects experiences DLT, the dose will be escalated to the next dose. Dose escalation will stop when the MTD or maximum administered dose (in case no MTD is determined) is reached, which will be based on emerging data.
  • DLT is defined as any of the following toxicity that occurs during the DLT observation period (Week 1 ⁇ Week 9) in each cohort for Phase 1, except for those that are clearly and incontrovertibly due to disease progression or extraneous causes.
  • the toxicities below are evaluated by NCI CTCAE 5.0:
  • subjects are still considered DLT evaluable subjects if they discontinue study treatment or withdraw from the study due to DLT.
  • the subject will not be included in the final DLT observation. If the above situation occurs on any subject, one more subject should be recruited in the same dose level to ensure the minimum number of DLT evaluable subjects.
  • Phase 1 Part A of this study is a non-randomized trial, and no randomization will be performed.
  • Phase 2 Depending on the results of safety and efficacy data of the Part A (Phase 1) , if 2 cohorts are selected for Part B (Phase 2) , study protocol will be amended to include the randomization method.
  • the study may be prematurely terminated or suspended if there is a sufficient reason. If the study is terminated or suspended early, the sponsor should submit a written notice stating the reasons for the early termination or suspension to the investigator, the regulatory authority and the relevant department of the country or region where the study site is located. The investigator must immediately report to the IRB/IEC and provide the corresponding reasons.
  • Criteria for termination of the study include but are not limited to the following conditions:
  • End of study is defined as the last subject having completed the last protocol-specified visit or the study being prematurely terminated for any reason (please refer to Section 3.3) .
  • BMI Body mass index
  • CIN Grade 2 or 3 The lesion of CIN Grade 2 or 3 is large enough for histopathologic biopsy at screening and during treatment.
  • Adequate hematologic, renal, and hepatic function are determined by the Investigator, based upon medical history, physical examination, and laboratory test results at screening:
  • Bone marrow function absolute neutrophil count ⁇ 1, 500/ ⁇ L, hemoglobin (HGB) ⁇ 90 g/L, and platelets ⁇ 100/L.
  • Renal function creatinine ⁇ 1.5 ⁇ institutional upper limit of normal (ULN) .
  • Hepatic function total bilirubin ⁇ 1.5 ⁇ ULN, Aspartate aminotransferase (AST) and/or alanine transaminase (ALT) ⁇ 1.5 ⁇ ULN.
  • WOCBP Women of child-bearing potential
  • AIS Cervical adenocarcinoma in situ
  • HSV herpes simplex virus
  • cardiovascular and cerebrovascular diseases including but not limited to serious cardiac rhythm or conduction abnormalities, such as ventricular arrhythmia requiring clinical intervention; repeated 12-lead ECG with QTcF interval ⁇ 470 msec; acute coronary syndrome, congestive cardiac failure, aortic dissection, stroke or other Grade 3 or above cardiovascular and cerebrovascular events within 6 months before the first administration; New York Heart Association (NYHA) cardiac function classification ⁇ Grade III or hypertension that cannot be clinically controlled (systolic blood pressure ⁇ 150 mmHg, diastolic blood pressure ⁇ 100 mmHg) .
  • NYHA New York Heart Association
  • Hereditary hemorrhagic tendency or coagulation dysfunction or a history of thrombosis or hemorrhagic disease, or requirement of continuous use of anticoagulants.
  • Immunosuppression due to treatment for concurrent disease or medical history HIV treatment, antirheumatic drugs, organ or bone marrow transplantation or relevant treatment.
  • Subjects are currently participating or have participated in a study with an investigational drug or device within 30 days of signing informed consent.
  • Subjects who are judged by the investigator to have a history of other serious systemic diseases, or not suitable for participating in the trial for any other reason the subject has mental illness, alcohol abuse, drug use or drug abuse that may affect her compliance with the trial or may interfere with the interpretation of the study results) .
  • Subject may be “withdrawn” (or “dropped out” ) if they no longer receive medication and evaluation and are lost to follow-up, although they do not explicitly propose to withdraw from the study. Effort must be made to obtain and record the reason for withdrawal.
  • Subject withdrawal from the study is defined by the inability to reach the subject after a minimum of 3 documented phone calls, text messages, faxes or emails (on different days) . All attempts should be documented in the subject’s eCRF and the source document. Data that would have been collected at subsequent visits will be considered missing including subject number and identification. All subjects withdrawn due to AEs will be followed until resolution of any AEs until the unresolved AEs are judged by the investigator to have stabilized, or the patient is lost to follow-up.
  • Subjects may withdrawal or drop out for any reason except for DLT occurrence during the study, this subject will be allowed to be replaced.
  • the subjects in Part A and Part B will receive RG002 injection intramuscularly at assigned dose level on Day 1, Day 15 and Day 29 for a total of 3 doses.
  • the injection sites are the lateral deltoid muscle area of the left and right upper arms in turn.
  • dose adjustment may not be performed for AEs of any grade considered unrelated to the investigational drug (e.g., fracture due to bony metastases) or for laboratory abnormalities considered not to be of clinical significance.
  • RG002 injection should be suspended, and supportive care should be given according to the local practice/guidelines.
  • the adjustable dose levels of RG002 injection are 25 ⁇ g, 75 ⁇ g, 150 ⁇ g. If the dose needs to be adjusted to ⁇ 25 ⁇ g, the vaccination will be permanently discontinued.
  • the rules of dose adjustment are specified as in Table 3.

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Abstract

The invention provides a HPV mRNA lipid nanoparticle vaccine, comprising an open reading frame encoding a fusion protein of E2, E6 and E7 antigens of a high-risk HPV antigen. The invention also provides a method for treating and preventing HPV infection related diseases such as cervical cancer, cervical precancerous lesions, high-grade intraepithelial lesions of the cervix, high-grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasia (CIN).

Description

HUMAN PAPILLOMA VIRUS (HPV) MRNA VACCINES AND USES THEREOF TECHNICAL FIELD
The present application relates to a polynucleotide molecule encoding HPV antigen peptide, a delivery body comprising the polynucleotide molecule, a vaccine formulation, and a method for treating or preventing HPV infection related diseases.
BACKGROUND
Human papillomavirus (HPV) is a virus that has a circular, double-stranded DNA molecule as its genome without envelope membrane, and there are about 200 genotypes of this virus (Virology 2013; 445: 2e10) . The HPV genome has eight genes encoding viral proteins, which are classified into early genes (E1, E2, E4, E5 E6 and E7) and late genes (L1 and L2) according to the stage of their expression in viral life cycle. Early genes regulate viral replication and transformation of infected cells to cancer cells, while L1 and L2 are structural proteins which form a virus particle capsid (J Clin Virol 2005; 32 (Suppl. 1) : S7e15) .
Cervical cancer is one of the leading causes of death among women worldwide. Major risk factors associated with the development of cervical cancer include high-risk human papillomavirus (hrHPV) infection, age, smoking, childbirth, use of oral contraceptives, and diet. With the development of clinical research on cervical cancer, the World Health Organization has confirmed that cervical cancer is a cancer attributed to infection, HPV DNA has been identified in more than 99.7%of tumors biopsy specimens, and HPV16 and HPV18 have been identified in all cervical biopsy specimens. Among the hrHPVs, HPV16 and HPV18 are the most prevalent types, responsible for 50-60%and 10-20%of cervical cancer cases, respectively.
