WO2023179548A1 - Construction de nano-anticorps s43 et son utilisation - Google Patents

Construction de nano-anticorps s43 et son utilisation Download PDF

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WO2023179548A1
WO2023179548A1 PCT/CN2023/082534 CN2023082534W WO2023179548A1 WO 2023179548 A1 WO2023179548 A1 WO 2023179548A1 CN 2023082534 W CN2023082534 W CN 2023082534W WO 2023179548 A1 WO2023179548 A1 WO 2023179548A1
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cov
sars
nanobody
seq
strain
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王奇慧
高福
刘红辉
刘博�
仵丽丽
韩鹏程
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中国科学院微生物研究所
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    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from 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, specifically to the construct and application of Nanobody S43, and more specifically to multivalent Nanobodies, Nanobody fusion proteins, and coding based on Nanobody S43 that specifically binds SARS-CoV-2 RBD.
  • Polynucleotides thereof, nucleic acid constructs containing the polynucleotides, expression vectors containing the nucleic acid constructs, transformed cells containing the above polynucleotides, nucleic acid constructs or expression vectors, and medicines containing any of the above products The composition and its application in the preparation of drugs for preventing or treating the new coronavirus, and its application in the preparation of reagents or kits for detecting the new coronavirus or diagnosing the new coronavirus infection.
  • the current COVID-19 outbreak has promoted the development of various vaccines and antiviral drugs.
  • Vaccination can effectively prevent the occurrence of serious infectious diseases.
  • vaccines are only suitable for uninfected people, and the development cycle is long and the clinical research process is complex. For confirmed patients, they can only be treated with antiviral drugs.
  • One of the antiviral drugs is therapeutic antibody drugs, which mainly refer to neutralizing antibodies.
  • Neutralizing antibody drugs mainly prevent the infection by binding to antigens on the surface of pathogenic microorganisms. Specific molecules expressed by pathogenic microorganisms bind to cell surface receptors to achieve a "neutralization" effect.
  • the SARS-CoV-2 virus has a glycosylated spike protein (S) on its surface. The S protein can interact with the host cell receptor protein ACE2 and trigger membrane fusion. Therefore, blocking the binding of the S protein to ACE2 is An effective way to treat COVID-19 infection.
  • S glycosylated spike protein
  • Nanobodies have attracted much attention as therapeutic drugs.
  • the nanobody drug Caplacizumab (Cablivi TM ) developed by Ablynx is used to treat acquired thrombotic thrombocytopenic purpura and is the first nanobody drug approved for marketing;
  • the nanobody drug candidate ALX-0171 is a trivalent form of nanobody used to treat respiratory syncytial virus (RSV) infection in children. It is administered by aerosol and has entered the clinical phase II stage (https://clinicaltrials .gov), these all suggest that nanobody drugs are safe and feasible.
  • RSV respiratory syncytial virus
  • nanobody drugs targeting the new coronavirus and suitable for respiratory mucosal immunity has potential clinical application value and prospects.
  • the purpose of this application is to provide constructs (including multivalent Nanobodies and Nanobody fusion proteins) based on Nanobodies S43 that specifically bind SARS-CoV-2 RBD, polynucleotides encoding them, and nucleic acids containing the polynucleotides.
  • the construct of the present application based on Nanobody S43 that specifically binds SARS-CoV-2 RBD can effectively inhibit SARS-CoV-2 infection and infection of its variant strains, and can Aerosol administration can directly reach the lungs, has a rapid onset of action and a long half-life, providing a more effective treatment strategy for infections caused by the new coronavirus and its mutant strains.
  • the present application provides a multivalent Nanobody, which includes more than two VHH chains of Nanobodies that specifically bind SARS-CoV-2 RBD, wherein the specific binding is SARS-CoV-2 RBD
  • the VHH chain of the Nanobody includes the following CDRs:
  • the amino acid sequence is CDR1 shown in SEQ ID NO:1 (i.e., GFTLDYYAIG),
  • CDR2 with the amino acid sequence shown in SEQ ID NO:2 (i.e., CISSNNSTYYADSVKG), and
  • the amino acid sequence is CDR3 shown in SEQ ID NO:3 (i.e., EPDYSGVYYYTCGWTDFGS).
