WO2022158497A1 - ANTICORPS VISANT LA PROTÉINE DE SPICULE DU SARS-CoV-2 - Google Patents

ANTICORPS VISANT LA PROTÉINE DE SPICULE DU SARS-CoV-2 Download PDF

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WO2022158497A1
WO2022158497A1 PCT/JP2022/001859 JP2022001859W WO2022158497A1 WO 2022158497 A1 WO2022158497 A1 WO 2022158497A1 JP 2022001859 W JP2022001859 W JP 2022001859W WO 2022158497 A1 WO2022158497 A1 WO 2022158497A1
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antibody
cov
sars
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健 浦野
裕子 成相
公一 森田
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株式会社mAbProtein
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/1002—Coronaviridae
    • 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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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

Definitions

  • antibodies that bind to the SARS-CoV-2 spike protein relate to antibodies and antigen-binding fragments thereof that bind to the region where the spike protein binds to the receptor, angiotensin converting enzyme-2 (ACE2), and have neutralizing activity. Further, it relates to methods of preventing, testing and treating coronavirus infection using antibodies or antigen-binding fragments thereof.
  • ACE2 angiotensin converting enzyme-2
  • the infectious disease COVID-19 caused by the new coronavirus (SARS-CoV-2), has also quickly expanded from an outbreak in Wuhan, China to a pandemic.
  • SARS-CoV-2 new coronavirus
  • the WHO recognized that it had reached a pandemic on March 11, 2020, the cumulative number of infected people was 118,381 and the cumulative number of deaths was 4292, but at the end of December 2020, The number of infected people is about 85.1 million, and the death toll is about 1.84 million. Ending the pandemic through the development and dissemination of diagnostic kits, therapeutic drugs, and prophylactic drugs is an urgent issue.
  • SARS-CoV-2 causes symptoms such as fever, dry cough, and malaise by binding the spike protein on the surface of virus particles to ACE2 on the surface of human cells as a virus receptor, invading cells and destroying cells. In severe cases, it can cause pneumonia and even death. In addition, it has been reported that aftereffects occur at a certain frequency even after recovery, and many restrictions are imposed on social activities thereafter.
  • neutralizing antibodies In order to prevent the spread of SARS-CoV-2 infection and bring it to an end as soon as possible, it is essential to develop kits for early diagnosis and medicines for treatment.
  • neutralizing antibodies By administering neutralizing antibodies at the early stage of infection, it is possible not only to prevent aggravation, but also to treat severely ill patients by administering them in combination with anti-cytokine antibodies. Expected. Furthermore, administration before infection can be expected to have an effect of preventing infection like a vaccine. Pending the development of effective small molecule drugs, neutralizing antibodies may be effective for treating infected individuals. Furthermore, neutralizing antibodies can be used as test reagents for detecting viruses and adsorbents for removing viruses.
  • Non-Patent Documents 1 and 2 The development of antibodies that neutralize SARS-CoV-2 is progressing, and there are several antibody drugs that are already undergoing clinical trials.
  • viruses are known to mutate, and many mutant strains have already been reported. Among them, it is known that some have a high affinity for the receptor ACE2 (Non-Patent Documents 1 to 3). Furthermore, it has also been reported that some mutants affect the affinity of existing neutralizing antibodies and reduce the efficacy of the antibodies (Non-Patent Document 4).
  • the present invention provides antibodies that have high neutralizing activity and high binding ability even to mutant strains that have high affinity for receptors, as well as pharmaceutical compositions, test agents, and virus adsorbents using the same. The task is to
  • the present invention includes the following antibodies, pharmaceutical compositions, detection kits, and adsorbents.
