WO2023125520A1 - NANOCORPS DÉRIVÉ DU CHAMEAU À HAUTE AFFINITÉ POUR DES SOUCHES MUTANTES α, β, γ ET δ DE SARS-COV-2 - Google Patents

NANOCORPS DÉRIVÉ DU CHAMEAU À HAUTE AFFINITÉ POUR DES SOUCHES MUTANTES α, β, γ ET δ DE SARS-COV-2 Download PDF

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WO2023125520A1
WO2023125520A1 PCT/CN2022/142281 CN2022142281W WO2023125520A1 WO 2023125520 A1 WO2023125520 A1 WO 2023125520A1 CN 2022142281 W CN2022142281 W CN 2022142281W WO 2023125520 A1 WO2023125520 A1 WO 2023125520A1
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antibody
antigen
cov
sars
protein
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PCT/CN2022/142281
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杨鹏远
王楷
刘兰兰
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中国科学院生物物理研究所
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/569—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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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

Definitions

  • the invention belongs to the fields of biotechnology, immunoassay and biomedicine, and in particular relates to broad-spectrum high-affinity antibodies or antigen-binding fragments and their applications in the detection, diagnosis, prevention and treatment of coronaviruses, especially SARS-CoV-2 Use in detection, diagnosis, prevention and treatment of Alpha mutant strain, Beta mutant strain, Gamma mutant strain and/or Delta mutant strain.
  • the novel coronavirus SARS-CoV-2 is an RNA virus of the genus Betacoronavirus.
  • the virus has the characteristics of strong transmissibility, high lethality and rapid mutation.
  • SARS-CoV-2 causes respiratory infections that lead to viral pneumonia and acute respiratory distress syndrome (ARDS) in some patients. At the same time, it can also trigger a cytokine storm and cause multiple organ damage.
  • ARDS acute respiratory distress syndrome
  • the original strain of the new coronavirus has been isolated so far, and new mutant viruses such as D614G mutant, B.1.1.7 mutant, B.1.351 mutant, B.1.429 mutant, and P.1 mutant have emerged continuously during the global spread.
  • strain, B.1.617.2 mutant strain, etc. not only greatly enhanced the transmissibility and lethality of the virus, caused repeated outbreaks of the epidemic in most countries around the world, but also caused the continuous reduction of vaccine protection.
  • Nanobody is a single-domain antibody that only contains the heavy chain antibody antigen-binding domain VHH. Compared with traditional polyclonal antibodies, monoclonal antibodies and single-chain antibodies, it has many obvious advantages, such as small size, and can pass through conventional antibodies. Into the tissues and organs (such as the sheath, spinal cord, brain, etc.); strong stability, no need for cold chain transportation and cold storage; low immunogenicity, easy to carry out humanization transformation.
  • the present invention takes SARS-CoV-2 virus surface spike protein (Spike protein, i.e.
  • S protein as the target, and develops a variety of SARS-CoV-2 virus mutations that can simultaneously recognize multiple SARS-CoV-2 virus mutations by constructing a phage display nanobody immune library and biological panning
  • the camel-derived high-affinity nanobody of the strain is of great significance for laying the foundation for the mechanism research, clinical diagnosis and treatment of new coronary pneumonia, as well as the strategic reserve for possible outbreaks of new coronaviruses in the future.
  • the technical problem to be solved by the present invention is to provide a broad-spectrum, high-affinity antibody against coronavirus, which can effectively detect, block, and treat coronavirus, especially the original strain of SARS-CoV-2 virus and its mutants .
  • anti-SARS-CoV-2 Nanobodies are provided, which can be combined with British mutant strain (Alpha; B.1.1.7), South African mutant strain (Beta; B.1.351), Brazilian mutant strain (Gamma; P.1) binds to the S1 subunit (also known as S1 protein) of the Indian mutant (Delta; B.1.617.2) S protein, and the affinity reaches nanomolar levels.
  • nanobodies against coronaviruses such as SARS-CoV-2 are provided, which can effectively block the infection of SARS-CoV-2 pseudoviruses to hACE2 overexpressed 293T cells, and neutralize them half effectively.