  • the VHH chain of the Nanobody that specifically binds to the SARS-CoV-2 RBD also includes 4 framework regions FR1-4, the FR1-4 and the CDR1, CDR2 and CDR3 are staggered in order;
  • amino acid sequences of the FR1-4 are respectively as SEQ ID NO:4 (i.e., QVQLQESGGGLVQPGGSLRLTCAPS), SEQ ID NO:5 (i.e., WFRQAPGKEREGVS), SEQ ID NO:6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA) and SEQ ID NO: 7 (i.e., WGQGTQVTVSS).
  • SEQ ID NO:4 i.e., QVQLQESGGGLVQPGGSLRLTCAPS
  • SEQ ID NO:5 i.e., WFRQAPGKEREGVS
  • SEQ ID NO:6 i.e., RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA
  • SEQ ID NO: 7 i.e., WGQGTQVTVSS
  • the VHH chain of the Nanobody that specifically binds to the SARS-CoV-2 RBD has an amino acid sequence as shown in SEQ ID NO:8, or is identical to the amino acid sequence as shown in SEQ ID NO:8 An amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity; preferably, the amino acid sequence of the VHH chain is as shown in the following SEQ ID NO: 8: Among them, the underlined parts are the framework regions FR1-4 respectively, and the black parts are CDR1, CDR2 and CDR3 of the heavy chain variable region respectively.
  • the multivalent Nanobody is composed of two or more, preferably three, VHH chains of the Nanobody that specifically binds SARS-CoV-2 RBD, connected through a Linker;
  • the multivalent Nanobody is a trivalent Nanobody and has the amino acid sequence shown in SEQ ID NO: 9:
  • the multivalent Nanobody is an IgM pentamer formed from the following fusion protein, and the structure of the fusion protein from the N-terminus to the C-terminus is shown in formula (I): ALB(I)
  • A is a single VHH chain of the Nanobody that specifically binds to the SARS-CoV-2 RBD, or a multivalent Nanobody as described in the preferred embodiment I above;
  • the Fc fragment of human IgM has a structure such as SEQ ID NO: 10 (i.e., VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKG VALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY), or an amino acid sequence having at least 95%, 96%, 97%, 98% or
  • the present application provides a Nanobody fusion protein.
  • the structure of the Nanobody fusion protein from the N-terminus to the C-terminus is as shown in formula (I): ALB(I)
  • the Fc fragment of human IgM is the Fc fragment of human IgM; preferably, the Fc fragment of human IgM has the amino acid sequence shown in SEQ ID NO: 10, or has at least 95%, 96% of the amino acid sequence shown in SEQ ID NO: 10 An amino acid sequence with %, 97%, 98% or 99% sequence identity;
  • the above fusion protein has the amino acid sequence shown in SEQ ID NO: 11.
  • the present application provides a polynucleotide encoding a multivalent Nanobody as described in the above first aspect, or encoding a Nanobody fusion protein as described in the above second aspect.
  • the polynucleotide is DNA or mRNA
  • the polynucleotide encodes a multivalent Nanobody according to the preferred specific embodiment 1 of the above-mentioned first aspect.
  • the polynucleotide includes as shown in SEQ ID NO: 12 (i.e., ) Nucleotide sequence;
  • the polynucleotide encodes a Nanobody fusion protein as described in the second aspect above.
  • the polynucleotide includes a protein such as SEQ ID NO: 13 (i.e., CAGGGTGCAGCTGCAGGAGAGCGGAGGAGGGCTGGTGCAGCCCGGAGGAAGCCTGAGACTGACCTGCGCCCCCAGCGGATTCACCCTGGATTATTATGCTATTGGCTGGTTTAGGCAGGCTCCCGGCAAAGAGAGAGGGGGTGTCATGCATTAGCAGCAATAACTCAACCTACTAC GCCGACAGCGTCAAGGGACGCTTCACCATTTCCAGGGACAACGC ) the nucleotide sequence shown.
  • the present application provides a nucleic acid construct comprising the polynucleotide as described in the third aspect above, and optionally, at least one expression control element operably linked to the polynucleotide.
  • at least one expression control element operably linked to the polynucleotide. For example, histidine tag, stop codon, etc.
  • the present application provides an expression vector comprising the nucleic acid construct described in the fourth aspect.
  • the present application provides a transformed cell, which includes the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, or the expression vector as described in the fifth aspect. .
  • the present application provides a pharmaceutical composition, which includes the multivalent Nanobody as described in the first aspect, the Nanobody fusion protein as described in the second aspect, and the nanobody fusion protein as described in the third aspect.
