WO2020143799A1 - Anticorps neutralisants a large spectre entierement humains - Google Patents

Anticorps neutralisants a large spectre entierement humains Download PDF

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WO2020143799A1
WO2020143799A1 PCT/CN2020/071524 CN2020071524W WO2020143799A1 WO 2020143799 A1 WO2020143799 A1 WO 2020143799A1 CN 2020071524 W CN2020071524 W CN 2020071524W WO 2020143799 A1 WO2020143799 A1 WO 2020143799A1
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antibody
variable region
light chain
heavy chain
chain variable
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PCT/CN2020/071524
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Chinese (zh)
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孙兵
孙晓玉
卢晓
凌志洋
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中国科学院分子细胞科学卓越创新中心
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
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    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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    • C07—ORGANIC CHEMISTRY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention belongs to the field of biomedicine. Specifically, the invention relates to two neutralizing monoclonal antibodies against influenza viruses.
  • Influenza is a highly contagious disease caused by influenza virus. It has always been a major threat to human health and caused significant economic losses. Around 5%-15% of the world’s population is infected with seasonal influenza virus each year, and 250,000-500,000 people die. In recent years, there have been frequent outbreaks of influenza in various places, seriously threatening people's health and production safety. At present, vaccines and antiviral drugs are the main methods for preventing and treating influenza, but the continuous emergence of new virus strains and drug-resistant virus strains makes the development of new drugs, broad-spectrum vaccines and broad-spectrum antibodies urgent.
  • oseltamivir and zanamivir, which are neuraminidase inhibitors that can inhibit the release of the virus.
  • Fapiravir developed by Fukuyama Chemicals, Japan
  • Fapiravir was approved for marketing, mainly acting on influenza virus polymerase, thereby inhibiting virus replication.
  • most influenza viruses are still sensitive to oseltamivir and zanamivir, but drug-resistant strains have gradually appeared in Japan and other regions. The emergence of drug-resistant strains has limited the large-scale of these small chemical drugs use.
  • Vaccination is the most effective way to prevent flu.
  • Seasonal vaccine components mainly include influenza A, H1N1, H3N2, and influenza B components.
  • WHO regularly monitors the epidemic strains in the northern and southern hemispheres and predicts the upcoming strains, and major manufacturers prepare influenza vaccines accordingly.
  • influenza viruses are highly susceptible to mutation, so this prediction is often inaccurate, leading to the pandemic of new influenza viruses.
  • the preparation cycle of the vaccine is very long. It takes about 9 months from the WHO announced the predicted strain results to the successful preparation of the vaccine, and this simply cannot quickly respond to the sudden outbreak of influenza virus.
  • the effect of vaccination also depends on the basic immunization conditions of the inoculated person. For the elderly and children and those with immunodeficiency, the flu vaccine does not induce good protective activity.
  • the purpose of the present invention is to provide a neutralizing monoclonal antibody of influenza virus and its application.
  • a heavy chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • V H -CDR1 shown in SEQ ID NO.1,
  • V H -CDR2 shown in SEQ ID NO. 2, and
  • V H -CDR3 shown in SEQ ID NO.3;
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 4.
  • an antibody heavy chain having the heavy chain variable region and the heavy chain constant region according to the first aspect of the present invention.
  • the constant region of the light chain is of human or murine origin.
  • a light chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • V L -CDR1 shown in SEQ ID NO.6,
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.9.
  • a light chain of an antibody having a light chain variable region and a light chain constant region according to the third aspect of the present invention.
  • the heavy chain constant region is of human or murine origin.
  • an antibody having:
  • the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
  • the antibody is an antibody specifically against HA protein of influenza virus.
  • the antibodies include: single chain antibodies (scFv), double chain antibodies, monoclonal antibodies, chimeric antibodies (such as human and mouse chimeric antibodies), murine antibodies, or humanized antibodies.
  • scFv single chain antibodies
  • monoclonal antibodies such as human and mouse chimeric antibodies
  • chimeric antibodies such as human and mouse chimeric antibodies
  • murine antibodies or humanized antibodies.
  • the antibody is a fully human antibody.
  • the antibody can bind all Group 2 influenza virus HA proteins and H1 subtype HA proteins.
  • the antibody can neutralize influenza virus strains of H1, H3, H7, H4 subtypes.
  • the antibody can inhibit the cleavage of HA0.
  • the antibody can inhibit the conformational change of HA induced by low pH.
  • the antibody recognizes the conformational epitope of HA.
  • the conformational epitope is located in HA2 Helix A.
  • the antibody recognizes the linear epitope of H3/H4/H14 HA.
  • the antibody recognizes the conformational epitopes of H7 and H1.
  • a recombinant protein is provided.
  • the recombinant protein has:
  • the tag sequence is selected from the group consisting of 6 ⁇ His tag, GGGS sequence, and FLAG tag.
  • the recombinant protein specifically binds to the HA protein of influenza virus.
  • a polynucleotide encoding a polypeptide selected from the group consisting of:
  • the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, and the light chain variable region according to the third aspect of the present invention such as The light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention; or
  • polynucleotide has the sequence shown in SEQ ID NO. 5, 10.
  • a vector containing the polynucleotide according to the seventh aspect of the present invention there is provided a vector containing the polynucleotide according to the seventh aspect of the present invention.
  • the vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a ninth aspect of the present invention there is provided a genetically engineered host cell containing the vector or genome of the eighth aspect of the present invention integrated with the polynucleotide of the seventh aspect of the present invention.
  • an immunoconjugate comprising:
  • Coupling moieties selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes.
  • the conjugate is selected from: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or capable of producing detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, pro Drug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or nanoparticles in any form, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • chemotherapeutic agents eg, cisplatin
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition is a spray.
  • the pharmaceutical composition is used to prepare a medicament for treating influenza virus infection.
  • a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, and a light chain according to the third aspect of the invention
  • Chain variable region, the light chain according to the fourth aspect of the invention, the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, or the tenth according to the invention The use of the immunoconjugate described in the aspect is for preparing a medicament, reagent, detection plate or kit.