Continuous infection of HR-HPV causes the transformation of cervical cells from normal state into cervical intraepithelial neoplasia (CIN) grades 1, 2, and 3, and then into cervical cancer. The natural outcome of CIN is in three directions: regression, persistence and progression. The pre-malignant cervical intraepithelial neoplasia 2 and 3 (CIN2/3) , in particular those positive for HPV16 or 18, are considered as high-grade lesions that have approximately an 18%～40%chance of developing into invasive cancer. CIN3 has a higher risk of progressing to cancer, and the model estimates that 15%to 23%of untreated CIN3 will develop invasive cancer over an average of 13 years, with a lifetime risk of 40%. A retrospective cohort study showed that 31%of patients with untreated CIN3 progressed to invasive cancer within 30 years. The risk of CIN2 is somewhere between CIN1 and CIN3. Studies show that in CIN2, 50%of patients will resolve naturally within two years, 32%will persist, and 18%will progress. In young women younger than 30 years, 60%had regression, 23%continued, and 11%progressed.
Current methods of managing CIN2/3 are typically excisional and ablative surgical, including CKC, LEEP/LEETZ, laser ablation, or conical resection.
However, surgical resection does not necessarily solve the problem of HPV infection, and according to a retrospective multi-institutional study, it was 8.1%and 4%recurrence rate after LEEP and laser conization, respectively. The frequency and severity of adverse sequelae increases with increasing cone depth and is higher for excision than it is for ablation and there are pregnancy-related complications, such as preterm delivery, low birth weight, premature rupture of membrane, higher neonatal intensive care unit (NICU) admission rate and perinatal mortality.
Currently available prophylactic HPV vaccines (such asand) have been shown to be effective in preventing health women from HPV infection, but do not treat pre-existing HPV infections, pre-malignant lesions, or malignant lesions.
Therefore, there is an urgent need for an effective therapeutic vaccine that can eradicate HPV-related neoplasia without surgical manipulation.
EFFECT OF THE APPLICATION
From our experiments, the immunogen containing HPV16/18 E6 and E7 with N-terminus and C-terminus domains could induce strong specific cellular immune responses in vivo. The addition of E2 conserved peptides could further enhance specific cellular immune responses and effectively induce humoral immune responses against E2.
According to the present application, it is possible to induce effective cellular immune response, and potentially benefic patients with high-risk HPV related lesions. Accordingly, it is possible to prevent and/or treat infections with human papillomavirus. According to the present application, it is also possible to prevent and/or treat diseases caused by infections with HPV, such as cervical intraepithelial neoplasia (CIN) , cervical cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1A and 1B are the schematic diagram of the phase 1 and phase 2 study designs.
DETAILED DESCRIPTION
Herein below, embodiments of the present application will be described in detail.
Some abbreviations and definitions used in the present application are summarized in table 1 below:
Table 1: ABBREVIATIONS AND DEFINITIONS


In the First aspect, the present application provides a method for treating or preventing human papillomavirus (HPV) infection related diseases, comprising administering to a human subject at least one dose of a vaccine comprising a lipid nanoparticle (LNP) that comprises a messenger ribonucleic acid (mRNA) , wherein the mRNA comprises an open reading frame encoding a fusion protein of high-risk HPV antigen (s) , the one dose of the vaccine comprises 0.01μg～500μg of the mRNA.
In some embodiments, the amount of the mRNA in the one dose is selected from the group consisting of: 0.01μg～500μg, 0.05μg ～500μg, 0.1μg ～500μg, 1μg ～500μg, 2.5μg～500μg, 5μg～500μg, 10μg～500μg, 10μg～480μg, 10μg～450μg, 12.5μg～450μg, 25μg～450μg, 25μg～400μg, 25μg～350μg, 25μg～250μg, 25μg～200μg, 25μg～150μg, 50μg～350μg, 75μg～350μg, 75μg～300μg, 75μg～200μg, and 75μg～150μg.
In some embodiments, the amount of the mRNA in the one dose is selected from the group consisting of: 0.01μg, 0.015μg, 0.1μg, 0.5μg, 1μg, 2μg, 2.5μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12.5μg, 14μg, 15μg, 17.5μg, 18μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, 100μg, 105μg, 110μg, 120μg, 125μg, 130μg, 135μg, 140μg, 145μg, 150μg, 155μg, 160μg, 165μg, 170μg, 175μg, 180μg, 185μg, 190μg, 195μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, 310μg, 320μg, 330μg, 340μg, 350μg, 360μg, 370μg, 380μg, 390μg, 400μg, 410μg, 420μg, 430μg, 440μg, 450μg, 460μg, 470μg, 480μg, 490μg, and 500μg.
In some embodiments, the method further comprises administering to the subject a second dose of the vaccine.
In some embodiments, the second dose of the vaccine is administered at least 2 weeks after the first dose is administered.
In some embodiments, the method further comprises administering to the subject a third dose of the vaccine.
In some embodiments, the third dose of the vaccine is administered at least 2 weeks after the second dose is administered.
In some embodiments, the amount of the mRNA in the second dose or the third dose is selected from the group consisting of: 0.01μg～500μg, 0.05μg ～500μg, 0.1μg ～500μg, 1μg ～500μg, 2.5μg～500μg, 5μg～500μg, 10μg～500μg, 10μg～480μg, 10μg～450μg, 12.5μg～450μg, 25μg～450μg, 25μg～400μg, 25μg～350μg, 25μg～250μg, 25μg～200μg, 25μg～150μg, 50μg～350μg, 75μg～350μg, 75μg～300μg, 75μg～200μg, and 75μg～150μg.
In some embodiments, the amount of the mRNA in the second dose or the third dose is selected from the group consisting of: 0.01μg, 0.015μg, 0.1μg, 0.5μg, 1μg, 2μg, 2.5μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12.5μg, 14μg, 15μg, 17.5μg, 18μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, 100μg, 105μg, 110μg, 120μg, 125μg, 130μg, 135μg, 140μg, 145μg, 150μg, 155μg, 160μg, 165μg, 170μg, 175μg, 180μg, 185μg, 190μg, 195μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, 310μg, 320μg, 330μg, 340μg, 350μg, 360μg, 370μg, 380μg, 390μg, 400μg, 410μg, 420μg, 430μg, 440μg, 450μg, 460μg, 470μg, 480μg, 490μg, and 500μg.
In some embodiments, the amount of the mRNA in the first dose , second dose or the third dose is selected from the group consisting of: 25μg, 75μg or 150μg.
In some embodiments, the subject is a 9-70 years old subject.
In some embodiments, the subject is an 18-70 years old subject.
In some embodiments, the subject is an 18-55 years old subject.
In some embodiments, the subject is an 18-45 years old subject.
In some embodiments, the high-risk HPV antigen encoded by the ORF of the mRNA has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the amino acid sequence of SEQ ID NO: 3; in some embodiments, the high-risk HPV antigen encoded by the ORF of the mRNA comprises or consists of the amino acid sequence of SEQ ID NO: 3, or a conserved substitution variant of SEQ ID NO: 3. In some embodiments, the encoding sequence of the high-risk HPV antigen comprises or consists of a polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the encoding sequence of the high-risk HPV antigen comprises or consists of a synonymous mutant of SEQ ID NO: 10.