  • the concentration reaches nanomolar level.
  • the establishment of various ELISA detection methods based on antigen/antibody reactions and the development of detection products can be carried out.
  • multivalent isogenetic engineering based on the same or multiple Nanobodies can be performed.
  • Nanobody against a coronavirus such as SARS-CoV-2
  • said Nanobody comprising the following amino acid sequence and functional properties:
  • the antibody can have the amino acid sequence of the hypervariable region CDR1 shown in any of SEQ ID NO: 9-15; any of SEQ ID NO: 16-22 The amino acid sequence of the hypervariable region CDR2 shown; and the amino acid sequence of the hypervariable region CDR3 shown in any one of SEQ ID NO:23-30;
  • Nanobodies have nanomolar affinity to coronaviruses such as SARS-CoV-2 virus Alpha mutants, Beta mutants, Gamma mutants and Delta mutants;
  • Nanobodies effectively block the infection of SARS-CoV-2 pseudoviruses to hACE2 overexpressed 293T cells.
  • the present invention also provides a biological material containing the nucleic acid molecule encoding the antibody, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, virus vector or engineering bacteria.
  • the present invention also provides any of the following applications of the antibody:
  • the enzyme label plate of the coronavirus such as the SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain antigen.
  • the amount of the added enzyme-labeled secondary antibody bound to the bound nanobody is small, and finally the substrate is added Liquid and chromogenic solution, the color reaction is shallow, and the OD value detected by the microplate reader is low; on the contrary, when the nanobody is combined with the solid-phase antigen, the measured OD value is high, according to the amount of nanobody added and The binding curves of Nanobodies and SARS-CoV-2 were drawn corresponding to the OD values of the wells.
  • the present invention provides the following technical solutions:
  • An antibody or antigen-binding fragment thereof the amino acid sequence of which comprises CDR1 shown in any of SEQ ID NO: 9-15, CDR2 shown in any of SEQ ID NO: 16-22, and CDR2 shown in SEQ ID NO: CDR3 shown in any one of 23-30;
  • the antigen-binding fragment is, for example, Fv, Fab, Fab', scFv, F(ab') 2 , multivalent or multispecific fragments.
  • the antibody or antigen-binding fragment is an antibody comprising a sequence obtained by truncating amino acids from the 1st to the 130th amino acid from the N-terminal of any of the sequences shown in SEQ ID NO: 1-8, or SEQ ID NO: Antibodies or antigen-binding fragments with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to any of the sequences shown in 1-8.
  • a genetically engineered antibody comprising the antibody or antigen-binding fragment described in item 1 or 2; preferably, the genetically engineered antibody is a humanized antibody, a chimeric antibody, a multivalent or multispecific antibody.
  • a fusion protein comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3; preferably, the fusion protein further comprises a tag polypeptide, a detection protein or an auxiliary protein.
  • a conjugate comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4; preferably, the conjugate further comprises a detectable Labels, contrast agents, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, liposomes, viral coat proteins or VLPs, or combinations thereof.
  • a nucleic acid molecule encoding the antibody or antigen-binding fragment as described in item 1-2, the genetically engineered antibody as described in item 3, the fusion protein as described in item 4, or the conjugated protein as described in item 5 A substance, wherein the nucleic acid molecule is RNA, DNA or cDNA.
  • An expression vector comprising the nucleic acid molecule described in item 6;
  • the expression vector may be a DNA, RNA, viral vector, plasmid, expression cassette, transposon, other gene transfer system, or a combination thereof;
  • the expression vectors include viral vectors, such as phage vectors, lentiviruses, adenoviruses, AAV viruses, retroviruses, other protein expression systems, or combinations thereof.
  • viral vectors such as phage vectors, lentiviruses, adenoviruses, AAV viruses, retroviruses, other protein expression systems, or combinations thereof.
  • a host cell comprising the expression vector described in item 7; wherein, the host cell is a host cell for expressing a foreign protein, such as a prokaryotic expression cell, a eukaryotic expression cell, or a transgenic cell line; preferably, the The host cells include prokaryotic cells, yeast cells, insect cells, plant cells, and animal cells.