  • a pharmaceutical composition which includes the multivalent Nanobody as described in the first aspect, the Nanobody fusion protein as described in the second aspect, and the nanobody fusion protein as described in the third aspect.
  • the pharmaceutical composition may be a nasal spray, oral formulation, suppository, or parenteral formulation form;
  • the nasal spray is selected from aerosols, sprays and powder sprays;
  • the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated agents, pellets, sublingual tablets and ointments;
  • the parenteral preparation is a transdermal preparation, an ointment, a plaster, a topical liquid, an injectable or a pushable preparation.
  • the dosage of the active ingredient of the pharmaceutical composition of the present application varies depending on the administration target, the target organ, symptoms, administration method, etc., and may take into consideration the type of dosage form, administration method, age and weight of the patient, The patient's symptoms, etc., are determined based on the doctor's judgment.
  • the present application provides a multivalent Nanobody as described in the first aspect, a Nanobody fusion protein as described in the second aspect, a polynucleotide as described in the third aspect,
  • the nucleic acid construct described in the fourth aspect, the expression vector described in the fifth aspect, the transformed cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect are used for the prevention and treatment of /or application in drugs to treat novel coronavirus infection.
  • the new coronavirus can be the original strain of SARS-CoV-2 and/or the mutant strain of SARS-CoV-2;
  • the SARS-CoV-2 variant strains are Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B. 1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain; further preferably, the SARS-CoV-2 The mutated strains are Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.
  • the present application provides a multivalent Nanobody as described in the first aspect, a Nanobody fusion protein as described in the second aspect, a polynucleotide as described in the third aspect,
  • the nucleic acid construct described in the fourth aspect, the expression vector as described in the fifth aspect, or the transformed cells as described in the sixth aspect are used in preparing reagents for detecting a new coronavirus or for diagnosing a new coronavirus infection. or application in a kit.
  • the new coronavirus can be the original strain of SARS-CoV-2 and/or the mutant strain of SARS-CoV-2;
  • the SARS-CoV-2 variant strains are Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B. 1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain; further preferably, the SARS-CoV-2 The mutated strains are Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.
  • the present application provides a novel coronavirus detection kit, which includes the multivalent Nanobody as described in the above first aspect, the Nanobody fusion protein as described in the above second aspect, and the above third aspect. described Polynucleotide, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, or the transformed cell as described in the sixth aspect.
  • This application is for the drug development of nanobody constructs for the new coronavirus.
  • the constructs based on Nanobody S43 of this application can bind to SARS-CoV-2 RBD with high affinity and can neutralize SARS-CoV-2 with high neutralizing activity.
  • Virus (SARS-CoV-2) Nanobodies Nanobodies.
  • the inventors have demonstrated through a series of experiments that the trivalent Nanobody (TS43) and IgM pentameric form (MS43) based on Nanobody S43 of the present application have significantly improved performance compared to its monomer (i.e., Nanobody S43). With its neutralizing activity and significantly extended half-life, it achieves mucosal immunity, can limit the reproduction of the virus and further cross the mucosal barrier, control the mucosal spread of the virus, and provides a potential aerosolized solution for the clinical prevention and treatment of the new coronavirus.
  • the new antibody drug administered can achieve sensitive and reliable detection of the new coronavirus.
  • Figure 1 is a schematic structural diagram of the Nanobody constructs TS43 and MS43 constructed in Example 1 of the present application;
  • Figure 2 is a diagram of the S43 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;
  • Figure 3 is a diagram of the TS43 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;
  • Figure 4 is a diagram of the MS43 protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;
  • Figure 5 is the SARS-CoV-2 RBD-his protein (A) described in Example 2 of the present application, the RBD-his protein (B) of the mutant strain Omicron (B.1.1.529) subtype BA.1 and SDS-PAGE identification results of RBD-his protein (C) of Omicron (B.1.1.529) subtype BA.2.
  • Figure 6 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 prototype strain pseudovirus infection.
  • Figure 7 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus infection.
  • Figure 8 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 pseudovirus infection.
  • Figure 9 is a schematic diagram of the effect of the three antibodies measured in Example 5 of the present application on neutralizing SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.2 pseudovirus infection.