  • the reagent, detection plate or kit is used to detect influenza virus.
  • the agent is used to treat or prevent influenza virus infection.
  • the reagent includes a chip and an antibody-coated immune particle.
  • a method for detecting influenza virus hemagglutinin HA protein in a sample there is provided a method for detecting influenza virus hemagglutinin HA protein in a sample.
  • a fourteenth aspect of the present invention there is provided a method for preparing a recombinant polypeptide, the method comprising:
  • the recombinant polypeptide is isolated from the culture, and the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.
  • an influenza virus epitope peptide is selected from the group consisting of:
  • the homology with the amino acid sequence shown in SEQ ID NO: 11 or 12 is ⁇ 90%, preferably ⁇ 95%, and is derived from a polypeptide of influenza virus HA2 Helix A region.
  • the epitope peptide is derived from the HA2 Helix A region of H3, H4, and H14 influenza viruses.
  • the epitope peptides include linear epitope peptides and conformational epitope peptides.
  • the epitope peptide may have a stereo conformation.
  • a sixteenth aspect of the present invention there is provided a nucleic acid molecule encoding the epitope peptide according to the sixteenth aspect of the present invention.
  • the nucleic acid molecule includes deoxyribonucleotides and messenger ribonucleotides.
  • the nucleic acid molecule includes naked form polynucleotide and wrapped form polynucleotide.
  • a vector which contains the nucleic acid molecule of the sixteenth aspect of the present invention.
  • an eighteenth aspect of the present invention there is provided a genetically engineered host cell containing the vector or chromosome of the seventeenth aspect of the present invention integrated with the nucleic acid of the sixteenth aspect of the present invention molecular.
  • a method of preparing the influenza virus epitope peptide of claim 15 comprising the steps of:
  • the method further includes mixing the epitope peptide obtained in (b), or mixing it with other influenza virus epitope peptides, thereby preparing an influenza virus epitope peptide combination.
  • the combination of epitope peptides can be a mixture or a combination of multiple epitope peptides connected in series.
  • composition comprising the influenza virus epitope peptide according to the fifteenth aspect of the present invention, the nucleic acid molecule according to the sixteenth aspect of the present invention, the present invention
  • the vector according to the seventeenth aspect of the invention, the host cell according to the eighteenth aspect of the invention, and a pharmaceutically acceptable carrier and/or adjuvant is provided.
  • the composition is a vaccine composition.
  • the vaccine is a broad-spectrum influenza vaccine.
  • the vaccine composition further contains an adjuvant.
  • the vaccine composition includes a nucleic acid vaccine composition.
  • the vaccine includes peptide vaccine, protein vaccine, mRNA vaccine and DNA vaccine.
  • an influenza virus epitope peptide according to the fifteenth aspect of the present invention, the nucleic acid molecule according to the sixteenth aspect of the present invention, and the seventeenth aspect of the present invention
  • the drugs include antibodies and therapeutic vaccines.
  • Figure 1 shows the results of volunteer serological testing.
  • A Detection of the binding activity of the serum of a volunteer on H3N2 and H1N1 HA protein. Coat A/HongKong/01/1968(H3N2) and A/California/06/2009(H1N1) HA protein at 4°C overnight, dilute serum samples at different multiples the next day, and load the samples for anti-human Fc HRP detection .
  • FIG. 2 shows a schematic diagram of the antibody acquisition experiment.
  • PBMC was isolated from a volunteer, labeled with IgG + CD19 + 1968H3N2 HA + single memory B cells that could specifically bind to HA were selected, cDNA was obtained by single-cell RT-PCR, and antibody variable region genes were obtained by nested-PCR Fragment, and construct the sequenced antibody gene to the corresponding heavy and light chain expression vector.
  • the heavy and light chain genes were co-transfected into CHO cells to express the antibody, and Protein G purified the antibody. Finally, the antibody with higher purity was obtained and used in the next functional experimental study.
  • Figure 3 shows the 12mAb amino acid sequence and germline gene source information.
  • A Amino acid sequence of 12mAb. The red font is the differential amino acid relative to the germline gene, and the gray shade is the CDR region.
  • Figure 4 shows that 12mAb can bind and neutralize the influenza viruses of Group1 and Group2.
  • A: 12mAb can combine different influenza viruses HA of Group1 and Group2. Use the OCTET RED 96 instrument to test the affinity of 12mAb for different HA. To use AHC sensor, follow the procedure below: baseline 120s-loading Ab 300s-baseline 240s-association 900s-disassociation 900s-regenaration 5s-baseline 5s-regenaration 5s-baseline 5s-regenaration 5s-baseline 5s.
  • the 12mAb antibody concentration is 20ug/ml, and the antigen concentration is set to six: 200nM-133nM-89nM-59nM-39nM-26nM. Calculate the Kd value.
  • 12mAb can neutralize the different influenza viruses of Group1 and Group2. After incubating the influenza virus with antibodies of different concentrations for 1h, they were added to MDCK cells that had been laid in advance, and the supernatant was removed after 24 hours of culture. The cells were fixed with 80% acetone for 10 minutes, and the virus titer was detected by anti-NP antibody by ELISA. Calculate IC 50 .
  • Figure 5 shows that 12mAb shows better ability to bind and neutralize H3N2 influenza virus than MEDI8852.
  • AI plated with 0.5ug/ml HA protein, blocked with 2% BSA, and then added 12mAb, MEDI8852, respectively, 3 times dilution from 10ug/ml, anti-human-IgG Fc detection, OD 450 read the value.
  • Figure 6 shows that 12mAb can protect mice from influenza virus infection.
  • mice were divided into 5 groups, 5 mice in each group, the control group was inoculated with PBS, and the experimental group was inoculated with 12mAb of 1mg/kg, 3mg/kg, 10mg/kg, and 30mg/kg, respectively.
  • Dose of influenza virus Weigh for 14 consecutive days.
  • mice were divided into 5 groups, 5 mice in each group, all with lethal dose of influenza virus, the control group was inoculated with PBS, and the experimental group was inoculated with 25mg/kg 12mAb on days 1, 2, and 3, respectively. Weigh for 14 consecutive days.