  • a method for preparing the antibody or antigen-binding fragment as described in item 1-2, the genetically engineered antibody as described in item 3, the fusion protein as described in item 4, or the conjugate as described in item 5, comprising Separating/recovering the target protein or polypeptide from the tissue sample or culture described in 9.
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5 as an active ingredient;
  • the pharmaceutical composition is an inhaled atomized drug, a drug for mucosal or epidermal application, a drug for subcutaneous injection, a drug for blood vessel transfusion, or a combination thereof; preferably, the drug also includes a pharmaceutical excipient or carrier .
  • the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5 is used in the preparation of prevention, treatment and/or Use in products or medicines for the diagnosis of coronavirus infection.
  • the coronavirus includes HCoV-NL63, SARS-CoV-1, SARS-CoV-2, HCoV-229E, MERS-CoV, HCoV-OC43, HCoV-HKU1 or other viruses with similar surface Coronavirus with S protein structure.
  • the mutant strains of SARS-CoV-2 virus include D614G mutant strain, B.1.1.7 mutant strain, B.1.351 mutant strain, B.1.429 mutant strain, P.1 mutant strain, B .1.617.2 mutant strains, etc.
  • functional polypeptides such as purification tags, detection tags, identification tags, conjugation tags, and functional verification tags included in the tag polypeptides, such as His tag, HA tag, Flag tag, c-Myc tag , Avi tags, etc.
  • the detection protein contained in the fusion protein includes functional proteins such as fluorescent protein, fluorescein-labeled protein, and peroxidase, such as FPs protein, HRP protein, Alexa Fluor-labeled protein, FITC-labeled protein, etc. protein etc.
  • functional proteins such as fluorescent protein, fluorescein-labeled protein, and peroxidase, such as FPs protein, HRP protein, Alexa Fluor-labeled protein, FITC-labeled protein, etc. protein etc.
  • the auxiliary protein contained in the fusion protein is a protein used for functions such as assisting folding, assisting expression, assisting dissolution, and shielding toxic proteins, such as GST protein, MBP protein, SUMO protein, NusA protein, etc. protein.
  • the antibody provided by the invention for anti-coronavirus such as SARS-CoV-2 effectively overcomes the shortcomings of current coronavirus such as SARS-CoV-2 recovered patients with less serum sources, high cost and unstable structure, and has high affinity and high sensitivity , high and high capacity, high output, high stability, low cost and capable of rapid mass production.
  • the antibody provided by the present invention can be used not only for initial infection blocking, early infection diagnosis, and middle and late infection treatment, but also for scientific research tools and in vitro rapid detection, such as the production of ELISA detection/diagnostic kits, colloidal gold detection/diagnostic reagents box.
  • the ELISA detection method established by the antibody of the present invention can accurately and sensitively detect whether a sample contains coronavirus such as SARS-CoV-2 virus.
  • the sample pretreatment process is simple, less time-consuming, and can detect a large number of samples at the same time, and the cost of sample detection is much lower than traditional nucleic acid detection methods.
  • the application of the antibody of the present invention to colloidal gold detection/diagnostic kits can quickly and accurately detect whether a sample contains coronaviruses such as SARS-CoV-2 virus, and is useful for solving large-scale crowd infections and environmental and cargo sample pollution screening and identification important practical significance.
  • Fig. 1 is the binding curve of Nanobody of the present invention and SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain S1 protein;
  • Fig. 2 is the affinity curve (taking antibody A1 as example) of Nanobody of the present invention and SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain S1 protein;
  • Fig. 3 is the neutralization inhibition curve of Nanobody of the present invention to SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain pseudovirus.
  • Figure 4 shows the sequence of the antibody of the present invention and its CDR region
  • FIG. 5 is a plasmid map of pComb3Xss used in Example 1.
  • FIG. 5 is a plasmid map of pComb3Xss used in Example 1.
  • the nanobody can be prepared as follows: the original strain SARS-CoV-2 protein is used as an immunogen to immunize camels of experimental animals, the total RNA of peripheral blood lymphocytes is extracted, and after inversion The nanobody heavy chain (VHH) gene fragment was cloned by recording and nested PCR, and the gene fragment was cloned into a phagemid vector by restriction enzyme digestion, and then transformed into Escherichia coli with high-efficiency electrotransformation, and the phage nanobody was constructed by assisting phage rescue.