  • Figure 10 is the neutralizing activity of the three antibodies measured in Example 7 of the present application against pseudovirus before and after aerosolization; Among them, A is the neutralizing activity result of Nanobody S43 against SARS-CoV-2 prototype strain pseudovirus before and after aerosolization, and B is the neutralizing activity of Nanobody construct TS43 against SARS-CoV-2 prototype strain pseudovirus. and activity results, C is the neutralizing activity result of Nanobody construct MS43 against SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus.
  • “Nanobody” is a “heavy chain single domain antibody”. This type of antibody only contains a heavy chain variable region (VHH, variable domain of heavy chain of heavy-chain antibody). Compared with other antibodies, the light chain is naturally missing. .
  • nanobodies Due to their biophysical advantages, nanobodies can be easily atomized and delivered directly to the lungs through inhalers to treat infections caused by respiratory viruses and are considered to be very promising antibody-based drugs.
  • binding refers to the determination of the presence or absence of said protein, such as the Nanobodies of the present application, in a heterogeneous population of proteins and/or other biological agents. Binding reaction with SARS-CoV-2 RBD protein. Thus, under specified conditions, a specific ligand/antigen binds to a specific receptor/antibody and does not bind in significant amounts to other proteins present in the sample.
  • reagents, enzymes, culture media, antibiotics, milk and other chemical materials used in the following examples of this application are all commercially available products.
  • TRIzol is purchased from Invitrogen
  • Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.
  • pCAGGS vector was purchased from MiaoLingPlasmid
  • 293F cells, HEK293T cells, etc. were purchased from ATCC
  • Series Sensor Chip SA chip Purchased from GE Healthcare
  • Vero cells were purchased from ATCC CCL81.
  • coding sequence of TS43 in this application was synthesized by Beijing Qingke Biotechnology Co., Ltd.
  • Example 1 Construction, expression and purification of antibodies based on the trivalent form (TS43) and IgM pentameric form (MS43) of Nanobody S43
  • the basic nanobody S43 used in this laboratory was obtained by immunizing alpacas with SARS-CoV-2S protein, constructing an antibody library, and screening using phage display technology; the amino acid sequence of the VHH chain of single-chain nanobody S43 is as SEQ ID As shown in NO: 8, it can bind SARS-CoV-2 RBD with high affinity and specificity (binding constant is 1.2E-10 ⁇ 1.4E-11M), and in pseudovirus neutralization experiments, it can neutralize with high Activity neutralizes SARS-CoV-2 pseudovirus, all of which indicate that S43 nanobody is a novel coronavirus (SARS-CoV-2) sheep that can bind to SARS-CoV-2 RBD with high affinity and has high neutralizing activity. Camel-derived nanobodies.
  • SARS-CoV-2 novel coronavirus
  • a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15) is connected to the 5' end of the coding sequence of the S43VHH chain of the above-mentioned single-chain Nanobody (as shown in SEQ ID NO: 14), and 6 histidine tags are connected to the 3' end ( hexa-His-tag) and the translation stop codon TGA, it was constructed into the pCAGGS vector (purchased from Invitrogen) through the restriction endonuclease sites EcoRI and XhoI, and then the resulting recombinant vector was transfected into 293F In cells (purchased from Invitrogen), S43-his protein was expressed.
  • the coding sequences of the three Nanobody S43VHH chains shown in SEQ ID NO: 14 are connected in a head-to-tail format (directly synthesized by Beijing Qingke Biotechnology Co., Ltd.), and in their 5
  • the 'end is connected to the signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15)
  • the 3' end is connected to the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon TGA, through the restriction endonuclease site EcoRI and XhoI were spotted and constructed into the pCAGGS vector (purchased from Invitrogen), and then the resulting recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express TS43-his protein.
  • the coding sequence of the Nanobody S43VHH chain (shown in SEQ ID NO:14) was connected to the coding sequence of the Fc of the human IgM antibody (shown in SEQ ID NO:16), and at 5 The 'end is connected to a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15), the 3' end is connected to the translation stop codon TGA, and it is constructed into the pCAGGS vector (purchased from Invitrogen) through the restriction endonuclease sites EcoRI and XhoI. The pCAGGS-S43-IgM Fc recombinant expression vector was obtained.
  • the coding sequence of the J chain (Joining chain) (such as SEQ ID NO:17
  • the translation stop codon TGA was connected to the 3' end of (shown), and it was constructed into the pCAGGS vector (purchased from Invitrogen) through the restriction endonuclease sites EcoRI and XhoI to obtain the pCAGGS-J chain recombinant expression vector.