  • an antibody that binds to the SARS-CoV-2 spike protein and has neutralizing activity wherein the heavy chain variable domain consists of the amino acid sequence GFTFNTHA (SEQ ID NO: 44), the CDRH1 region, IRSKTYNYAR (SEQ ID NO: 45); a CDRH2 region consisting of VRDLGSGYSYAMDY (SEQ ID NO: 46), a CDRL1 region consisting of the light chain variable domain consisting of the amino acid sequence ESVDDFGISF (SEQ ID NO: 47), a CDRL2 region consisting of AAS (SEQ ID NO: 48), QQSKEVPFT (SEQ ID NO: 48) 49), an anti-SARS-CoV-2 spike protein antibody characterized by comprising a CDRL3 region consisting of (2) an antibody that binds to the SARS-CoV-2 spike protein and has neutralizing activity, wherein the heavy chain variable domain consists of the amino acid sequence G
  • the anti-SARS-CoV-2 spike protein antibody according to (3) wherein the amino acid sequence of the H chain variable region is SEQ ID NO:32 and the amino acid sequence of the L chain variable region is SEQ ID NO:34.
  • the anti-SARS-CoV-2 spike protein antibody according to (4) wherein the amino acid sequence of the H chain variable region is SEQ ID NO:36 and the amino acid sequence of the L chain variable region is SEQ ID NO:38.
  • a gene comprising the CDR sequence of any one of (1) to (5) or the gene encoding the variable region of any one of (6) to (10) as an open reading frame.
  • a pharmaceutical composition comprising, as an active ingredient, at least one of the antibody of (13) or an antigen-binding fragment of the antibody of (12).
  • kits To detect and/or quantify SARS-CoV-2 containing at least one of the antibody according to any one of (1) to (10) or the antigen-binding fragment of the antibody according to (12) kit.
  • kit The kit according to (15), wherein the kit is an immunochromatographic test strip or ELISA.
  • SARS-CoV-2 adsorption characterized by immobilizing at least one antigen-binding fragment of the antibody according to any one of (1) to (10) or the antibody according to (12). agent.
  • the adsorbent according to (17), wherein the antibody according to any one of (1) to (10) is a low-molecular-weight antibody.
  • a sanitary material, filter or carrier comprising the adsorbent according to (17) or (18).
  • (20) A method for testing whether a subject is infected with SARS-CoV-2, comprising collecting a sample from the subject, (1) to (10) using at least one or more antibodies against a protein in the sample A testing method characterized by detecting antibody reactivity.
  • (21) A method of treating a subject infected with SARS-CoV-2, comprising administering the pharmaceutical composition of (14).
  • (22) The treatment method according to (21), wherein the subject is examined by the examination method of (20), and if the subject is positive for SARS-CoV-2 infection, treatment is performed.
  • a diagram showing an example of an antibody selection method by immunoprecipitation The figure which shows the result of having evaluated the antibody by the western blotting method. The figure which shows the result of having evaluated the antibody by the immunoprecipitation method. This shows that the resulting monoclonal antibody recognizes the three-dimensional structure of the spike protein of SARS-CoV-2, which is an antigen. The figure which shows the result of having evaluated the antibody by the immunoprecipitation method. The obtained monoclonal antibody recognizes the spike protein Y453F mutant.
  • a diagram showing the evaluation of the binding properties of antibodies to various mutant strains by immunoprecipitation The left figure schematically shows the sites where the spike protein is mutated in each mutant strain. The right figure shows the analysis result by immunoprecipitation method. The figure which shows the result of having evaluated the antibody by ELISA. The figure which shows an example of the evaluation result of the monoclonal antibody by a virus neutralization activity. Schematic showing evaluation of monoclonal antibodies by competitive ELISA assay.
  • the antibody refers to a monoclonal antibody or derivative that specifically binds to the following SARS-CoV-2 spike protein receptor binding domain (RBD). Also intended to include antigen-binding fragments of such antibodies that exhibit substantially the same antigen specificity as the original antibody.
  • the antigen-binding fragment is Fab, Fab', F(ab') 2 , single chain antibody (scFv), disulfide-stabilized V region fragment (dsFv), miniaturized antibody (Fv-clasp, Non-Patent Document 5) or CDR A region with a sequence necessary for an antibody to exert its function, such as a peptide containing
  • humanized antibodies such as humanized chimeric antibodies, humanized CDR-grafted antibodies, and humanized antibodies using gene recombination technology, monoclonal antibodies that specifically bind to RBD shown in the following examples, humanized using genetically modified mice
  • Antibodies are also included in the antibodies of the present invention.