  • VHH nanobody heavy chain
  • the prepared nanobody molecule is small, highly soluble, high temperature resistant, easy to purify, and easy to express.
  • the SARS-CoV-2 virus wild type original strain S protein and RBD protein are used as immunogens
  • the SARS-CoV-2 virus wild type original strain S1 protein, SARS-CoV-2 virus Alpha Mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein were used as coated antigens, all of which were purchased from Beijing Sino Biological Co., Ltd.
  • the microtiter plate is a 96-well microtiter plate, and the coating concentration of the coated antigen is 1 ug/mL.
  • the enzyme-labeled secondary antibody is an anti-HA tag antibody labeled with horseradish peroxidase, and the concentration is 0.1 ⁇ g/mL.
  • Abcam Company item number: ab1265.
  • the chromogenic solution A is prepared from 1 g of carbamide peroxide, 10.3 g of citric acid, 35.8 g of Na 2 HPO 4 ⁇ 12H 2 O, 100 ⁇ L of Tween-20 and 1000 mL of distilled water, with a pH value of 5.
  • chromogenic liquid B liquid is formulated by tetramethylbenzidine 700mg, DMSO 40mL, citric acid 10.3g and distilled water 1000mL, pH value 2.4.
  • the reaction termination solution is 2M sulfuric acid solution.
  • Leukocytes were isolated from peripheral blood after the fifth immunization, total RNA was extracted, and the VHH gene was cloned by reverse transcription PCR and nested PCR (the systems and parameters of reverse transcription PCR and nested PCR were as follows)
  • the fragment was modified with the restriction endonuclease SfiI, and the VHH gene fragment was connected to the phagemid pComb3Xss by T4 ligase (a gift from the laboratory of Professor Bruce D Hammock of UC Davis), and efficiently transformed into Escherichia coli ER2738 (laboratory Preservation, also commercially available, such as purchased from British NEB company), constructing a phage nanobody library of SARS-CoV-2.
  • the reverse transcription kit uses PrimeScript TM RT-PCR Kit, purchased from Takara Company, product number: AK2701.
  • the reverse transcription system is as follows:
  • Nested PCR (purchased from TAKATA company, item number: 6210A)
  • the reaction system is as follows:
  • the reaction system is as follows:
  • GSP-RT CGCCATCAATRTACCAGTTGA (SEQ ID NO: 31)
  • R represents base A/G
  • W represents base A/T
  • K represents base G/T
  • the amplified product is subjected to the next round of screening, ensuring that the addition amount of each round of screening is the same, the antigen coating concentration and the S protein competition elution concentration are reduced by 2 times, the titer of each round is calculated, and a single clone is selected for amplification and ELISA identification. Positive single clones were obtained after 3 rounds of panning.
  • Embodiment 3 the expression of SARS-CoV-2 Nanobody
  • the positive monoclonal plasmid was extracted, transformed into Escherichia coli TOP10F' competent cells (purchased from Thermo Fisher), and spread on solid medium after recovery for overnight culture. The next day, a single clone was picked and cultured in SB-carboxybenzyl medium, and IPTG was added to induce overnight expression; the next day, the cells were lysed with a high-pressure homogenizer, filtered with a filter membrane, and purified with a nickel column, that is, using a histidine tag and The affinity chromatography of nickel chloride in the nickel column is used to separate and purify the nanobodies to obtain high-purity anti-SARS-CoV-2 nanobodies, namely antibodies A1-A8. After amino acid sequencing analysis, the amino acid sequence of the obtained nanobodies is shown in SEQ ID NO:1-8.