  • Figure 7 shows that the 12mAb mainly recognizes the conformational epitope of Helix A of HA and its vicinity.
  • Figure 8 shows that 12mAb recognizes the linear epitope of H3 evolutionary branch HA.
  • A-D Detection of the binding capacity of 12mAb to H3 subtype denatured HA protein.
  • E-G 12mAb binding ability of H4/H14 subtype denatured HA protein.
  • H-I 12mAb binding ability of H7/H1 subtype denatured HA protein.
  • AI 0.5ug/ml HA protein and denatured HA protein plate, 2% BSA blocked, and then add different antibodies, 5ug/ml, anti-human-IgG Fc detection, OD 450 read the value.
  • J, K, M 0.5ug/ml of different peptide coated plates, blocked with 2% BSA, and then added different antibodies separately, different antibodies starting from 10ug/ml, 3 times gradient dilution, anti-human-IgG Fc detection, OD 450 Read the value.
  • Figure 9 shows that 12mAb inhibits the fusion of the host cell membrane with the viral envelope by inhibiting HA0 digestion into HA1 and HA2, and inhibiting the conformational changes of HA induced by low pH, thereby blocking the virus from invading cells.
  • A HAI activity detection of 12mAb. 4HA units/25ul of influenza virus were incubated with different concentrations of antibodies in equal volumes at 37°C for 1h, and the antibody was diluted 2-fold from 100ug/ml. Then add to 1% chicken red blood cells, wait 30 minutes at room temperature, and observe the blood clotting.
  • B 12mAb inhibits the ability to detect membrane fusion.
  • HA protein and TPCK were digested at 1:50 ratio at 0, 30, 60, 90, and 120 min, and Western Blot was used to detect HA0.
  • D: 12mAb inhibits the conformational change of HA induced by low pH.
  • Figure 10 shows that H3 Helix A linear peptide can better induce broad-spectrum antibodies.
  • A Detection of the binding activity of 12mAb to H1 and H3 VLP. 0.5ug/ml VLP plate overnight, 2% BSA blocked for 2h, antibody 5ug/ml loaded, incubated for 2h, goat-anti human Fc-HRP secondary antibody diluted 8000 times and loaded for 1h, TMB color development, detection of OD 450 .
  • BC Detection of the binding activity of immune serum on H1 and H3 polypeptides. 0.5ug/ml polypeptide was plated overnight, blocked with 2% BSA for 2h, serum was started from 600 times, 3 times diluted to load, incubated for 2h, goat-anti mouse Fc-HRP secondary antibody was diluted 6000 times to load and incubated for 1h, TMB showed Color, detect OD 450 .
  • DH detection of the binding activity of immune serum to different HA proteins. 0.5ug/ml polypeptide was plated overnight, 2% BSA was blocked for 2h, serum was started from 600 times, 3 times diluted to load, incubated for 2h, goat-anti mouse Fc-HRP secondary antibody was diluted 6000 times to load and incubated for 1h, TMB showed Color, detect OD 450 .
  • the mechanism study shows that 12mAb further inhibits the fusion of host cell membrane and viral envelope by inhibiting the conformational change of HA induced by low pH value, thereby inhibiting the virus from invading the host cell.
  • Epitope studies have shown that 12mAb can recognize both the conformational epitope of HA protein and the linear epitope of HA. This will provide new candidate therapeutic drugs for conquering influenza, and also enhance the understanding of the mechanism of influenza antibody binding to antigens, and provide new ideas for the design of broad-spectrum influenza vaccines.
  • Influenza viruses belong to the single-stranded RNA virus of the Orthomyxoviridae family and can be divided into three subtypes of A, B, and C (A, B, and C). Influenza A viruses can be further divided into different subtypes according to the antigenicity of hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. Currently, there are 18 HA subtypes (H1–H18) and 9 NA subtypes (N1–N9) of influenza A viruses. The genome of influenza A virus consists of 8 separate single-stranded RNA fragments, each of which encodes one or more proteins.
  • HA hemagglutinin
  • NA neuraminidase
  • the HA protein plays an important role in binding cell surface receptors and mediating the fusion of the viral envelope with the host cell membrane. Before infecting cells, HA will be hydrolyzed into HA1 and HA2. Among them, the area near the receptor binding domain (RBD) on HA1 is highly variable among different influenza virus strains (RBD itself is more conservative), which is important Most of the neutralizing epitopes are located in the area around the RBD of HA1 protein, such as S139/1 and 5j8, but most of these antibodies are not neutralizing different subtypes. Or the ability of different strains of influenza virus of the same subtype.
  • a WHO survey shows that about 5-10% of adults and 20-30% of children worldwide suffer from seasonal influenza each year, resulting in 3-5 million hospitalizations and 250,000-500,000 deaths.
  • Seasonal epidemic strains are mainly H1N1, H3N2 and influenza B viruses.
  • the above data does not include millions of deaths caused by several famous influenza pandemics, and outbreaks caused by avian influenza viruses such as H7N9, H5N1, H5N6 and even H1N1.
  • the course of influenza virus infection is about 14 days. In severe cases, the disease deteriorates rapidly to death within 20 days after the onset.
  • specific antiviral drugs such as Tamiflu are also given for treatment, due to the rapid replication of the virus later in the disease The titer is too high, and Duffy’s inhibitory effect is limited and cannot save lives.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced disulfide bonds in the chain. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain parts of the variable region of an antibody differ in sequence, which forms the binding and specificity of various specific antibodies for their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669) (1991)).
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity involved in antibodies.
  • immunoglobulins can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of its constant region. According to the amino acid sequence of the constant region of its heavy chain, immunoglobulins can be divided into different types. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • the term "monoclonal antibody (mAb)” refers to antibodies obtained from a substantially homogeneous population, that is, the individual antibodies contained in the population are the same, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigen site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (usually with different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of antibodies and is obtained from a homogeneous antibody population. This should not be interpreted as requiring any special method to produce antibodies.