In some embodiments, the polynucleotide molecules further include coding sequences of immunostimulatory factors or their functional domains. In some embodiments, the coding sequence of the immunostimulating factor or its functional domain is located at the 3' or 5' end of the coding sequence of the HPV antigen peptide. In some embodiments, the immunostimulatory factor is Flt3L. In some embodiments, the polypeptide sequence of the immunostimulatory factor comprises at least an amino acid sequence of SEQ ID NO: 1, a conserved substituent variant of SEQ ID NO: 1, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 1. In some embodiments, the polypeptide sequence of the immunostimulating factor consists of the amino acid sequence of SEQ ID NO: 1, or a conserved substitution variant of SEQ ID NO: 1, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 1. In some embodiments, the encoding sequence of the immunostimulatory factor comprises or consists of a polynucleotide sequence of SEQ ID NO: 9. In some embodiments, the encoding sequence of the immunostimulating factor comprises or consists of a synonymous mutant of SEQ ID NO:9.
In some embodiments, the polynucleotide molecule further comprises a signal peptide encoding sequence. In some embodiments, the signal peptide encoding sequence is located at the 5' end of the coding sequence of the HPV antigen. In some embodiments, the signal peptide is a human secretory signal peptide. In some embodiments, the secretory signal peptide is tPA-SP. In some embodiments, the polypeptide sequence of the secretory signal peptide comprises an amino acid sequence of SEQ ID NO: 2, a conserved substituent variant of SEQ ID NO: 2, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 2. In some embodiments, the polypeptide sequence of the secretory signal peptide consists of an amino acid sequence of SEQ ID NO: 2, or a conserved substituent variant of SEQ ID NO: 2, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 2. In some embodiments, the encoding sequence of the secretory signal peptide comprises or consists of a polynucleotide sequence of SEQ ID NO: 8. In some embodiments, the encoding sequence of the secretory signal peptide comprises or consists of a synonymous mutant of SEQ ID NO: 8.
In some embodiments, the high-risk HPV antigen encoded by the ORF of the mRNA has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the high-risk HPV antigen encoded by the ORF of the mRNA comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a conserved substituent variant of SEQ ID NO: 4.
In some embodiments, the mRNA or polynucleotide molecule encoding the high-risk HPV antigen has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of a synonymous mutant of SEQ ID NO: 11.
In some embodiments, the mRNA or polynucleotide molecule further comprises a 5'UTR structure. In some embodiments, the mRNA or polynucleotide molecule comprises a 3'UTR structure. In some embodiments, the mRNA or polynucleotide molecule further comprises a 5'UTR structure and a 3'UTR structure. In some embodiments, the 5’ UTR structure comprises at least a polynucleotide sequence of SEQ ID NO: 5, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 5. In some embodiments, the 5’UTR structure consists of a polynucleotide sequence of SEQ ID NO: 5, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 5. In some embodiments, the 3'UTR structure comprises at least a polynucleotide sequence of SEQ ID NO: 6, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 6. In some embodiments, the 3'UTR structure consists of a polynucleotide sequence of SEQ ID NO: 6, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 6.
In some embodiments, the mRNA or polynucleotide molecule further comprises a 5'cap structure, preferably the 5'cap structure is m7G (5') ppp (5') (2'-OMeA) pG.
In some embodiments, the mRNA or polynucleotide molecule further comprises a poly (A) tail; preferably, the poly (A) tail sequence comprises at least 50, at least 60, or at least 100 A nucleotides; preferably, the poly (A) tail comprises or consists of a polynucleotide sequence as shown in SEQ ID NO: 7, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 7.
From the 5' end to the 3' end, the mRNA molecule of the present application sequentially comprises 5’ cap, 5'UTR, ORF, 3'UTR, and poly A tail, wherein the5’ cap, 5'UTR, ORF, 3'UTR, and poly A tail are operably connected to each other.
In some embodiments, the mRNA or polynucleotide molecule encoding the high-risk HPV antigen has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of the amino acid sequence of SEQ ID NO: 12.
In some embodiments, the mRNA or polynucleotide molecule encoding the high-risk HPV antigen comprises or consists of a synonymous mutant of SEQ ID NO: 12.
In some embodiments, part or all of the uridines in the mRNA molecule are chemically modified uridines, preferably 1-methyl-pseuduridine modification.
In some embodiments, the lipid nanoparticle (LNP) comprises ionizable lipid (s) , phospholipids, cholesterol, and polyethylene glycol (PEG) -lipids.
In some embodiments, the lipid nanoparticles comprise ionizable lipid (s) , phospholipids, cholesterol, and polyethylene glycol (PEG) –lipids; wherein the content of the ionizable lipid (s) is 35mol%-65mol%, the content of the sum of the phospholipids and cholesterol is 35mol%-65mol%, the content of the PEG–lipids is 0.5mol%-5mol%.
In some embodiments, the lipid nanoparticles comprise 40mol%-50mol%ionizable lipid (s) , 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 1.5mol%-2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 48mol%-49mol%ionizable lipid (s) , 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 1.5mol%-2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 15mol%phospholipids, 42.5mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 15mol%phospholipids, 43.5mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 48.5mol%ionizable lipid (s) , 5mol%phospholipids, 45mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 44.3mol%ionizable lipid (s) , 9.3mol%phospholipids, 45mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 5mol%phospholipids, 43mol%cholesterol, and 2.1mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 12.5mol%phospholipids, 45mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 46mol%ionizable lipid (s) , 11mol%phospholipids, 41mol%cholesterol, and 2mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 47.5mol%ionizable lipid (s) , 5mol%phospholipids, 45mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 13mol%phospholipids, 45mol%cholesterol, and 2mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 12.5mol%phospholipids, 35mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 8mol%phospholipids, 39.5mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 42.3mol%ionizable lipid (s) , 15mol%phospholipids, 40.3mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 46mol%ionizable lipid (s) , 11mol%phospholipids, 41mol%cholesterol, and 2mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 47.5mol%ionizable lipid (s) , 15mol%phospholipids, 35mol%cholesterol, and 2.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 48.5mol%ionizable lipid (s) , 15mol%phospholipids, 35mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 50mol%ionizable lipid (s) , 5mol%phospholipids, 43.5mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 40mol%ionizable lipid (s) , 15mol%phospholipids, 43.5mol%cholesterol, and 1.5mol%polyethylene glycol (PEG) –lipids.
In some embodiments, the lipid nanoparticles comprise 48mol%ionizable lipid (s) , 10.3mol%phospholipids, 39.6mol%cholesterol, and 2.1mol%polyethylene glycol (PEG) –lipids.
In the lipid nanoparticle composition of the present application, the molar ratio of the ionizable lipids, the sum of the phospholipids and the structural lipids, and the PEG-lipids is (35-65) : (35-65) : (0.5-5) ; wherein the 35-65 can be any value between 35-65, for example, it can be 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; the 0.5-5 can be any value between 0.5-5, for example, it can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.
In the embodiments of the present application, the above mol %is calculated based on the total moles of the lipids of lipid nanoparticle composition.
Preferably the lipid nanoparticle (LNP) futher comprises Ca2+, Mg2+, Zn2+ and /or Mn2+.
In some embodiments, the PEG –lipid is DMG-PEG2000, and the phospholipid is selected from DSPC or DOPE; preferably, the phospholipid is DSPC.
In some embodiments, the administration is intratumoral, perilymphatic, intradermal (ID) , or intramuscular injection; preferably intramuscular injection.
In some embodiments, the vaccine is administered to the subject once, twice, thrice, 4 times, 5 times, 6 times, 7 times, 8 times 9 times, 10 times or until the disease is alleviated or cured.
In some embodiments, the vaccine is administered to the subject for a total of 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses or 10 doses.
In some embodiments, the vaccine is administered to the subject every 7 days, every 10 days, every 14 days, every 21 days, every 28 days, every 35 days, every 60 days, every 90 days, or every 120 days.
In some embodiments, the vaccine is administered to the subject every 7 days, every 14 days, every 21 days or every 28 days.