  • Embodiment 4 the binding curve of nanobody and SARS-CoV-2 virus S1 protein
  • the SARS-CoV-2 virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein (Beijing Yiqiao Shenzhou Biological Co., Ltd.) were respectively coated on 96-well microtiter plates, The coating concentration of each well is 1ug/mL, and react overnight at 4°C; the next day, shake off the liquid in the well, wash 3 times with PBST containing 0.05% Tween, and invert the microplate on absorbent paper to pat dry; Add blocking solution, incubate at 37°C for 30 minutes, shake off the liquid in the well, wash 3 times with 0.05% PBST, invert the plate on absorbent paper and pat dry; Antibody solution, incubate at 37°C for 30 minutes; shake off the liquid in the well, wash 3 times with PBST, invert the plate on absorbent paper and pat dry; add enzyme-labeled secondary antibody (horseradish peroxidase-labeled anti-HA
  • Embodiment 5 the affinity curve of Nanobody and SARS-CoV-2 virus S1 protein
  • Affinity detection uses an avidin probe, which is detected using an Octecred 96 instrument.
  • the affinity detection method is a routine technical operation in the art, and the specific operations are as follows. Add 0.02% Tween-20 PBST to the 8 wells of the first column of a black non-binding 96-well plate; then add a concentration of 15ug/ml biotin-labeled SARS-CoV- 2 Virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein.
  • the balanced probe is immersed in the fourth row of nanobody dilution solution to carry out specific binding of antigen and antibody for 3 minutes;
  • the results are shown in Figure 2 and Table 1.
  • the results show that the eight Nanobodies have an affinity range of 0.28-0.82nM for the S1 protein of the Alpha mutant strain of SARS-CoV-2; and a range of 0.25-0.68nM for the S1 protein affinity of the Beta mutant strain;
  • the range of affinity for Gamma mutant S1 protein is: 0.27-0.95nM
  • the range of affinity for Delta mutant S1 protein is: 0.47-0.99nM.
  • Embodiment 6 the neutralization ability detection of nanobody to SARS-CoV-2 pseudovirus infection
  • the eight kinds of nanobodies described in the present invention are diluted to 10 concentration gradients with DMEM medium, and the final volume of each concentration is 50ul, wherein, only DMEM medium is contained in the 10th gradient and the nanobody concentration is 0 , and it was used as a control group, and then 3 ul of SARS-CoV-2 Alpha mutant strain pseudovirus capable of producing about 1x10 5 RLUs (related luciferase activity) (gifted by researcher Wang Haikun from Shanghai Pasteur Institute, Chinese Academy of Sciences, or also Can be purchased from Beijing Yunling Biotechnology Co., Ltd.) added to the nanoantibody diluent, mixed and incubated at 37 degrees for 60 minutes, and then 50ul containing 10,000 HEK293T-hACE2 cells (gifted by Wang Haikun Research Institute, Shanghai Pasteur Institute, Chinese Academy of Sciences, Alternatively, it can also be purchased from Nanjing Novizan Biotechnology Co., Ltd.) and added to the virus-antibody complex, mixed thoroughly and then added to
  • the neutralization EC 50 range for the Alpha mutant pseudovirus is 0.22-4.46nM
  • the neutralization EC 50 range for the Beta mutant pseudovirus is 0.81-10.33nM
  • the neutralizing EC 50 range of Delta mutant pseudovirus is 1.49-11.54nM.

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Abstract

La présente invention concerne un nanocorps dérivé du chameau qui se lie de manière spécifique à une protéine S de SARS-CoV-2 et un fragment de liaison à l'antigène de celui-ci, et concerne en particulier un nanocorps dérivé du chameau ou un fragment de liaison à l'antigène de celui-ci capable de se lier à des protéines S de surface de souches mutantes du coronavirus telles que SARS-CoV-2 Alpha (B.1.1.7), Bêta (B.1.351), Gamma (P.1) et Delta (B.1.617.2) avec une affinité élevée. Le nanocorps ou le fragment de liaison à l'antigène de celui-ci peut être utilisé pour prévenir, détecter, diagnostiquer ou traiter des infections provoquées par un coronavirus, en particulier le virus SARS-CoV-2.
PCT/CN2022/142281 2021-12-31 2022-12-27 NANOCORPS DÉRIVÉ DU CHAMEAU À HAUTE AFFINITÉ POUR DES SOUCHES MUTANTES α, β, γ ET δ DE SARS-COV-2 WO2023125520A1 (fr)

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