  • the present invention also includes a monoclonal antibody having the corresponding amino acid sequence of the anti-influenza virus HA protein monoclonal antibody, a monoclonal antibody having the anti-influenza virus HA protein monoclonal antibody variable region chain, and having these chains Of other proteins or protein conjugates and fusion expression products.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a hypervariable region (complementarity determining region, CDR), as long as the The hypervariable region is the same as or at least 90% homologous to the hypervariable regions of the light chain and heavy chain of the present invention, preferably at least 95% homologous.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules and the anti-influenza virus HA protein monoclonal Conjugates formed by the binding of antibodies or fragments thereof.
  • the invention also includes cell surface markers or antigens that bind to the anti-influenza virus HA protein monoclonal antibody or fragments thereof.
  • the present invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity, such as Fab or (Fab") 2 fragments; antibody heavy chain; antibody light chain.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes the following three complementarity determining region CDRs:
  • V H -CDR1 the amino acid sequence of which is SEQ ID NO.1;
  • V H -CDR2 the amino acid sequence of which is SEQ ID NO. 2;
  • V H -CDR3 the amino acid sequence of which is SEQ ID NO.3.
  • amino acid sequence of the heavy chain variable region (V H ) is SEQ ID NO. 4, and the encoding nucleotide sequence is SEQ ID NO. 5.
  • the heavy chain of the antibody includes the above heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
  • the light chain variable region of the antibody according to the present invention has a complementarity determining region CDR selected from the group consisting of:
  • V L -CDR1 the amino acid sequence of which is SEQ ID NO. 6;
  • V L -CDR2 the amino acid sequence of which is SEQ ID NO.7;
  • V L -CDR3 the amino acid sequence of which is SEQ ID NO. 8;
  • amino acid sequence of the light chain variable region (V L) of SEQ ID NO.9 which encodes the nucleotide sequence of SEQ ID NO.10.
  • the light chain of the antibody includes the above light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin.
  • antibody of the present invention refers to antibodies that specifically bind to influenza virus, for example, having a heavy chain variable region (such as SEQ ID The protein or polypeptide of the amino acid sequence shown in NO. 4) and/or the light chain variable region (such as the amino acid sequence shown in SEQ ID NO. 9). They may or may not contain starting methionine.
  • the antibody is a mouse or human mouse chimeric monoclonal antibody against influenza virus HA protein, and its heavy chain constant region and/or light chain constant region may be humanized heavy chain constant Region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, IgG2, or the like.
  • the invention also provides other proteins or fusion expression products with the antibodies of the invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having heavy and light chains containing variable regions, as long as the variable region and the antibody of the present invention
  • the variable regions of the heavy chain and the light chain are the same or at least about 90% homologous, preferably at least about 95% homologous.
  • variable regions which divide this segment into 4 framework regions (FR)
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the ⁇ sheets formed by the FRs in between are close to each other in space structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because at least part of them are involved in binding antigen. Therefore, the present invention includes those molecules having light chain and heavy chain variable regions of monoclonal antibodies with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably 98%) of the CDRs identified here Above).
  • the present invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the antibody of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues May or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of a polypeptide, for example (Polyethylene glycol) fusion polypeptide, or (iv) the additional amino acid sequence is fused to the polypeptide sequence to form the polypeptide (such as the leader sequence or secretion sequence or the sequence used to purify the polypeptide or proprotein sequence, or 6His tag fusion protein).
  • the antibody of the present invention refers to a polypeptide having the HA protein binding activity of influenza virus, including the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides containing the above CDR regions that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , Insertion and/or substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybridized with DNA encoding the antibody of the present invention under high or low stringency conditions
  • the encoded protein and the polypeptide or protein obtained by using antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the invention also includes fragments of the antibodies of the invention.
  • the fragment has at least about 50 consecutive amino acids, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the invention.
  • “conservative variant of the antibody of the present invention” refers to at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 compared to the amino acid sequence of the antibody of the present invention Amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservatively variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the present invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO. 5, 10 or a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence that encodes the same amino acid sequence as the polypeptide of the present invention, but differs from the coding region sequences shown in SEQ ID NOs. 5 and 10.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optionally additional coding sequences) and a non-coding sequence .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide further including additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) Only the identity between the two sequences is at least 90%, more Fortunately, hybridization occurs only when it is over 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptides shown in SEQ ID NO.4 and SEQ ID NO.9.
  • the full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use synthetic methods to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • the present invention also relates to vectors containing the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • transformation can also be carried out by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by a conventional method and express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the cultivation is carried out under conditions suitable for the growth of host cells.
  • the selected promoter is induced by an appropriate method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method may be expressed in a cell, on a cell membrane, or secreted out of the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibody of the present invention may be used alone, or may be combined or coupled with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modified portion, or a combination of any of these.
  • a detectable marker for diagnostic purposes
  • a therapeutic agent for therapeutic purposes
  • a PK (protein kinase) modified portion or a combination of any of these.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or are capable of producing detectable products Of enzymes.
  • Therapeutic agents that can be coupled include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptides, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.
  • Radionuclides Karl et al., 2005, Cancer Metastasis Reviews 24, 539
  • Biological toxicity Choaudhary et al. , 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy
  • Nanoparticles/nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 5. Virus particles (Peng et al. , 2004, Gene therapy 11,1234); 6. Liposomes (Mamot et al., 2005, Cancer research 65,11631); 7. Nanomagnetic particles; 8. Prodrug activating enzymes (eg , DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind influenza virus HA protein molecules, and thus can be used to extend the half-life of the drug, in addition, other therapeutic agents can be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and pharmaceutical Acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 10 mg/kg body weight per day.
  • the polypeptide of the present invention can be used together with other therapeutic agents
  • a safe and effective amount of an immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases does not exceed about 8 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of skilled physicians.
  • the antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, it can be obtained from the memory B cells in the peripheral blood of volunteers by flow-single cell PCR technology; obtained by the phage display system; obtained by immunizing transgenic humanized mice.
  • Monoclonal antibodies can be appropriately separated by conventional immunoglobulin purification processes such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity And chromatography.