  • humanized antibodies and human antibodies When administered to humans, humanized antibodies and human antibodies have fewer side effects than non-human animal antibodies, and their therapeutic effects last for a long time.
  • when used as a pharmaceutical composition it also includes an antibody that has been modified to enhance the effect of an antibody drug by means of a technique for adjusting the half-life of the antibody, a technique for enhancing ADCC activity, or the like.
  • epitope refers to a portion of an antigen recognized by an antibody (here, it refers to the RBD of the spike protein of SARS-CoV-2), and the domain comprising the antibody variable region disclosed herein is It means the site on the antigen that binds.
  • the antibody recognizes either a linear amino acid sequence or a three-dimensional structure, so an epitope can be defined by the amino acid sequence or the structure of the antigen. can.
  • antibodies that mainly recognize three-dimensional structures are selected and obtained.
  • kit refers to not only ELISA, Western blotting, and immunoprecipitation shown in the following examples, but also detection and quantification of SARS-CoV-2 using the antibodies disclosed herein.
  • a packaged combination consisting of necessary reagents, instruments, etc.
  • an immunochromatographic test strip on which the antibody disclosed herein is immobilized an extract solution for extracting an antigen from a specimen such as saliva or a nasopharyngeal swab, and a test that can rapidly test for infection.
  • I mean kit.
  • kit when used by being fixed to an adsorbent, it refers to a packaged combination of components such as a filter and the like necessary for exchanging the adsorbent.
  • testing refers to detecting the presence of SARS-CoV-2 in a subject's sample. In particular, it refers to detecting the presence of SARS-CoV-2 in a subject sample. Furthermore, it refers to estimating the detected mutant strain.
  • Samples used for SARS-CoV-2 infection testing such as saliva, nasopharyngeal swab, and sputum, can be suitably used. Also, not limited to this, any sample that can contain SARS-CoV-2 can be used.
  • a pharmaceutical composition is formulated to be compatible with the route of administration.
  • the route of administration is generally intravenous administration, subcutaneous administration, or parenteral administration such as a spray administered into the respiratory tract of the nasal cavity or throat, but is not limited thereto.
  • Pharmaceutical compositions can contain compounds generally used in the art, such as carriers, excipients, solvents, and diluents suitable for dosage forms. In the case of administration by intravenous injection or subcutaneous injection, it is possible to administer at a maximum dose of 0.1 gr/kg, depending on the symptoms.
  • antibodies can be used as adsorbents for equipment and materials related to epidemic prevention.
  • SARS-CoV-2 can be captured by immobilizing a neutralizing antibody against SARS-CoV-2 on sanitary materials such as masks, filters of air conditioners and air purifiers.
  • sanitary materials such as masks, filters of air conditioners and air purifiers.
  • ECMO extracorporeal membrane oxygenation
  • the virus will be captured by the antibodies, making it possible for healthcare workers to safely carry out ECMO.
  • ECMO extracorporeal membrane oxygenation
  • a low-molecular-weight antibody Fv-Clasp
  • Fv-Clasp low-molecular-weight antibody
  • a method of immobilizing an antibody on a carrier by ionic bonding by immersion in an aqueous solution of the antibody a method of introducing an aldehyde group into a carrier having a specific functional group and covalently bonding the aldehyde group and the antibody, a method of covalently bonding the aldehyde group and the antibody, Examples include a method of ionically bonding an antibody to a carrier, and a method of coating a carrier with a polymer having a specific functional group and then introducing an aldehyde group to covalently bond the aldehyde group and the antibody.
  • the RBD positions 319-589, 271 amino acids, SEQ ID NO: 1 of the SARS-CoV-2 spike protein (UniProt P0DTC2) was added with its own secretion signal, and the C-terminal G196 tag sequence (DLVPR, SEQ ID NO: 2) and His Synthesize an artificial gene fusing 6 sequences (HHHHHH, SEQ ID NO: 3), incorporate it into an expression vector (hereinafter, this expression vector is referred to as a spike protein RBD expression vector), express it as a secretory protein using animal cells, Ni - Purified using NTA agarose (QIAGEN).