  • the above two recombinant expression vectors pCAGGS-S43-IgM Fc and pCAGGS-J chain were co-transfected into 293F cells (purchased from Invitrogen) to express the S43-IgM Fc fusion protein and J chain, and the two were then self-assembled.
  • MS43 protein forming the IgM form of MS43 protein.
  • the obtained MS43 protein was purified by HiTrap TM IgM Purification HP (GE Healthcare) and Superose TM 6 increase 10/300GL (GE Healthcare), and then identified by SDS-PAGE.
  • the SDS-PAGE identification size of MS43 protein is about 70KD, and the results are shown in Figure 4.
  • the coding sequence of the RBD protein (the amino acid sequence of which is shown in SEQ ID NO: 19) of the SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529 )
  • the 3' end of the coding sequence of the RBD protein of subtype BA.2 (the amino acid sequence of which is shown in SEQ ID NO: 20) is connected to the coding sequence and translation of 6 histidine tags (hexa-His-tag). Stop codon TGA, expressed by bac-to-bac baculovirus expression system (Invitrogen).
  • bacmids Transform the pFastbac1 plasmid containing the target gene into DH10Bac competent cells to generate recombinant bacmids (bacmids).
  • the recombinant bacmid bacmids were transfected into Sf9 cells for virus amplification, and protein expression was performed in Hi5 cells. After 48 h of expression, the Hi5 cell supernatant was collected, and the soluble protein was purified by nickel affinity chromatography using HisTrap TM excel (GE Healthcare).
  • the cell culture medium containing the target protein is purified by the nickel ion affinity chromatography column HisTrap TM excel (GE Healthcare) and the gel filtration chromatography Superdex TM 200 Increase 10/300GL column (GE Healthcare), a relatively pure target protein can be obtained.
  • SARS-CoV-2 RBD-his protein, RBD-his protein of mutant strain Omicron (B.1.1.529) subtype BA.1, and RBD-his protein of Omicron (B.1.1.529) subtype BA.2 The SDS-PAGE identification size is about 30KD, as shown in Figures 5A to C respectively.
  • Example 3 Surface plasmon resonance technology detects the binding ability of each antibody to the RBD protein of the original strain of SARS-CoV-2 and its mutant strains
  • the single-chain Nanobody S43 and its constructs TS43 and MS43 prepared in the above examples were biotinylated respectively, and then immobilized on the Series Sensor Chip SA chip (Cytiva Life Sciences); PBST buffer (2.7mM KCl, 137mM NaCl, 4.3mM Na 2 HPO 4 , 1.4mM KH 2 PO 4 , 0.05% Tween) respectively.
  • PBST buffer 2.7mM KCl, 137mM NaCl, 4.3mM Na 2 HPO 4 , 1.4mM KH 2 PO 4 , 0.05% Tween
  • the RBD proteins of the original SARS-CoV-2 strain and its mutant strains prepared in the Example were diluted multiple times and loaded onto the chip one by one from low concentration to high concentration. Calculation of binding kinetic constants was performed using BIAevaluation software 8K (Biacore, Inc.).
  • the equilibrium dissociation constant (K D ) between each antibody and each RBD is shown in Table 1.
  • the results in Table 1 show that the single-chain nanobody S43 and its constructs TS43 and MS43 can all interact with the SARS-CoV-2 prototype strain. And the RBD proteins of mutant strains Omicron subtype BA.1 and Omicron subtype BA.2 bind with higher affinity.
  • the packaging steps for SARS-CoV-2 original strain and mutant strain pseudovirus are as follows:
  • HEK293T cells purchased from ATCC CRL-3216
  • the culture medium was DMEM medium containing 10% FBS.
  • Transfection Take the expression plasmid of each S protein in the above step 2), and use PEI to transfect 30 ⁇ g plasmid/10cm cell culture dish. Mix the target plasmid and PEI in a ratio of 1:3 before transfection, and transfect after 4-6 hours. Culture medium (DMEM medium containing 10% FBS), culture at 37°C for 24 hours.
  • Toxin addition Add the pseudovirus packaging skeleton virus G*VSV-delG (purchased from Wuhan Shumi Brain Science and Technology Co., Ltd.) to the above-transfected HEK293T cells, incubate at 37°C for 2 hours, and change the culture medium (containing 10% FBS) DMEM medium), and add VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank), and continue to culture in the incubator for 30 h.