In some embodiments, the vaccine is administered to the subject every 1 week or every 2 weeks, every 3 weeks or every 4 weeks.
In some embodiments, the vaccine is administered intramuscularly at assigned dose level, with administration frequency of every 2 weeks (D1, D15, and D29) for a total of 3 doses.
In some embodiments, the vaccine is administered intramuscularly at assigned dose level of 25μg, 75μg or 150μg mRNA respectively, with administration frequency of every 2 weeks (D1, D15, and D29) for a total of 3 doses.
In some embodiments, the method further comprises administering immune stimulating factors, chemotherapy, radiation therapy, and/or targeted therapy to an individual.
It is possible to express the E6 and E7 antigens of human papillomavirus in vivo or in vitro using the particle of the present invention. Therefore, the present invention provides a method of expressing the E6 and E7 antigens of human papillomavirus in vitro, comprising introducing into cells a composition containing the above-described lipid particle. Further, the present invention also provides a method of expressing the E6 and E7 antigens of human papillomavirus in vivo, comprising administering to a mammal a composition containing the above-described lipid particle. By expressing the E6 and E7 antigens of human papillomavirus in vivo, it is possible to induce immune response to human papillomavirus. As a result, it becomes possible to prevent and/or treat human papillomavirus infections.
In the Second aspect, the present application provides a use of any one of the vaccines described above in the manufacture of a medicament for inducing an immune response in a subject.
The present invention also provides a method of inducing immune response to human papillomavirus, comprising administering to a mammal a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) .
In the Third aspect, the present application provides a use of any one of the vaccines described above in treating or preventing HPV type 16 and/or 18HPV infection related disease, comprising administering to a mammal a composition containing the above-described mRNA, vaccine or the lipid nanoparticle (LNP) .
In some embodiments, the HPV type 16 and/or 18 HPV infection related disease is selected from the group consisting of: cervical cancer, cervical precancerous lesions, high-grade intraepithelial lesions of the cervix, high-grade squamous intraepithelial lesions (HSIL) , cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN2/3) , cervical intraepithelial neoplasia (CIN) grade 1 (CIN1) .
In some embodiments, the HPV type 16 and/or 18HPV infection related disease is cervical cancer.
In some embodiments, the HPV type 16 and/or 18 HPV infection related disease is Human Papillomavirus (HPV) 16 or 18 Associated Cervical Intraepithelial Neoplasia Grade 2 or 3 (CIN2/3) .
In the Fourth aspect, the present application provides a composition for use in the method described above.
The lipid particle encapsulating a nucleic acid molecule according to the present invention may be prepared by various methods, such as a thin film method, a reverse phase evaporation method, an ethanol injection method, an ether injection method, a dehydration-rehydration method, a detergent dialysis method, a hydration method, a freezing-thawing method, and so forth. For example, the lipid particle encapsulating a nucleic acid molecule may be prepared by the methods described in WO2015/005253. Alternatively, the lipid particle encapsulating a nucleic acid molecule according to the present invention can also be prepared by mixing a nucleic acid solution and a solution of lipids in a micro flow channel. For example, the lipid particle may be prepared with NanoAssemblrTM from Precision NanoSystems, according to the method described in the attached protocol.
The mean particle size of the particle of the present invention may be 30 nm to 300 nm, preferably 30 nm to 200 nm, and more preferably 30 nm to 100 nm.
The particle of the present invention may be used as a pharmaceutical drug or an experimental reagent. The particle of the present invention is usually added to a carrier (such as water, buffer, saline, etc. ) , and the resultant formulation (composition) may be introduced into a cell (in vitro) or administered to a mammal (in vivo) . When the composition is administered to a mammal, the carrier may be a pharmacologically acceptable carrier (e.g., saline) . Further, the particle of the present invention may also be prepared into such formulations as cream, paste, ointment, gel, lotion or the like that comprise fat, fatty oil, lanolin, vaseline, paraffin, wax, resin, plastic, glycols, higher alcohol, glycerol, water, emulsifier, suspending agent, and the like as base materials.
The particle of the present invention may be administered to a mammal such as human, mouse, rat, hamster, guinea pig, rabbit, pig, monkey, cat, dog, goat, sheep, cattle, etc. orally or parenterally through various routes such as intramuscular, intravenous, rectal, transdermal, transmucosal, subcutaneous or intradermal administration.
When the particle of the present invention is administered to a human, the mRNA in the particle may be administered, for example, at an approximate dose of 0.01μg～500μg per adult per administration either once or several times by intramuscular injection, subcutaneous injection, intradermal injection, intravenous infusion or intravenous injection. The dose and the number of times of administration may be changed appropriately depending on the type and symptoms of the disease, the age of the patient, administration route, etc.
When the particle of the present invention is used as an experimental reagent, it is possible to express the E6 and E7 antigens of human papillomavirus in vitro by introducing the particle into a cell in which expression of the E6 and E7 antigens of human papillomavirus is desired [e.g., HEK293 cells and cells derived therefrom (HEK293T cells, FreeStyle 293 cells, Expi293 cells, etc. ) , CHO cells, C2Cl2 mouse myoblast cells, immortalized mouse dendritic cells (MutuDC1940) , or the like] . The expression of the E6 and E7 antigens of human papillomavirus may be analyzed by detecting the E6 and E7 antigen proteins of human papillomavirus in samples based on Western blotting or by detecting peptide fragments specific to the E6 and E7 antigens of human papillomavirus based on mass spectrometry.
As used herein, the term “treat” refers to recovery, amelioration, relaxation and/or delaying the progression of clinical symptoms of diseases in patients who are developing infections with viruses or bacteria or diseases caused by such infections (e.g., precancerous lesion or cancer) .
As used herein, the term “prevent” refers to reducing the incidence rate of diseases caused by infections with viruses or bacteria. “Prevent” encompasses lowering the risk of progression of diseases caused by infections with viruses or bacteria, or reducing exacerbation of such diseases. Since the particle of the present invention induces protective immune response, the particle of the present invention shows effectiveness on prevention and/or treatment of the above-described diseases.
DETAILED DESCRIPTION OF THE STUDY
1. Introduction to RG002 Injection
1.1 Strengths of RG002 Injection
RG002 is the active ingredient as a non-self-amplifying mRNA, which is formulated in LNPs to ensure that RG002 is stabilized and encapsulated, and to facilitate its uptake into cells and release into the cytosol. The mRNA is designed to express the E2, E6, and E7 antigens of high-risk HPV16 and HPV18 types as a therapeutic vaccine when administrated by intramuscular injection.
1.2 Basic information of RG002 Injection
Product name: RG002 injection;
Strength: 75 μg/0.5 mL;
Route of administration: intramuscular injection;
Dose regimen: RG002 injection is administered intramuscularly as a series of 3 doses with a two-week interval;
Properties: colorless or milky clear liquid;
Solubility: soluble in water;
Concentration: 1.5 to 2.5 mg/mL;
Molecular structure: RG002 is a non-self-amplifying mRNA. The theoretical length of the mRNA sequence is 3518nt, the nucleotide sequence is shown as SEQ ID NO: 12; the fusion protein sequence of RG002 contained a total of 1071 amino acid residues, the amino acid sequence is shown as SEQ ID NO: 4. RG002 mRNA is encapsulated in an LNP composed of four lipids: ionizable lipid, DMG-PEG 2000, cholesterol, and DSPC. The lipid nanoparticles comprise 40mol%-55mol%ionizable lipid, 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 0.5mol%-2.5mol%PEG–lipids.
