WO2023280220A1 - Variant de protéine s du coronavirus et son utilisation - Google Patents

Variant de protéine s du coronavirus et son utilisation Download PDF

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WO2023280220A1
WO2023280220A1 PCT/CN2022/104158 CN2022104158W WO2023280220A1 WO 2023280220 A1 WO2023280220 A1 WO 2023280220A1 CN 2022104158 W CN2022104158 W CN 2022104158W WO 2023280220 A1 WO2023280220 A1 WO 2023280220A1
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protein
protein variant
composition
nucleic acid
coronavirus
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PCT/CN2022/104158
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Chinese (zh)
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WO2023280220A9 (fr
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林金钟
卢静
谭舒丹
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复旦大学
上海蓝鹊生物医药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the field of biomedicine, in particular to a coronavirus S protein variant and its application in the preparation of vaccines.
  • the present application provides a coronavirus S protein variant, as well as nucleic acid molecules (including DNA and/or RNA) encoding it, which can stimulate the body to generate an immune response.
  • the S protein variant of the present application has a higher expression level, and when used to stimulate an immune response, it can produce higher total antibodies, and at the same time, protective antibodies/neutralizing antibodies also have Great improvement.
  • the S protein variants of the present application and their nucleic acid molecules can be prepared as vaccines.
  • mRNAs encoding S protein variants and lipid nanoparticles comprising them can be prepared as vaccines.
  • the vaccine of the present application can greatly improve the immune response, reduce the dosage and reduce side effects.
  • the present application provides a variant of the S protein, which does not contain a complete cytoplasmic tail domain compared with the S protein of the wild-type coronavirus.
  • the S protein variant comprises the amino acid sequence shown in any one of SEQ ID NO:8, 21, 24, 27, 30, 33, 35, 38, 41, 45, 59 and 61 .
  • the S protein variant comprises the amino acid sequence shown in SEQ ID NO:8.
  • the signal peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 1-2.
  • the present application provides an isolated nucleic acid molecule comprising a polynucleotide encoding the S protein variant described in the present application.
  • the nucleic acid molecule comprises at least one modified nucleotide.
  • the present application provides a vector comprising the nucleic acid molecule described in the present application.
  • the present application provides a cell comprising the nucleic acid molecule, and/or the vector.
  • the present application provides a composition comprising (1) mRNA encoding the S protein variant described in the present application, and (2) a delivery vehicle.
  • the delivery vehicle comprises cationic lipids.
  • the molar ratio of the cationic lipid is about 45% to about 55%.
  • the cationic lipid can be Dlin-MC3-DMA.
  • the chemical structural formula of the Dlin-MC3-DMA can be:
  • the delivery vehicle includes cholesterol.
  • the present application provides a pharmaceutical composition, the pharmaceutical composition comprising the S protein variant described in the present application, the nucleic acid molecule, the vector, the cell and/or the composition, and optionally a pharmaceutically acceptable carrier.
  • Figure 1A shows the in vitro expression results of the S protein variant mRNA transfected into 293T cells.
  • truncated cytoplasmic tail domain generally refers to a cytoplasmic tail domain in which there is an amino acid deletion relative to the intact cytoplasmic tail domain.
  • SARS severe Acute Respiratory Syndrome
  • SARS-CoV severe Respiratory Syndrome
  • SM102 generally refers to an ionizable amino lipid that has been combined with other lipids to form lipid nanoparticles.
  • the molecular structure of SM102 can be found in CAS 2089251-47-6.
  • the cytoplasmic tail domain of the S protein variant may be missing about 40 to about 60 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be missing about 30 to about 40 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be missing about 20 to about 30 amino acids at the C-terminus.
  • the coronavirus may be SARS-CoV virus.
  • the cytoplasmic tail domain of the S protein variant may be deleted at the C-terminus by about 14, about 15, about 16, about 17, About 18, about 19 or about 20 amino acids.
  • the cytoplasmic tail domain of the S protein variant may be deleted at the C-terminus by about 10, about 20, about 30, about 40, About 50 or about 60 amino acids.
  • the cytoplasmic tail domain of the S protein variant may be missing about 22 to about 64 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 22 to about 60 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 30 to about 60 amino acids at the C-terminus.
  • the cytoplasmic tail domain of the S protein variant may be missing about 40 to about 60 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 30 to about 40 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 20 to about 30 amino acids at the C-terminus.
  • the cytoplasmic tail domain of the S protein variant may be missing about 22 to about 64 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 22 to about 60 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 30 to about 60 amino acids at the C-terminus.
  • the cytoplasmic tail domain of the S protein variant may be missing about 40 to about 60 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 30 to about 40 amino acids at the C-terminus. In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be missing about 20 to about 30 amino acids at the C-terminus.
  • the nucleic acid molecule may be RNA.
  • the nucleic acid molecule may be mRNA.
  • the mRNA may include a 5' cap, a 5' untranslated region (5'UTR), an open reading frame, a 3' untranslated region (3'UTR) and a poly A tail.
  • said poly A can comprise the nucleotide sequence shown in SEQ ID NO:66.
  • the mRNA can include a 5' cap, a 5' untranslated region (5'UTR), an open reading frame, a 3' untranslated region (3'UTR) and a poly A tail; wherein the 5'UTR can be Comprising the nucleotide sequence shown in SEQ ID NO:64, the 5'UTR can include the nucleotide sequence shown in SEQ ID NO:65, and the poly A can include the nucleoside shown in SEQ ID NO:66 acid sequence, the open reading frame can comprise the nucleotide sequence shown in SEQ ID NO:7; the mRNA can comprise the nucleotide sequence shown in SEQ ID NO:67.
  • the lipid nanoparticles may comprise cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, pegylated lipids and/or structured Lipid, or a combination of the above
  • the lipid nanoparticle comprises one or more RNAs described herein, such as mRNA, and for example, mRNA encoding a S protein variant.
  • the nanolipid particles may comprise one or more (eg 1, 2, 3, 4, 5, 6, 7 or 8) non-cationic lipids.
  • the non-cationic lipids may include anionic lipids.
  • Anionic lipids suitable for lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine , N-glutarylphosphatidylphosphoethanolyl, and other neutral lipids with attached anionic groups.
  • the nano-lipid particles may also contain cholesterol.
  • the molar proportion of cholesterol in the lipid nanoparticles is about 5-15%, for example, about 6-14%, about 7-13%, about 8-12%, or about 9-11% %. In certain embodiments, the molar proportion of cholesterol in the lipid nanoparticles may be about 10%.
  • the present application provides a cell comprising the nucleic acid molecule, and/or the vector.
  • the cells may be prokaryotic cells, for example, Escherichia coli.
  • the cells may be eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells.
  • the cells may be mammalian cells, such as mouse cells, human cells and the like.
  • the vaccine may be a protein vaccine, which may contain the S protein variant.
  • the present application provides a method for producing antibodies against coronavirus, the method comprising administering the S protein variant, the nucleic acid molecule, the carrier, the cell, the composition, the drug Composition and/or said vaccine.
  • SARS-Cov-2 virus S protein (code name: SDC60Alb) containing Alb signal peptide and SARS virus S protein (SARS S).
  • SARS-Cov-2 virus SDC60 Alb protein has the following variants: full-length protein (FL), full-length protein (2P) containing K986P and V987P mutations, and the C-terminus of the full-length protein is truncated by 10, 18, Proteins with 20, 40, 60, and 67 amino acids (DC10, DC18, DC20, DC40, DC60, DC67);
  • the SARS S protein variants of SARS virus include: full-length protein (FL), full-length C-terminal truncated by 10 , 18, 40 amino acid proteins (DC10, DC18, DC40).