  • G*VSV-delG purchased from Wuhan Shumi Brain Science and Technology Co., Ltd.
  • Toxin collection Collect the supernatant and centrifuge it at 3000rpm for 10 minutes, filter it through a 0.45 ⁇ m sterile filter in a clean workbench. Remove cell debris, aliquot, and freeze in -80°C refrigerator.
  • SARS-CoV-2 prototype strain SARS-CoV-2WT
  • variant strains Delta B.1.617.2
  • Omicron B.1.1.529
  • subtypes BA.1 and Omicron B.1.1. 529) Pseudovirus of subtype BA.2.
  • the purified single-chain Nanobody S43 and its constructs TS43 and MS43 (prepared from Example 1) were diluted 5-fold to the ninth gradient (2.56pg/mL), and the dilution was mixed with 1.6 ⁇ 10 4 TCID 50
  • a series of pseudoviruses of the original SARS-CoV-2 strain and variant strains obtained in Example 4 were mixed separately, mixed and incubated at 37°C for 1 hour, and then added to 96 cells pre-inoculated with Vero cells (purchased from ATCC CCL81) in the orifice plate. After incubation for 18 to 20 hours, it was detected by CQ1 Confocal Quantitative Image Cytometer (Yokogawa).
  • IC 50 ( ⁇ g/mL) is the half inhibitory concentration of the antibody. *Indicates that the highest concentration of 10 mg/mL has not yet reached 100% inhibition rate.
  • CPE cytopathic effect
  • the purified single-chain Nanobody S43 and its TS43 and MS43 (prepared in Example 1) were diluted 2-fold to the 11th gradient, with 4 replicate wells for each gradient, 50 ⁇ L per well, and the diluent was Incubate with an equal volume of 100 TCID 50 of the original SARS-CoV-2 strain or its variant strain Delta and Omicron subtype BA.1 at 37°C. After 1 hour, the mixture was added to the suspended Vero cells and incubation was continued at 37°C for 3 days. Observe and record cell lesions. IC50 of Nanobodies and their constructs were calculated using GraphPad Prism 7.0. The experiments were all conducted in the biosafety level three laboratory (BSL3) of the Chinese Center for Disease Control and Prevention.
  • BSL3 biosafety level three laboratory
  • the live virus neutralizing effects of the single-chain nanobody S43 and its TS43 and MS43 on the original strain of the new coronavirus and its mutant strains are shown in Table 3.
  • the results in Table 3 show: The effect of TS43 and MS43 on the original strain and the mutant strain of the new coronavirus. Live viruses all have good inhibitory effects.
  • IC 50 ( ⁇ g/mL) is the half inhibitory concentration of the antibody.
  • Example 7 Detection of antibody stability before and after atomization
  • the single-chain Nanobody S43 and its constructs TS43 and MS43 were atomized using an Aerogen Solo (Aerogen Inc., Chicago, USA) nebulizer, respectively, and then used in an all-glass SKC (Eighty Four, PA, USA) containing 20 mL of PBS. ) Collect the aerosolized antibodies and conduct a pseudovirus neutralization test as described in Example 5.
  • the construct of the present application based on the nanobody S43 that specifically binds to the SARS-CoV-2 RBD can effectively inhibit SARS-CoV-2 infection and its variant strain infection, can be administered by aerosol, can directly reach the lungs, and has a rapid onset of effect. , and has a long half-life, providing a more effective treatment option for the clinical prevention or treatment of infections caused by the new coronavirus and its mutant strains.

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Abstract

L'invention concerne une construction (comprenant un nano-anticorps multivalent et une protéine de fusion de nano-anticorps) basée sur un nano-anticorps S43 qui se lie de manière spécifique au RBD de SARS-CoV-2, un produit associé à la construction, et une utilisation associée. La construction peut inhiber de manière efficace une infection par le SARS-CoV-2 et une infection par une souche de variant du SARS-CoV-2, peut être administrée par nébulisation, peut atteindre directement le poumon, prend effet rapidement, a une longue demi-vie, et fournit une stratégie de traitement plus efficace pour la prévention ou le traitement clinique du nouveau coronavirus et d'une infection par une souche de variant de celui-ci.
PCT/CN2023/082534 2022-03-21 2023-03-20 Construction de nano-anticorps s43 et son utilisation WO2023179548A1 (fr)

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