2. STUDY OBJECTIVES AND ENDPOINTS
2.1 Study Objectives
2.1.1 Part A (Phase 1 Stage)
2.1.1.1 Primary Objective of Part A
To assess the safety and tolerability of RG002 Injection in adult female subjects with HPV16/18 associated CIN2/3, and to determine the maximum tolerated dose (MTD) and/or RP2D of RG002 Injection.
2.1.1.2 Secondary Objectives of Part A
1) To assess the preliminary efficacy of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
2) To assess the immunogenicity of RG002 Injection in subjects HPV16/18 associated CIN2/3.
3) To assess the exposure levels of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
4) To assess the anti-drug antibody (ADA) to polyethylene glycol (PEG) of RG002 injection.
2.1.2 Part B (Phase 2 Stage)
2.1.2.1 Primary Objective of Part B
To assess the primary efficacy of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
2.1.2.2 Secondary Objectives of Part B
1) To assess the secondary efficacy of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
2) To assess the immunogenicity RG002 Injection in subjects with HPV16/18 associated CIN2/3.
3) To assess the exposure levels of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
4) To assess the ADA to PEG of RG002 injection.
5) To assess the safety and tolerability in subjects with HPV16/18 associated CIN2/3.
6) To optionally assess the biomarker of RG002 Injection in subjects with HPV16/18 associated CIN2/3.
2.2 Study Endpoints
2.2.1 Part A (Phase 1 Stage)
2.2.1.1 Primary Endpoints of Part A:
1) Incidence of adverse events.
2) Clinically significant changes in vital signs, physical examination, laboratory tests, 12-lead electrocardiogram (ECG) .
3) MTD and/or RP2D of RG002 Injection.
2.2.1.2 Secondary Endpoints of Part A:
2.2.1.2.1 Efficacy endpoints:
1) The proportion of subjects with histopathological regression to either Grade 1 Cervical Intraepithelial Neoplasia (CIN1) or normal in 36 weeks.
2) The proportion of subjects with clearance of HPV16 or HPV18, or both in 36 weeks.
3) The proportion of subjects with histopathological regression to CIN1 or normal and clearance of HPV16 or HPV18, or both in 36 weeks.
4) The histopathological regression over time for 36 weeks.
5) The clearance of HPV16 or HPV18 over time for 36 weeks.
2.2.1.2.2 Immunogenicity endpoints:
1) The level of cellular immune response in PBMC as measured by IFN-γELISPOT.
2) The immuno-phenotyping including the proportion of CD3+CD4+CD137+and CD3+CD8+CD137+ T lymphocytes in whole blood as measured by flow cytometry.
3) The expression of cytokines in serum, i.e., IL-1β, IL-1ra, IL-2, IL-6, IL-10, GM-CSF, TNF-α and IFN-γ measured byAssays.
4) Serum levels of anti-HPV16 and anti-HPV18 IgG antibodies as measured by Luminex.
2.2.1.2.3 Exposure endpoints: the plasma exposure level of RG002 Injection including mRNA as measured by RT-qPCR and the plasma exposure level of cationic lipids as measured by LC-MS/MS.
2.2.1.2.4 PEG-ADA endpoints: the serum titer of anti-PEG IgG and anti-PEG IgM as measured by ELISA.
2.2.2 Part B (Phase 2 Stage)
2.2.2.1 Primary Endpoint (Part B) :
Primary efficacy endpoint: the proportion of subjects with histopathological regression to either CIN1 or normal in 36 weeks.
2.2.2.2 Secondary Endpoints (Part B) :
2.2.2.2.1 Secondary efficacy endpoints:
1) The proportion of subjects with clearance of HPV16 or HPV18, or both in 36 weeks.
2) The proportion of subjects with histopathological regression to CIN1 or normal and clearance of HPV16 or HPV18, or both in 36 weeks.
3) The histopathological regression over time for 36 weeks.
4) The clearance of HPV16 or HPV18 over time for 36 weeks.
2.2.2.2.2 Safety endpoints:
1) Incidence of adverse events.
2) Clinically significant changes in vital signs, physical examination, laboratory tests, 12-lead ECG.
2.2.2.2.3 Immunogenicity endpoints:
1) The level of cellular immune response in PBMC as measured by IFN-γELISPOT.
2) The immuno-phenotyping including the proportion of CD3+CD4+CD137+and CD3+CD8+CD137+ T lymphocytes in whole blood as measured by flow cytometry.
3) The expression of cytokines in serum, i.e., IL-1β, IL-1ra, IL-2, IL-6, IL-10, GM-CSF, TNF-α and IFN-γ, measured byAssays.
4) Serum levels of anti-HPV16 and HPV18 total IgG antibodies as measured by Luminex.
2.2.2.2.4 Exposure endpoints: the plasma exposure level of RG002 Injection including mRNA as measured by RT-qPCR and the plasma exposure level of cationic lipids as measured by LC-MS/MS.
2.2.2.2.5 PEG-ADA endpoints: the serum titer of anti-PEG IgG and anti-PEG IgM as measured by ELISA.
2.2.2.2.6 Optional Biomarker endpoints:
1) The level of potential biomarkers of RG002 Injection, i.e., CD45, CD3, CD4, CD8, CD25, FOXP3, PD-1, and CD137.
2) The level of infiltrating T cells (CD3, CD8, FOXP3, TIM3, Tbet, PD-1, DAPI) and myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI) in the lesions.
3. STUDY DESIGN
3.1 Overall Design
This is a phase 1/2, open-label clinical study to evaluate the safety, tolerability, immunogenicity, and efficacy of RG002 Injection in subjects with HPV16/18 associated CIN2/3. The study has two parts (see Figures 1A-1B) . Part A is a phase 1 dose escalation study. Part B is a phase 2 proof-of-concept (PoC) study. The available data of Part A including safety, tolerability, immunogenicity, exposure levels and preliminary efficacy of RG002, will be reviewed to determine the RP2D. In Part B, one or two dose levels including RP2D will be evaluated to optimize the efficacy of RG002 Injection in the subjects with HPV16/18 associated CIN2/3. The schematic diagram of the phase 1/2 study design is shown in Figures 1A-1B.
3.1.1 Part A of Study (Phase 1 Stage)
In this part, dose escalation will start from 25 μg and the present 3 dose cohorts are 25 μg (Cohort A1) , 75 μg (Cohort A2) and 150 μg (Cohort A3) .
Part A will include 3 cohorts of 3 to 6 subjects each based on a “3+3” dose-escalation study design. A total of 9 to 18 subjects are planned to be enrolled. All subjects will receive three RG002 Injections at assigned dose level intramuscularly every 2 weeks (D1, D15, and D29) . Subjects will be evaluated for a minimum of 30 min for injection site reaction (such as pain, tenderness, erythema, and induration) after each vaccination, and they will be instructed to report any local or systemic AEs on diary cards after each dose. The DLT observation period of each cohort will be from the first dose administration to 4 weeks after the last dose administration (Week 1～Week 9) . Safety and efficacy will be evaluated up to 36 weeks from the first dose of vaccination according to the Schedule of Activities.
In order to decide on the escalation to the next dose level, all safety data, including injection site reactions, AEs and abnormal laboratory test results will be carefully reviewed by the sponsors and investigators from the first vaccination to 2 weeks after the last vaccination (Week 1～Week 7) .