  • mice were immunized with the purified protein (hereinafter referred to as purified protein) according to a conventional method, and the following hybridomas were selected and established by ELISA using the purified protein.
  • the isotype of the antibodies produced by the hybridomas was confirmed using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Sigma). Genetic recombination experiments for protein expression and preparation of antibodies were applied to Shimane University and the Minister of Education, Culture, Sports, Science and Technology, and the necessary approvals were obtained before implementation.
  • the main purpose of using antibodies is to detect viruses, which are antigens, to inhibit SARS-CoV-2 cell infection, and to develop adsorbents. Antibodies were further screened by immunoprecipitation.
  • immunoprecipitation of the purified protein was performed according to the standard method. Immunoprecipitates were detected by Western blotting (WB) using a home-made rabbit polyclonal antibody that recognizes the G196 tag sequence. As a positive control, one-tenth the amount of purified protein used for immunoprecipitation was used. An example of analysis results is shown in FIG. IgH stands for immunoglobulin heavy chain and IgL for immunoglobulin light chain.
  • the 4-8.2, 10-7.2, and 11-4.1 antibodies efficiently immunoprecipitated the purified protein.
  • the 4-4.1 antibody could not be immunoprecipitated.
  • the 4-8.2, 10-7.2, and 11-4.1 antibodies are monoclonal antibodies that can specifically recognize and capture the three-dimensional structure of the purified protein and efficiently immunoprecipitate it. . 14 types of hybridomas shown in the table below were selected by immunoprecipitation screening (Table 1).
  • the purified RBD protein was immunoprecipitated according to the standard method using each of the 9 types of purified antibodies cultured and purified by high-density culture of antibody-producing cells (Fig. 3). Immunoprecipitated products were detected by Western blotting using a homemade rabbit polyclonal antibody that recognizes the G196 tag sequence. As a positive control, one-fifth the amount of purified RBD protein used for immunoprecipitation was used. A monoclonal antibody G196 that recognizes the G196 tag sequence was used as a positive control antibody for immunoprecipitation. A home-made anti-B-Raf purified monoclonal antibody was used as a negative control antibody (control mAb) for immunoprecipitation.
  • control mAb negative control antibody
  • the anti-B-Raf purified monoclonal antibody control mAb
  • the 9 types of purified antibodies subjected to immunoprecipitation specifically recognized the purified RBD protein and could efficiently immunoprecipitate it.
  • Immunoprecipitation of the purified protein was performed according to the standard method using each of the 9 types of purified antibodies cultured and purified by the high-density culture method of antibody-producing cells. Immunoprecipitated products were detected by Western blotting using a homemade rabbit polyclonal antibody that recognizes the G196 tag sequence. As a positive control, one-fifth the amount of purified protein used for immunoprecipitation was used (Fig. 4). A monoclonal antibody G196 that recognizes the G196 tag sequence was used as a positive control antibody for immunoprecipitation. An anti-B-Raf purified monoclonal antibody was used as a negative control antibody (control mAb) for immunoprecipitation.
  • the seven purified antibodies other than the 4-8.2 and 5-3.6 antibodies immunoprecipitated the purified protein (Y453F mutant) as efficiently as or more efficiently than the existing G196 antibody.
  • Two purified antibodies, the 4-8.2 and 5-3.6 antibodies were slightly less efficient at immunoprecipitating the purified protein (Y453F mutant).
  • the anti-B-Raf purified monoclonal antibody (control mAb) could not be immunoprecipitated.
  • the 7 types of purified antibodies other than the 4-8.2 and 5-3.6 antibodies are mutant purified proteins (Y453F mutant), they can specifically recognize and efficiently immunize in the same way as the wild type. It was found to be able to settle.
  • the mutant spike Immunoprecipitation of the purified RBD protein of each virus was performed to determine whether the protein could be recognized and captured.