  • the invention provides a monoclonal antibody against influenza virus, especially a monoclonal antibody against influenza virus HA protein.
  • the antibody of the present invention is a fully human antibody, to avoid immune rejection caused by clinical mouse antibody.
  • the antibodies of the present invention can broadly neutralize a variety of Group 1 and Group 2 influenza A viruses, effectively responding to the variation of influenza viruses and the alternating prevalence of different subtypes.
  • the recognition epitope of the antibody of the present invention is unique, that is, it can recognize the HA conformation epitope of the H3 evolutionary branch strain, and can also recognize its linear epitope.
  • the antibody of the present invention recognizes a linear epitope of H3 evolutionary branched strain HA, namely HA2 37-55 Helix A.
  • This epitope is a dominant epitope that can induce the production of a broad-spectrum antibody, which is a broad-spectrum for influenza Vaccine design provides new ideas.
  • experimental materials used in the embodiments of the present invention can be obtained from commercially available channels.
  • mice Female BalB/c mice aged 6-8 weeks were purchased from Lingchang Company.
  • Fully human antibody expression vectors IgG1, Ig ⁇ and Ig ⁇ (respectively expressing antibody heavy chain, kappa chain, lambda chain) were donated by Patrick Wilson laboratory, the vector sequence is NCBI GenBank: FJ475055 (IgG1), FJ475056 (Ig ⁇ ) and FJ517647 (Ig ⁇ ) ; A/HongKong/01/1968 (H3N2) HA sequence and A/Washington/20172011 (H1N1) HA sequence were purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; DH5 ⁇ chemical competence was purchased from Qingke Biotechnology Co., Ltd.
  • the experimental cells human embryonic kidney epithelial cell line HEK293T and horse-Daer canine kidney cell line MDCK, are kept by the laboratory for themselves (originally purchased from ATCC).
  • Chinese hamster ovary cells CHO were purchased from Thermo Fisher Scientific.
  • PrimeSTAR HS DNA Polymerase and Ligase Solution I were purchased from Takara; EcoRI, KpnI and other DNA restriction enzymes were purchased from Thermo Fisher Scientific; AgeI, SalI and BsiWI and other DNA restriction enzymes were purchased from NEB; Marker II and Marker IV Purchased from Beijing Transgen Biotechnology Co., Ltd.; Ordinary DNA spin column type PCR product purification/gel DNA recovery dual-purpose kit purchased from Qingke Biotechnology Co., Ltd.; FavorPrepTM spin column type plasmid small extraction kit purchased from Favorgen; plasmid large The extraction kit Nucleobond xtra Maxiplus was purchased from MACHEREY-NAGELMN; the RNA extraction kit Rneasy MiNi Kit was purchased from Qiagen; the inversion kit ReverTra RT Kit purchased from TOYOBO; purified cDNA kit SV Gel and PCR Clean-Up System available from Promega; KOD Fx Neo Polymerase was purchased from TOYOBO Company, Taq Ex Polymerase purchased from Takara Company
  • MEDI8852 (Kallewaard et al., 2016, Cell), FI6v3 (Davide Corti et al., 2011, Science), F10 (Jianhua Sui et al., 2009, NS&MB), CR8020 (Damian C. Ekiert et al., 2011, Science), S139 /1 (Peter S. Lee et al., 2012, PANS), 5j8 (Jens C. Krause et al., 2011, Journal of virology) antibody heavy and light chain variable region amino acid sequences were obtained from the corresponding articles above, and through The gene sequence was obtained by codon optimization, and the whole gene was synthesized by Jerry Biotechnology Co., Ltd. ()()()()()()()().
  • LB broth Tryptone (peptone) 10g, Yeast Extract (yeast) 5g, NaCl 10g, add ddH 2 O to a total volume of 1000mL, 121 °C, 20min.
  • LB solid plate medium Tryptone (peptone) 10g, Yeast Extract (peptone) 5g, NaCl 10g, Agar (agar) 15g, dd H 2 O was added to a total volume of 1000mL, 121 °C, 20min. Cool to room temperature and add antibiotics.
  • ELISA blocking solution 2% BSA/PBS (wt/vol), 2g bovine serum albumin into 100mL PBS, mix well.
  • ELISA washing solution add 500 ⁇ L of Tween-20 to 1L of PBS and mix well.
  • ELISA color developing solution 1 ⁇ TMB ELISA substrate solution (ELISA Substrate Solution), purchased from ebioscience.
  • SDS gel loading buffer 50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 1% ⁇ -mercaptoethanol and 0.1% bromophenol blue.
  • 10 ⁇ SDS-PAGE electrophoresis buffer Tris base (trimethylolaminomethane) 30g, glycine (Glycine 144g), SDS 10g, constant volume to 1L.
  • 10 ⁇ transfer buffer 10 ⁇ SDS-PAGE electrophoresis buffer 100 mL, 200 mL methanol, add ddH 2 O to 1 L, mix and use.
  • TBST buffer 50 mM Tris, 150 mM NaCl and 0.1% Tween-20, pH 7.5.
  • Binding buffer 20 mM sodium phosphate buffer (Sodium Phosphate Buffer), pH 7.0; 4.372g Na 2 HPO 4 ⁇ 7H 2 O, first dissolved in 800 mL of distilled water, adjusted to pH 7.0 with 1M HCl or 1M NaOH, and then added Distilled water to a volume of 1L.
  • Elution buffer 0.1M Glycine (glycine), pH 2.8, 500mL 3.75g Glycine is dissolved by adding 400mL of distilled water, and then about 1.4mL of concentrated HCl is added to adjust the pH to 2.8, and then bring the volume to 500mL with distilled water.
  • Neutralization buffer 1M Tris, PH 9.0, 12.1g Tris base was first dissolved in 80mL of distilled water, adjusted with 1M HCL to pH 9.0, and then added distilled water to bring the volume to 100mL.
  • TPCK-Trypsin 12.5mg is added to 50ml PBS to make 100*2.5ug/ml TPCK-Trypsin solution. Trypsin needs to be protected from light.