3.1.2 Part B of Study (Phase 2 Stage)
After all subjects in Part A completed DLT observations, the investigator and the sponsor will review all available safety data, immunogenicity data, and efficacy data from Part A, to determine the recommended dose level and immunization schedule for Part B. Part B is to further evaluate the efficacy and safety of RG002 Injection in subjects with HPV16/18 associated CIN2/3. Part B is planned to enroll 15 or 30 subjects with 1 or 2 dose levels according to the results of Part A.
Part B comprises the vaccination period (Week 1 ～Week 5) and follow-up period (Week 6 ～ Week 37) . In the vaccination period, eligible subjects will receive RG002 Injection at assigned dose level intramuscularly every 2 weeks, and complete safety and immunogenicity evaluation. From Week 13 to Week 37, all subjects will return to the research site regularly for follow-up evaluation of safety, immunogenicity and efficacy.
3.1.3 Rules for dose escalation study of Phase 1
For dose escalation of Phase 1, three subjects will be treated in each dose group, and the occurrence of DLT will be evaluated during the observation period, from the first vaccination to 4 weeks after the last vaccination. If ≥ 2/3 subjects in any group experience DLT, dose escalation will be terminated, and the lower next dose level will be determined as MTD; if 1/3 subjects experience DLT, 3 more subjects will be enrolled in the same dose group; if ≥ 1/3 of the 3 newly enrolled subjects experience DLT (i.e. ≥ 2/6 in total) , dose escalation will be terminated, and the lower next dose level will be determined as MTD; if none of the additional 3 subjects experiences DLT, the dose will be escalated to the next dose. Dose escalation will stop when the MTD or maximum administered dose (in case no MTD is determined) is reached, which will be based on emerging data.
3.1.4 DLT Observation (Phase 1)
3.1.4.1 DLT Definition
DLT is defined as any of the following toxicity that occurs during the DLT observation period (Week 1～Week 9) in each cohort for Phase 1, except for those that are clearly and incontrovertibly due to disease progression or extraneous causes. The toxicities below are evaluated by NCI CTCAE 5.0:
3.1.4.1.1 Hematologic toxicities:
1) Grade ≥ 3 neutropenic fevers should be DLT regardless of duration.
2) Grade 4 neutropenia (without fever) for >5 days.
3) Any other Grade 4 hematologic toxicities.
3.1.4.1.2 Non-hematologic toxicities:
Grade ≥ 3 non-hematologic toxicities should be considered DLTs with the following exceptions:
1) Grade 3 fatigue <7 days.
2) Grade 3 nausea, vomiting, or diarrhea if well controlled within 72 hours.
3) Laboratory abnormalities that are asymptomatic and not requiring urgent intervention.
3.1.4.2 DLT evaluable subjects
During the DLT observation period, subjects are still considered DLT evaluable subjects if they discontinue study treatment or withdraw from the study due to DLT.
If the dose received by a subject during the DLT observation period is less than 100%of the scheduled dose in total for reasons other than toxicity, the subject will not be included in the final DLT observation. If the above situation occurs on any subject, one more subject should be recruited in the same dose level to ensure the minimum number of DLT evaluable subjects.
3.2 Randomization and Blinding
3.2.1 Randomization
Phase 1: Part A of this study is a non-randomized trial, and no randomization will be performed.
Phase 2: Depending on the results of safety and efficacy data of the Part A (Phase 1) , if 2 cohorts are selected for Part B (Phase 2) , study protocol will be amended to include the randomization method.
3.2.2 Blinding
This study is an open-label trial and is not blinded.
3.3 Premature termination of the study
The study may be prematurely terminated or suspended if there is a sufficient reason. If the study is terminated or suspended early, the sponsor should submit a written notice stating the reasons for the early termination or suspension to the investigator, the regulatory authority and the relevant department of the country or region where the study site is located. The investigator must immediately report to the IRB/IEC and provide the corresponding reasons.
Criteria for termination of the study include but are not limited to the following conditions:
1) Unexpected, significant or unacceptable risks to subjects are found.
2) Major errors are found in the protocol during the implementation of the trial.
3) The investigational drug/trial treatment is ineffective, or it is meaningless to continue the trial.
4) The sponsor decides to terminate the study due to reasons such as serious delay in the selection of subjects or frequent protocol deviations.
3.4 Definition of End of Study
End of study is defined as the last subject having completed the last protocol-specified visit or the study being prematurely terminated for any reason (please refer to Section 3.3) .
3.5 Early Discontinuation due to CIN Progression
According to the central pathologist penal review of colposcopy and/or biopsy results at Week 21 and Week 37, subjects with sufficient pathological evidence of CIN progression to invasive disease at Week 21 or Week 37 will undergo surgical resection of the lesion via a LEEP or CKC at the investigator’s discretion after evaluating the overall benefit and risk of RG002 Injection for the subject. The subjects will be discontinued from the study if surgical resection is required.
4. STUDY POPULATION
4.1 Inclusion Criteria
Subjects must meet all of the following criteria to be included in the study:
1) Written informed consent in accordance with study site guidelines.
2) Female 18～45 years of age when signing the ICF for Part A, and 18～55 years of age when signing the ICF for Part B.
3) Body mass index (BMI) ≤30 kg/m2.
4) Pathological diagnosis of CIN Grade 2 or 3 as confirmed by central pathological reviewers within 12 weeks prior to administration of first study vaccination.
5) The lesion of CIN Grade 2 or 3 is large enough for histopathologic biopsy at screening and during treatment.
6) Has a satisfactory colposcopy at screening, i.e., the entire lesion as well as the entire squamocolumnar junction (type 1 or 2 transformation zone) is visualizable by colposcopy;
7) Confirmed high-risk HPV infection with HPV16+ and /or HPV18+ by a commercially available high-risk DNA assay (e.g., HPV test from Roche) .
8) Adequate hematologic, renal, and hepatic function are determined by the Investigator, based upon medical history, physical examination, and laboratory test results at screening:
a) Bone marrow function: absolute neutrophil count ≥1, 500/μL, hemoglobin (HGB) ≥ 90 g/L, and platelets ≥ 100/L.
b) Renal function: creatinine ≤ 1.5 × institutional upper limit of normal (ULN) .
c) Hepatic function: total bilirubin ≤ 1.5 × ULN, Aspartate aminotransferase (AST) and/or alanine transaminase (ALT) ≤ 1.5 × ULN.
9) Women of child-bearing potential (WOCBP) agree to remain sexually abstinent, use medically effective contraception (i.e., complex contraception, male condom and spermicide, contraceptive patches, barrier methods, spermicide, etc. ) , from enrollment to 9 months after the last injection or have a partner who is sterile (i.e., vasectomy) .
10) Able and willing to comply with all study procedures.
4.2 Exclusion Criteria
Subjects who meet any of the following criteria will be excluded from the study:
1) Cervical adenocarcinoma in situ (AIS) , or atypical endometrial or glandular cells, or evidence of invasive cervical carcinoma on cervical biopsy within 12 weeks prior to administration of first study vaccination.
2) High-grade intraepithelial neoplasia or invasive carcinoma of vulva, vagina or anus.
3) History of severe allergy to any vaccine or serious hypersensitivity reaction to a known ingredient (e.g., PEG) of RG002 injection judged by the investigator.
4) Active infection with herpes simplex virus (HSV) .
5) Positive serological test at screening for HIV virus, active syphilis infection, or positive hepatitis B virus surface antigen (HbsAg) and the number of copies of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) ≥ 500 IU/mL (or 2500 copies, or the lower limit of the positive detection value of the study site) at screening, or HbsAg (-) , hepatitis B core antibody (HbcAb) (+) and the number of copies of HBV DNA ≥ 500 IU/mL (or 2500 copies, or the lower limit of the positive detection value of the study site) after treatment of HBV infection, or positive hepatitis C antibody (HCV-Ab) and hepatitis C virus (HCV) ribonucleic acid (RNA) ≥ ULN of the study site.