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Abstract

La présente invention concerne un variant de protéine S, qui ne contient pas de domaine de queue cytoplasmique complet par comparaison avec une protéine S d'un coronavirus de type sauvage. L'invention concerne en outre une molécule d'acide nucléique codant pour le variant de protéine S, ainsi que l'utilisation du variant de protéine S et de la molécule d'acide nucléique dans la préparation d'un vaccin.
PCT/CN2022/104158 2021-07-09 2022-07-06 Variant de protéine s du coronavirus et son utilisation WO2023280220A1 (fr)

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WO2024183687A1 (fr) * 2023-03-03 2024-09-12 上海蓝鹊生物医药有限公司 Vaccin à base de protéine ou d'arnm contre le nouveau coronavirus, son procédé de préparation et son utilisation

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XIONG HUA-LONG, WU YANG-TAO, CAO JIA-LI, YANG REN, LIU YING-XIA, MA JIAN, QIAO XIAO-YANG, YAO XIANG-YANG, ZHANG BAO-HUI, ZHANG YA-: "Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells", EMERGING MICROBES & INFECTIONS, vol. 9, no. 1, 1 January 2020 (2020-01-01), pages 2105 - 2113, XP055926484, ISSN: 2222-1751, DOI: 10.1080/22221751.2020.1815589 *
ZHANG YUFEI, HUANG KUN, WANG TING, DENG FEI, GONG WENXIAO, HUI XIANFENG, ZHAO YA, HE XINLIN, LI CHENGFEI, ZHANG QIANG, CHEN XI, LV: "SARS-CoV-2 Rapidly Adapts in Aged BALB/c Mice and Induces Typical Pneumonia", JOURNAL OF VIROLOGY, vol. 95, no. 11, 10 May 2021 (2021-05-10), US , pages 1 - 19, XP093018632, ISSN: 0022-538X, DOI: 10.1128/JVI.02477-20 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023236468A1 (fr) * 2021-07-09 2023-12-14 复旦大学 Variant de protéine s de coronavirus et son utilisation
WO2024183687A1 (fr) * 2023-03-03 2024-09-12 上海蓝鹊生物医药有限公司 Vaccin à base de protéine ou d'arnm contre le nouveau coronavirus, son procédé de préparation et son utilisation

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