  • pH4.8 citric acid First, citric acid is formulated with PBS into a very high concentration solution, and then citric acid is added dropwise to the rotating PBS to adjust the pH to 4.8.
  • Coomassie brilliant blue staining solution Coomassie brilliant blue R-250 0.75g, 100% ethanol 135ml, glacial acetic acid 30ml, ddH 2 O 135ml, stir well.
  • Coomassie Brilliant Blue Decolorizing Solution 100ml of glacial acetic acid and 100ml of methanol, make up to 1L with dd H 2 O, and mix well for use.
  • Constant temperature incubator purchased from Shanghai Experimental Instrument Factory Co., Ltd.
  • High temperature heater (GL-150): purchased from its Limbel Instrument Manufacturing Co., Ltd.
  • Constant temperature shaking incubator (HZ-2210): purchased from Taicang Hualida Experimental Equipment Company.
  • Oscillating mixer (VORTEX-5): purchased from its Limber Bell Instrument Manufacturing Co., Ltd.
  • Ultra-low temperature refrigerator (-80°C): purchased from SANYO company.
  • pH meter (delta 320pH Meter): purchased from Mettler Toledo.
  • Ultraviolet-visible spectrophotometer (NanoDrop ND1000): purchased from Thermo Company.
  • Microplate reader (Multiskan MK3): purchased from Thermo Company.
  • Octet RED 96 protein interaction workstation using the public platform instrument of Shanghai Institute of Biochemistry and Cell, Chinese Academy of Sciences, purchased from ForteBio.
  • the carbon dioxide constant temperature cell incubator was purchased from Thermo.
  • the carbon dioxide constant temperature shaking cell incubator was purchased from Thermo.
  • the HA of A/HongKong/1968 H3N2 is biotin labeled, so that biotin-HA can be combined with Cy3-Streptavidin to achieve the purpose of labeling HA with Cy3.
  • Peripheral blood was drawn from the volunteers and subjected to conventional Ficoll-Paque density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • B cells that specifically bind to HA were obtained by flow cytometry to a 96-well RT-PCR plate, one cell per well.
  • the obtained single memory B cells were obtained by RT-PCR to obtain cDNA, and then the antibody gene variable region was obtained by nested-PCR, run agarose nucleic acid gel, recover the gel block that the heavy and light chains can match and connect it to the T- On the vector, sequenced and retrieved the antibody gene sequence through the IgBLAST website (https://www.ncbi.nlm.nih.gov/projects/igblast/). Next, the antibody genes were linked to the corresponding IgG1, Ig ⁇ and Ig ⁇ expression vectors through AgeI and SalI restriction sites, AgeI and BsiwI restriction sites, AgeI and XhoI restriction sites.
  • ExpiCHO-S TM cells were sorted to a final density of 3 ⁇ 10 ⁇ 6–4 ⁇ 10 ⁇ 6 viable cells/mL, allowing the cells to grow overnight. The next day (day 0), the viable cell density and the percentage of survival were determined. The cell density should reach approximately 7 ⁇ 10 ⁇ 6–10 ⁇ 10 ⁇ 6 viable cells/mL. The survival rate should be 95–99% before continuing transfection. The cells were diluted to a final density of 6 ⁇ 10 ⁇ 6 viable cells/mL. ExpiFectamine TM CHO/plasmid DNA complex was prepared using pre-chilled reagent (4°C).
  • Protein G Agarose 4FF media to purify antibodies.
  • the collected CHO cell suspension was centrifuged at 4000 rpm and 4°C for 30 min, and the collected supernatant was filtered again with 0.45 um filter to be purified.
  • Take a gravity spin column add Protein G Agarose 4FF packing, stabilize the packing with 3 times the column volume of 20% ethanol, then equilibrate the column with 5 times the column volume of binding buffer, then load the sample, and then use 10 times the column volume of the binding
  • the column was equilibrated with buffer, and finally the column was eluted with 3 times the column volume of elution buffer.
  • the neutralized buffer was added to the eluted antibody solution to make the eluted sample pH around 7.5.
  • the antibody solution was dialyzed 3 times in 5L 1XPBS, and the antibody was concentrated and stored at -80°C.
  • ELISA was used to detect whether the expressed antibodies recognized influenza virus HA proteins of different subtypes. Coated with HA protein ELISA plate, 5 ⁇ g/mL, 100 ⁇ L per well, overnight at 4°C. The next day PBST washes the plate 3 times. Blocked with 2% BSA, 200 ⁇ L per well, 37°C, 2h. Wash the plate 3 times with PBST again. Sample loading, 100 ⁇ L per well, 37°C, 2h. Wash the plate 3 times with PBST. Goat Anti-Human IgG (Fc specific)-Peroxidase antibody, diluted 1:8000, 100 ⁇ L per well, 37°C, 1h. Wash the plate 3 times with PBST. Add substrate TMB 100 ⁇ L/well for color development. Add 2M H 2 SO 4 to stop the reaction, 50 ⁇ L per well. Measure OD 450 and perform data processing.
  • MDCK was plated on 96-well plates (1-11 columns, AH rows), and grown to 90% after 12 hours. After 12h, the virus stock solution was diluted with DMEM + 0.2% BSA + 1 ⁇ g/ml TPCK in 96-well plates (1-10 column 10-1 ⁇ 10-10, 8 repeats in AH row). MDCK cells aspirated the culture solution, washed once with PBS, 100 ⁇ l of the corresponding dilution virus was added to each well of column 1-10. Column 11 is a blank control, and DMEM + 0.2% BSA + 1 ⁇ g/ml TPCK is added. AH line 8 repeats. Incubate at 37°C, 5% CO 2 for 20 h.
  • MDCK was plated on 96-well plates and grown to 90% after 12 hours.
  • the antibody 12 mAb was diluted from 100 ug/ml, and diluted eight times with DMEM + 0.2% BSA, with a final volume of 50 ⁇ l/well. Add 50 ⁇ l of virus containing 100 TCID 50 to each well (pre-dilute to 100 TCID 50 /50 ⁇ l with DMEM + 0.2% BSA).