6) Subjects with a concurrent condition of fatty liver disease at screening.
7) Subjects with poorly controlled diabetes (fasting blood glucose ≥ 10mmol/L) after drug treatment at screening.
8) History of serious cardiovascular and cerebrovascular diseases, including but not limited to serious cardiac rhythm or conduction abnormalities, such as ventricular arrhythmia requiring clinical intervention; repeated 12-lead ECG with QTcF interval ≥ 470 msec; acute coronary syndrome, congestive cardiac failure, aortic dissection, stroke or other Grade 3 or above cardiovascular and cerebrovascular events within 6 months before the first administration; New York Heart Association (NYHA) cardiac function classification ≥ Grade III or hypertension that cannot be clinically controlled (systolic blood pressure ≥ 150 mmHg, diastolic blood pressure ≥ 100 mmHg) .
9) Major surgery (except for surgery for diagnostic purposes) within 4 weeks before the first administration, or expected to undergo major surgery (except for surgery for diagnostic purposes) during the study period; If major surgery occurred >4 weeks prior to the first administration of the study, individual must have recovered adequately from the toxicity and/or complications from the intervention prior to the first administration of the study.
10) Hereditary hemorrhagic tendency or coagulation dysfunction, or a history of thrombosis or hemorrhagic disease, or requirement of continuous use of anticoagulants.
11) Female subjects in pregnancy or lactation, or a positive result on a serum human chorionic gonadotropin (HCG) test at screening (Visit 1) or a positive urine pregnancy test pre-vaccination at Visit 2 (and at subsequent vaccination visits) .
12) Currently receiving or has received treatment with systemic steroids in the following dosages prior to administration of the first study vaccination.
a) Long-term corticosteroids: ≥0.5 mg/kg/day of oral prednisolone or equivalent, within 30 days prior to administration of the first study vaccination.
b) Sporadic corticosteroids: ≥1 mg/kg/day of oral prednisolone or equivalent for 2 or more short courses of > 3 days.
Note: Current or recent use of eye drop or inhaled glucocorticoid therapy is acceptable.
13) Immunosuppression due to treatment for concurrent disease or medical history: HIV treatment, antirheumatic drugs, organ or bone marrow transplantation or relevant treatment.
14) Systemic treatment for malignancy within 2 years of enrollment.
15) Administration of any blood product within 3 months of enrollment.
16) Administration of any vaccine within 4 weeks of enrollment.
17) A history of any therapeutic HPV vaccination (commercially approved prophylactic HPV vaccination is acceptable) .
18) Other condition or prior therapy that, in the opinion of the investigator, compromises the subject’s welfare or may confound study results.
19) Tattoos, scars, or active lesions/rashes within 2 cm of the intended site of vaccination (deltoid muscle) or any implantable leads that may affect the safety observation.
20) Adverse events that do not recover to grade 1 during the screening period.
21) Subjects are currently participating or have participated in a study with an investigational drug or device within 30 days of signing informed consent.
22) Subjects who are judged by the investigator to have a history of other serious systemic diseases, or not suitable for participating in the trial for any other reason (the subject has mental illness, alcohol abuse, drug use or drug abuse that may affect her compliance with the trial or may interfere with the interpretation of the study results) .
4.3 Discontinuation Criteria
Reasons for investigational drug discontinuation include but are not limited to the following:
1) Non-compliance with the eligibility criteria but wrongly enrolled.
2) The subject received forbidden concomitant treatment.
3) The investigator decides the treatment is unfavorable for the subject (e.g., pregnancy) .
4) AEs or SAEs evaluated by the investigator as not appropriate for the subject to continue the trial.
5) Poor compliance to the study procedures.
6) The subject refuses further treatment with study vaccine.
For all subjects in the study, discontinuation from study treatment will not result in automatic withdrawal from the study. Subjects that discontinue study treatment due to any reason except withdrawal of consent including safety follow-ups will need to visit sites as outlined below.
1) Study treatment discontinuation in Part A: The subject needs to complete the 4-week DLT observation after the last dose, followed by the follow-up period originally planned from Week 13 to Week 37.
2) Study treatment discontinuation in Part B: The subject needs to complete the 1-week safety observation after the last dose, followed by the follow-up period originally planned from Week 6 to Week 37.
The reason (s) a subject discontinues treatment will be recorded on the electronic case report form (eCRF) . Once a subject discontinues treatment with the study vaccine, the subject will not be permitted to be re-treated.
4.4 Withdrawal Criteria
A subject will be withdrawn from the study for any of the following reasons:
1) Lost to follow-up.
2) Withdrawal of consent for follow-up.
3) The Sponsor discontinues the study.
Subject may be “withdrawn” (or “dropped out” ) if they no longer receive medication and evaluation and are lost to follow-up, although they do not explicitly propose to withdraw from the study. Effort must be made to obtain and record the reason for withdrawal.
Subject’s withdrawal from the study is defined by the inability to reach the subject after a minimum of 3 documented phone calls, text messages, faxes or emails (on different days) . All attempts should be documented in the subject’s eCRF and the source document. Data that would have been collected at subsequent visits will be considered missing including subject number and identification. All subjects withdrawn due to AEs will be followed until resolution of any AEs until the unresolved AEs are judged by the investigator to have stabilized, or the patient is lost to follow-up.
Subjects may withdrawal or drop out for any reason except for DLT occurrence during the study, this subject will be allowed to be replaced.
5. STUDY TREATMENTS
5.1 Information of RG002 Injection
Table 2: Information of RG002 Injection
5.2 Vaccination and route of administration of RG002 Injection
The subjects in Part A and Part B will receive RG002 injection intramuscularly at assigned dose level on Day 1, Day 15 and Day 29 for a total of 3 doses. The injection sites are the lateral deltoid muscle area of the left and right upper arms in turn.
5.3 Dose adjustment of Phase 1
At the discretion of the investigator, dose adjustment may not be performed for AEs of any grade considered unrelated to the investigational drug (e.g., fracture due to bony metastases) or for laboratory abnormalities considered not to be of clinical significance.
During the study, if the subject has experienced any AE ≥ CTCAE v5.0 Grade 3 or/and intolerable toxicity, including DLTs that are not related to the disease or is judged by the investigator as being related to progressive disease, treatment with RG002 injection should be suspended, and supportive care should be given according to the local practice/guidelines. The adjustable dose levels of RG002 injection are 25 μg, 75 μg, 150 μg. If the dose needs to be adjusted to ＜ 25 μg, the vaccination will be permanently discontinued. The rules of dose adjustment are specified as in Table 3.
Table 3: The Dose Adjustment of RG002 Injection
*Notes: The general criteria are that all toxicities at least possibly related to study drug should recover to Grade ≤ 1 prior to re-dosing. Exceptions may be made as for the definition of DLT (see the Section 3.1.4) .
Sequencing Listing







Claims (32)

  1. A method for treating or preventing human papillomavirus (HPV) infection related diseases, comprising administering to a human subject at least one dose of a vaccine comprising a lipid nanoparticle (LNP) that comprises a messenger ribonucleic acid (mRNA) , wherein the mRNA comprises an open reading frame encoding a fusion protein of high-risk HPV antigen (s) , the one dose of the vaccine comprises 0.01μg～500μg of the mRNA.