  • an artificial gene was prepared in the same manner as described above and incorporated into an expression vector. Specifically, an artificial gene is synthesized by adding its own secretion signal to the sequence of positions 319 to 589 of each spike protein, and fusing the G196 tag sequence and His 6 sequence to the C-terminus, and incorporating it into an expression vector. Protein was expressed.
  • SARS-CoV-2 is an Omicron strain (SEQ ID NOS: 4 and 5, even numbers are amino acid sequences, odd numbers are gene sequences.
  • alpha strains SEQ ID NOS: 6 and 7
  • beta strains SEQ ID NOS: 8, 9
  • sequences of gamma strains SEQ ID NOS: 10, 11
  • delta strains SEQ ID NOS: 12, 13
  • lambda strains SEQ ID NOS: 14, 15
  • mu strains SEQ ID NOS: 16, 17
  • coronavirus NL63 used the amino acid sequences from positions 481 to 677 (SEQ ID NOs: 18 and 19) and incorporated them into an expression vector to purify the RBD protein.
  • Immunoprecipitated products were detected by Western blotting using a homemade rabbit polyclonal antibody that recognizes the G196 tag sequence. As a positive control for Western blotting, one-tenth the amount of purified protein used for immunoprecipitation was used (Fig. 5).
  • Antibody 7-2.1 was able to immunoprecipitate the purified RBD protein of the conventional strain, but was unable to immunoprecipitate all other purified RBD proteins, including that of the spike protein of NL63.
  • Antibodies 10-2.1 and 10-7.2 were able to immunoprecipitate various purified RBD proteins of all novel coronaviruses studied, including the purified RBD protein of the Omicron strain. However, the purified RBD protein of the spike protein of NL63 could not be immunoprecipitated. In other words, it was revealed that they specifically recognize the spike protein of SARS-CoV-2.
  • Antibody 5-6.1 was able to immunoprecipitate various purified RBD proteins of novel coronavirus, except Omicron, Mu and NL63.
  • Antibody 11-4.1 was able to immunoprecipitate various purified RBD proteins of novel coronavirus, except Omicron strain and NL63.
  • antibody 10.2.1 or 10.7.2 all variants mapped as VOC or VOI can be detected.
  • an enzyme-labeled anti-mouse IgG antibody Jackson ImmunoResearch Laboratories was added, the absorbance (wavelength 450 nm, background wavelength 620 nm) was measured with a plate reader, and the antibody titer was determined by the turning point method.
  • Anti-B-Raf purified monoclonal antibody IgG1 was used as a negative control antibody. Color development time was 15 minutes at 25° C. in the dark, and all other conditions were the same (FIG. 6).
  • the 5-6.1 antibody showed an extremely high antibody titer of 5.4 ⁇ 10 4 times and the 7-2.1 antibody 1.8 ⁇ 10 4 times.
  • the antibody titer of each purified antibody was evaluated by measuring the virus-neutralizing activity in an infection inhibition experiment.
  • SARS-CoV-2 strain JPN/TY-WK-521, obtained from National Institute of Infectious Diseases, GenBank: LC522975.1 infection inhibition experiments on Vero E6 cells were performed at containment level P3.
  • Each purified antibody (1 mg/mL) was serially diluted 2-fold from 10-fold to 10,240-fold, and the inhibitory activity was evaluated based on the dilution ratio at which the number of plaques during virus-only infection decreased by 50%.
  • Inhibitory activity was evaluated by 50% Plaque Reduction Neutralization Test (PRNT 50 ) (Fig. 7, Table 2). In 7-2.1, PRNT 50 exhibited an extremely high inhibitory activity of 2,560-fold.
  • Antigen-antibody reaction was performed by adding 0, 100, and 1000 ng of unlabeled purified antibody per well to the SARS-CoV-2 spike protein antibody measurement plate. After washing, each enzyme-labeled purified antibody was added using the Peroxidase Labeling Kit (Dojindo Laboratories), and the absorbance (wavelength 450 nm, background wavelength 620 nm) was measured with a plate reader. Color development time was 15 minutes at 25° C. in the dark, and all other conditions were the same.