  • Virus control (11 columns A-D) 50 ⁇ l DMEM+0.2% BSA+50 ⁇ l virus;
  • NC Cell control: (11 columns E-H) 100 ⁇ l DMEM + 0.2% BSA.
  • MDCK cells were aspirated from the culture medium, washed twice with PBS, and added 100 ⁇ l of DMEM + 0.2% BSA + 2 ⁇ g/ml TPCK. All 100 ⁇ l of the serum-virus incubation mixture was transferred to MDCK cells. Incubate at 37°C and 5% CO 2 for 20h. Aspirate the supernatant, wash the cells once with PBS, and fix the pre-chilled 80% acetone-PBS solution with 100 ⁇ l/well for 10 min. The absorbance was measured by anti-NP antibody by ELISA. analyze data.
  • mice 6-8 weeks old BalB/c female mice were divided into 5 groups, 4-6 mice/group, and the mice in the 5 groups on day 0 were inoculated with 12mAb of 30mg/kg, 10mg/kg, 3mg/kg, 1mg/kg respectively Antibodies and PBS.
  • mice were anesthetized with 0.5% pentobarbital sodium, and lethal doses of influenza virus were challenged nasally by the mice (PR8-A/HongKong/01/1968(H3N2), PR8-A/Shanghai/02/2013( H7N9), A/Sichuan/01/2009 (H1N1)).
  • the mice were then weighed for 14 consecutive days. When the body weight of the mouse drops by 25%, the mouse is considered dead, and the mouse is killed according to animal ethical principles.
  • Transfected cells express HA protein:
  • the FUGW-HA plasmid system is transfected as follows:
  • Plasmid 80ul opti-MEN + 2ug/well plasmid
  • Lipo 80ul opti-MEN + 2ul/well Lipofectamine 2000 (liposome 2000),
  • Antibody incubation remove supernatant, wash cells twice, incubate 12mAb antibody and control antibody and control without antibody group, antibody concentration is 100ug/ml, incubate for 1 hour; enzyme digestion treatment: collect antibody, wash cells twice and then use 2.5ug/ml TPCK incubate the cells for 10min, 500ul/well; antibody incubation: wash the cells twice after enzyme digestion, and then conduct antibody incubation, incubate 12mAb antibody and control antibody and control without antibody group, the antibody concentration is 100ug/ml, Incubate for 1 hour; acid treatment: after washing the cells twice, incubate the cells with a pH 4.8 citric acid solution for 10 min at 500 ul/well; culture fusion: after washing the cells twice, add DMEM + 10% FBS + P/S culture solution Incubate for 2h; observe whether cells are confluent after 2h; fixation: wash cells with PBS after 2h, try to remove dead cells, then fix cells with 4% PFA for half an hour, fix
  • the binding activity of 12mAb to normal HA and denatured HA was detected by ELISA.
  • Denatured protein treatment method dilute the protein in a small volume, then add 0.1% SDS and 50mM DTT, and cook at 100 degrees for 5 minutes. Cover the plate with 1ug/ml normal HA protein and denatured HA protein overnight; wash the plate 3 times with PBST, block with 2% BSA buffer for 2h; load, 12mAb, 5ug/ml, incubate for 2h; wash the plate 3 times with PBST, anti-human -Fc-HRP 8000-fold dilution loading test, TMB substrate color development, detection OD 450 reading.
  • centrifuge tubes are prepared and placed on the sample collection rack; loading: suck the sample with a syringe and drive away Bubble into the instrument, about 40-50min; collect samples: collect samples according to the peak map, mark the sample tube; rinse the column with water, the flow rate is set to 0.5ml/min, at least one column volume; the collected samples are run SDS- PAGE, Coomassie brilliant blue staining to identify protein purity.
  • the inventors selected the HA protein of H3N2 (Group2) and H1N1 (Group1), and detected the antibody titer in the serum by ELISA, the results showed The serum of this volunteer had antibodies that recognized the HA of H3N2 and H1N1 ( Figure 1, A).
  • the neutralizing titer of the serum was detected by the virus micro-neutralization experiment. The results showed that there were indeed antibodies in the serum of the volunteers that could neutralize H3N2 and H1N1 ( Figure 1, B).
  • Single cell technology is used to screen antigen-specific memory B cells. Nested-PCR technology is used to obtain antibody genes and successfully express purified antibodies on CHO cells.
  • the inventors obtained a gene that specifically recognized the antibody of influenza virus HA protein by single-cell RT-PCR technology, and expressed and purified it in vitro.
  • the specific method was as follows ( Figure 2): 20ml of peripheral blood of the volunteer was drawn and isolated PBMC, to isolate memory B cells that specifically bind to influenza virus HA by flow cytometry; single-cell reverse transcription PCR to obtain cDNA, and then to obtain heavy and light chain variable region genes of single cell antibody genes by two rounds of nested-PCR ; Sequence the heavy and light chains of the paired antibody genes to obtain the base sequences of the above antibody variable region genes, and construct them on an antibody expression vector with antibody constant regions; transiently transfect CHO cells to express antibodies; collect CHO cell supernatants Use Protein G to purify antibodies by affinity chromatography to obtain antibodies with higher purity; perform functional evaluation on the obtained antibodies to screen out effective antibodies.
  • the 12mAb amino acid sequence and germline genes were analyzed. It was found that the 12 mAb heavy chain V region was derived from germline IGHV3-48 * 02, and the light chain V region was derived from IGKV1-12 * 01. The heavy and light chain genes have 8 or 9 somatic mutations relative to germline genes. Another special place is the 12mAb heavy chain CDR3 (ie VH- CDR3 or HCDR3) up to 23 amino acids ( Figure 3, A, B), which is very rare in all antibodies, which suggests that HCDR3 may be at 12mAb The process of recognizing antigen HA plays an important role.