  2. The method of claim 1, wherein the amount of the mRNA in the one dose is selected from the group consisting of: 0.01μg～500μg, 0.05μg ～500μg, 0.1μg ～500μg, 1μg ～500μg, 2.5μg～500μg, 5μg～500μg, 10μg～450μg, 12.5μg～450μg, 25μg～450μg, 25μg～400μg, 25μg～350μg, 25μg～250μg, 25μg～200μg, 25μg～150μg, 50μg～350μg, 75μg～350μg, 75μg～300μg, 75μg～200μg, and 75μg～150μg.
  3. The method according to claim 2, wherein the amount of the mRNA in the one dose is selected from the group consisting of: 0.01μg, 0.015μg, 0.1μg, 0.5μg, 1μg, 2μg, 2.5μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12.5μg, 14μg, 15μg, 17.5μg, 18μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, 100μg, 105μg, 110μg, 115μg, 120μg, 125μg, 130μg, 135μg, 140μg, 145μg, 150μg, 155μg, 160μg, 165μg, 170μg, 175μg, 180μg, 185μg, 190μg, 195μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, 315μg, 330μg, 350μg, 365μg, 380μg, 400μg, 415μg, 430μg, 450μg, 465μg, 480μg, and 500μg.
  4. The method of any one of claims 1-3, further comprising administering to the subject a second dose of the vaccine.
  5. The method of claim 4, wherein the second dose of the vaccine is administered at least 2 weeks after the first dose is administered.
  6. The method of claim 4 or 5, further comprising administering to the subject a third dose of the vaccine.
  7. The method of claim 6, wherein the third dose of the vaccine is administered at least 2 weeks after the second dose is administered.
  8. The method of any one of claims 4-7, wherein the amount of the mRNA in the second dose or the third dose is selected from the group consisting of: 0.01μg～500μg, 0.05μg ～500μg, 0.1μg～500μg, 1μg～500μg, 2.5μg～500μg, 5μg～500μg, 10μg～450μg, 12.5μg～450μg, 25μg～450μg, 25μg～400μg, 25μg～350μg, 25μg～250μg, 25μg～200μg, 25μg～150μg, 50μg～350μg, 75μg～350μg, 75μg～300μg, 75μg～200μg, and 75μg～150μg.
  9. The method according to claim 8, wherein the amount of the mRNA in the second dose or the third dose is selected from the group consisting of: 0.01μg, 0.015μg, 0.1μg, 0.5μg, 1μg, 2μg, 2.5μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12.5μg, 14μg, 15μg, 17.5μg, 18μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, 100μg, 105μg, 110μg, 115μg, 120μg, 125μg, 130μg, 135μg, 140μg, 145μg, 150μg, 155μg, 160μg, 165μg, 170μg, 175μg, 180μg, 185μg, 190μg, 195μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, 315μg, 330μg, 350μg, 365μg, 380μg, 400μg, 415μg, 430μg, 450μg, 465μg, 480μg, and 500μg.
  10. The method of any one of claims 1-9, wherein the high-risk HPV antigen encoded by the ORF of the mRNA has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the amino acid sequence of SEQ ID NO: 4.
  11. The method of claim 10, wherein the high-risk HPV antigen encoded by the ORF of the mRNA comprises or consists of the amino acid sequence of SEQ ID NO: 4.
  12. The method of any of claims 1-11, wherein the mRNA further comprises a 5'UTR structure and a 3'UTR structure.
  13. The method of any of claim 12, wherein the 5’ UTR structure comprises or consists of a polynucleotide sequence of SEQ ID NO: 5, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 5.
  14. The method of any of claim 12, wherein the 3'UTR structure comprises or consists of a polynucleotide sequence of SEQ ID NO: 6, or a polynucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 85%, or 80%sequence identity with SEQ ID NO: 6.
  15. The method of any one of claims 1-14, wherein the mRNA further comprises a 5'cap structure, preferably the 5'cap structure is m7G (5') ppp (5') (2'-OMeA) pG.
  16. The method of any one of claims 1-15, wherein the mRNA further comprises a poly (A) tail; preferably, the poly (A) tail sequence comprises at least 50, at least 60, or at least 100 A nucleotides; preferably, the poly (A) tail comprises or consists of a polynucleotide sequence as shown in SEQ ID NO: 7, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity with SEQ ID NO: 7.
  17. The method of any one of claims 1-16, wherein the mRNA encoding the high-risk HPV antigen has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%sequence identity to the polynucleotide sequence of SEQ ID NO: 12.
  18. The method of claim 17, wherein the mRNA encoding the high-risk HPV antigen comprises or consists of the amino acid sequence of SEQ ID NO: 12, or comprises or consists of a synonymous mutant of SEQ ID NO: 12.
  19. The method of any one of claims 1-18, wherein part or all of the uridines in the mRNA molecule are chemically modified uridines, preferably 1-methyl-pseuduridine modification.
  20. The method of any one of claims 1-19, wherein the lipid nanoparticle comprises ionizable lipid (s) , phospholipids, cholesterol, and polyethylene glycol (PEG) -lipids.
  21. The method of claim 20, wherein the lipid nanoparticles comprise 40mol%-55mol%ionizable lipid (s) , 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 1.5mol%-2.5mol%polyethylene glycol (PEG) –lipids;
    preferably 48mol%-49mol%ionizable lipid (s) , 10mol%-15mol%phospholipids, 35mol%-45mol%cholesterol, and 1.5mol%-2.5mol%polyethylene glycol (PEG) –lipids.
  22. The method of any one of claims 20-21, wherein the PEG –lipid is DMG-PEG2000, and the phospholipid is selected from DSPC or DOPE; preferably, the phospholipid is DSPC.
  23. The method of any one of claims 1-22 wherein the administration is intratumoral, perilymphatic, intradermal (ID) , or intramuscular injection; preferably intramuscular injection.
  24. The method according to any of claims 1-23, wherein the vaccine is administered to the subject twice, thrice, 4 times, 5 times, 6 times, 7 times, 8 times 9 times, or 10 times .
  25. The method according to any one of claims 1-24, wherein the vaccine is administered to the subject every 7 days, every 10 days, every 14 days, every 21 days, every 28 days, every 35 days, every 60 days, every 90 days, or every 120 days.
  26. The method according to any one of claims 1-25, further comprising administering immune stimulating factors, chemotherapy, radiation therapy, and/or targeted therapy to an individual.
  27. Use of the vaccine of any one of claims 1-26 in the manufacture of a medicament for inducing an immune response in a subject.
  28. Use of the vaccine of any one of claims 1-26 for treating or preventing HPV type 16 and/or 18 HPV infection related disease.
  29. The use of claim 28, wherein the HPV type 16 and/or 18HPV infection related disease is selected from the group consisting of: cervical cancer, cervical precancerous lesions, high-grade intraepithelial lesions of the cervix, high-grade squamous intraepithelial lesions (HSIL) , cervical intraepithelial neoplasia (CIN) grade 1, cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN2/3) .
  30. The use of claim 29, wherein the HPV type 16 and/or 18HPV infection related disease is cervical cancer.
  31. The use of claim 29, wherein the HPV type 16 and/or 18HPV infection related disease is Human Papillomavirus (HPV) 16 or 18 Associated Cervical Intraepithelial Neoplasia Grade 2 or 3 (CIN2/3) .
  32. A composition for use in the method of any one of claims 1-26 or in the use of any one of claims 27-31.
PCT/CN2025/073829 2024-01-30 2025-01-22 Human papilloma virus (hpv) mrna vaccines and uses thereof Pending WO2025162105A1 (en)

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