  • the antigen-binding site is already occupied by the unlabeled antibody added first, and if the binding site of the labeled antibody added later is the same, it cannot bind. Thus, the more unlabeled antibody added initially, the less labeled antibody will bind. In fact, as shown in the upper panel of FIG. 8, the larger the amount of unlabeled 10-2.1 antibody initially added, the less the labeled 10-2.1 antibody can bind to the plated antigen protein. , OD is low.
  • the antibody gene sequences and amino acid sequences of the hypervariable regions of the synthesized cDNA were determined by direct sequencing using H chain and L chain primer sets.
  • the primer sequences used are as follows. The underlined portion indicates the recognition site of the restriction enzyme.
  • Hybridoma 7-2.1 antibody H chain amino acid sequence (SEQ ID NO: 24), nucleotide sequence (SEQ ID NO: 25), L chain amino acid sequence (SEQ ID NO: 26), nucleotide sequence (SEQ ID NO: 27), hybridoma 10-2 .1 antibody H chain amino acid sequence (SEQ ID NO: 28), nucleotide sequence (SEQ ID NO: 29), L chain amino acid sequence (SEQ ID NO: 30), nucleotide sequence (SEQ ID NO: 31), hybridoma 5-6.1 antibody H chain amino acid sequence (SEQ ID NO: 32), nucleotide sequence (SEQ ID NO: 33), L chain amino acid sequence (SEQ ID NO: 34), nucleotide sequence (SEQ ID NO: 35), H chain amino acid of hybridoma 11-4.1 antibody Sequence (SEQ ID NO: 36), nucleotide sequence (SEQ ID NO: 37), L chain amino acid sequence (SEQ ID NO: 38), nucleotide sequence (SEQ ID NO: 39), H chain amino
  • sequence of the primer part is deleted from the base sequence in the sequence list.
  • the amino acid sequence of the L chain of the 7-2.1 antibody (SEQ ID NO: 26) and the amino acid sequence of the L chain of the 5-6.1 antibody (SEQ ID NO: 34) were identical, but the nucleotide sequences (SEQ ID NOS: 27 and 35 ) differed by one base.
  • the amino acid sequences and base sequences of the complementarity determining regions (CDRs) of each antibody are summarized below.
  • the antibody with the above CDRs binds to the SARS-CoV-2 spike protein with high sensitivity and specificity and has neutralizing activity. Furthermore, it maintains strong binding to the Y453 mutant, which exhibits strong binding to the receptor human ACE2, as well as to mutant spike proteins classified as VOC and VOI by WHO and calling attention to them. there is In addition, as shown in FIG. 8, antibodies with different epitopes have been obtained, so there is a high possibility that other mutant strains can also be treated. Therefore, it can be widely used as a pharmaceutical composition, test drug, and virus adsorbent in the aspects of treatment, examination, and prevention.

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Abstract

Dans cette invention, un hybridome est préparé en utilisant, comme antigène, une protéine dont le signal de sécrétion est ajouté au domaine de liaison au récepteur (RBD, positions 319 à 589) de la protéine de spicule de SARS-CoV-2 et la séquence marqueur G196 (DLVPR) avec la séquence His6 (HHHHH) fusionnée à l'extrémité C, pour obtenir une pluralité d'anticorps monoclonaux reconnaissant la structure tridimensionnelle du RBD. Les anticorps monoclonaux obtenus ont une activité de neutralisation élevée contre le virus, et peuvent être utilisés pour une composition pharmaceutique, un réactif d'essai et un matériau d'élimination de virus. En outre, étant donné que des anticorps ayant une capacité de liaison différente contre divers variants sont obtenus, les anticorps peuvent également être utilisés pour identifier les variants.
PCT/JP2022/001859 2021-01-22 2022-01-19 ANTICORPS VISANT LA PROTÉINE DE SPICULE DU SARS-CoV-2 WO2022158497A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
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