  • VH- CDR3 or HCDR3 up to 23 amino acids
  • the strains selected in the experiment are: A/HongKong/01/1968(H3N2), A/Shanghai/02/2013(H7N9), A/Sichuan/01/2009(H1N1), of which H3N2 and H7N9 are PR8
  • H3N2 and H7N9 are PR8
  • the recombinant strains constructed by the HA and NA genes of A/HongKong/01/1968 (H3N2) and A/Shanghai/02/2013 (H7N9) were recombined.
  • BalB/c mice were divided into 5 groups, 5 in each group, the control group was inoculated with PBS, and the experimental group was inoculated with 12mAb of 1mg/kg, 3mg/kg, 10mg/kg, 30mg/kg, and the lethal dose of influenza was challenged after 24h virus. Weighed for 14 consecutive days, and plotted the weight change curve and survival curve of the prevention experiment.
  • the antigen protein was denatured to investigate whether 12mAb can bind HA linear epitope.
  • Denature the HA protein by adding DTT and SDS and boil at 100 degrees, linearize it, and wrap the plate to the ELISA plate, and compare with the undenatured HA.
  • ELISA detects 12mAb to denature H3/H4/H14 (Group2, H3clade) , H7 (Group2, H7clade), and H1 (Group1) HA and unmodified H3/H7/H1 HA binding activity.
  • Other antibodies have been reported as experimental controls.
  • the previous results show that 12mAb can recognize both the conformational epitope of HA and the linear epitope of HA.
  • the conformational epitope is mainly Helix A. Does the recognized linear epitope are also located on Helix A? H3 HA2 37-55 amino acid polypeptide, and the binding activity of 12mAb to the above two peptides was detected by ELISA.
  • H3 and H1 HA2 37-55 amino acid comparison found that a total of 6 amino acids are different ( Figure 8, L), and single-point mutation of H3 HA2 37-55 polypeptide to amino acid on H1.
  • the results showed that 12mAb could not bind to the N49T mutant polypeptide at all, and the binding activity of the 12mAb to the L52V mutant polypeptide was also significantly reduced (Figure 8, N), indicating that N49 and L52 are the key amino acids for the 12mAb to recognize the H3N2 linear epitope.
  • 9,12mAb inhibits the fusion of host cell membrane and viral envelope by inhibiting HA0 digestion into HA1 and HA2, and the conformational change of HA induced by low pH, thereby blocking the virus from invading the cell and clearing the infected virus.
  • HA is located on the surface of influenza virus and mainly mediates the binding process of the virus and the sialic acid receptor of the host cell and the membrane fusion process of the viral envelope and the host cell membrane.
  • the mechanism of 12mAb neutralizing influenza virus may be one of the above two mechanisms. First, in order to investigate whether the 12mAb inhibits the binding process of the virus to the sialic acid receptor of the host cell, the virus was incubated with different concentrations of 12mAb for 1h and added to 1% chicken red blood cells to detect whether the chicken red blood cells agglomerated.
  • HA is digested by trypsin-like proteases produced by respiratory epithelial cells into HA1 and HA2, induced by the acidic environment in the body , HA undergoes a conformational change, exposing the fusion peptide and bringing the fusion peptide closer to the host cell membrane, thereby inducing membrane fusion.
  • trypsin-like proteases produced by respiratory epithelial cells into HA1 and HA2
  • HA undergoes a conformational change, exposing the fusion peptide and bringing the fusion peptide closer to the host cell membrane, thereby inducing membrane fusion.
  • H3N2 and H1N1 HA0 were digested at different times with the addition of 12mAb and control antibody, and the presence of HA0 was detected by Western Blot.
  • H3 Helix A linear peptide can better induce broad-spectrum antibodies
  • the aforementioned research proves that 12mAb recognizes the HA2 37-55 linear peptide of H3 HA, namely Helix A, but cannot recognize the H1 HA2 37-55 linear peptide. Whether the H3 polypeptide can be used as a new target for vaccine design.
  • the 37-55 polypeptides of H3 and H1 were recombinantly constructed into HBV core protein, and the recombinant VLP was expressed by E. coli expression system.
  • Therapeutic antibodies have been reported for the treatment of influenza and other viral infectious diseases.
  • the case of antiserum for the treatment of SARS and severe H5N1 avian influenza virus infection has proved the important role of antibodies in the treatment of viral infections.
  • the therapeutic antibody against influenza virus with broad-spectrum neutralizing activity has the following potential advantages. On the one hand, it can block the binding of the virus to the target cells. On the other hand, through the action of complement and effector cells such as T cells and NK cells, it will The cells infected by the virus are killed.
  • Murolib-CD3 murmonabCD3, hoclone OKT3
  • HAMA human anti-mouse antibodies in the human body
  • MEDI8852 developed by Medimmune has the best effect. MEDI8852 can neutralize almost all Group1 and Group2 influenza viruses. Ferrets show good protection. The US FDA granted this antibody fast lane status and is currently in clinical phase II. Most broad-spectrum influenza virus antibodies are mainly targeted at the stalk region of influenza virus hemagglutinin, mainly including fusion subdomains, fusion peptides, and outer ⁇ -sheets. This part of the region is a relatively conserved position of hemagglutinin. It's not very prone to mutation. Previously reported antibody epitopes are mostly conformational epitopes, but the discovery of linear epitopes is also very important for the design of influenza broad-spectrum vaccines.
  • Antibody of the invention 12mAb
  • the present invention screened a single human broad-spectrum neutralizing antibody, 12 mAb, by single cell RT-PCR technology.
  • the V region of the antibody heavy chain is derived from the IGHV3-48 * 02 germline.
  • H3 HA2 37-55 recombinant polypeptide VLP can induce broad-spectrum antibodies, but the H1 polypeptide is not. This suggests that H3 HA2 37-55 polypeptide is a potentially dominant epitope.
  • recognition epitopes of 12mAb are very special, and the discovery of new epitopes provides new targets and ideas for the design of influenza vaccines.

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Abstract

Font l'objet de la présente invention des anticorps monoclonaux dirigés contre le virus de l'influenza ayant une activité de fixation à un antigène du virus de l'influenza.
PCT/CN2020/071524 2019-01-10 2020-01-10 Anticorps neutralisants a large spectre entierement humains WO2020143799A1 (fr)

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