WO2022068895A1 - Anticorps monoclonal dirigé contre le domaine extracellulaire de la protéine de spicule du sars-cov-2, et son utilisation - Google Patents

Anticorps monoclonal dirigé contre le domaine extracellulaire de la protéine de spicule du sars-cov-2, et son utilisation Download PDF

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WO2022068895A1
WO2022068895A1 PCT/CN2021/121803 CN2021121803W WO2022068895A1 WO 2022068895 A1 WO2022068895 A1 WO 2022068895A1 CN 2021121803 W CN2021121803 W CN 2021121803W WO 2022068895 A1 WO2022068895 A1 WO 2022068895A1
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amino acid
seq
variable region
acid sequences
chain variable
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PCT/CN2021/121803
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Chinese (zh)
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丁莉丹
刘培
陈晖�
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南京金斯瑞生物科技有限公司
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Priority to CN202180067438.XA priority Critical patent/CN116419971A/zh
Publication of WO2022068895A1 publication Critical patent/WO2022068895A1/fr

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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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention belongs to the field of virus detection and diagnosis, and relates to a monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane.
  • the present invention also relates to the application of the monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane.
  • Severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) belongs to the beta genus of coronavirus, with a diameter of 60nm to 140nm, with a capsule, and the particles are round or oval, often polymorphic Its genetic characteristics are significantly different from SARSr-CoV and MERSrCoV. SARS-CoV-2 is the seventh coronavirus discovered so far that can infect humans. The disease caused by the virus is called novel coronavirus disease 2019 (COVID-19), and as of August 13, 2020, more than 20.52 million cases of COVID-19 have been reported to WHO, according to Worldometer and nearly 740,000 deaths.
  • COVID-19 novel coronavirus disease 2019
  • SARS-CoV-2 also utilizes its highly glycosylated spike protein (Spike protein, S protein, S protein) to complete host cell receptor binding and virus infection in the form of trimers.
  • ECD extra cellular domain
  • the RBD (receptor-binding domain) region of the S1 subunit can recognize and bind to the angiotensin-converting enzyme 2 (human angiotensin-converting enzyme 2, hACE2) of the host cell, and the S2 subunit mediates the membrane fusion between the virus and the host cell.
  • angiotensin-converting enzyme 2 human angiotensin-converting enzyme 2, hACE2
  • nucleic acid testing requires relatively high throat or nasopharyngeal swab sampling. Improper sample collection techniques, storage conditions, and PCR operations may result in false negative and false positive results, resulting in significant delays in early diagnosis and follow-up management. Prompt life support treatment and preventive quarantine pose serious challenges.
  • the present invention provides a monoclonal antibody against the outer membrane region of the SARS-CoV-2 spike protein or a functional fragment thereof, the antibody or functional fragment thereof comprising a heavy chain variable region and a light chain variable region ,in
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3,
  • the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118 or 124 or the same as shown in Variants with up to three (eg, one, two, or three) amino acid mutations in the amino acid sequence;
  • the HCDR2 comprises a variant selected from the group consisting of SEQ ID NOs: 35, 41, 47, 53, 59, 65, 71, 77, 83 , 89, 95, 101, 107, 113, 119, or 125, or a variant with up to three amino acid mutations to the amino acid sequence shown;
  • the HCDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 36, 42, 48 , 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 or 126 or variants with up to three amino acid mutation
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3,
  • the LCDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, or 127 or the amino acid sequence shown in Variants with up to three amino acid mutations are shown in the amino acid sequence;
  • the LCDR2 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110,
  • the LCDR3 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 39, 45, 51, 57, 63, 69, 75 , 81, 87, 93, 99, 105, 111, 117, 123 or 129 or variants with up to
  • the HCDR1 sequence comprises a sequence selected from SEQ ID NO: 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, or 124
  • the amino acid sequence of the HCDR2 sequence comprising amino acids selected from the group consisting of SEQ ID NOs: 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119 or 125
  • the HCDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 or 126
  • the LCDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the sequence:
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 37, 38 and 39 or variants having up to three amino acid mutations respectively from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 43, 44 and 45 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 49, 50 and 51 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 52, 53 and 54, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 55, 56 and 57 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 61, 62, and 63 or variants with up to three amino acid mutations, respectively, from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 64, 65 and 66 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 67, 68 and 69 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 70, 71 and 72, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 73, 74 and 75 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 76, 77 and 78 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 79, 80 and 81 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 82, 83 and 84, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 comprise, respectively, The amino acid sequences shown in SEQ ID NOs: 85, 86 and 87 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 88, 89 and 90, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 91, 92 and 93 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 94, 95 and 96, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 97, 98 and 99 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 100, 101 and 102 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 103, 104 and 105 or variants with up to three amino acid mutations respectively from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 106, 107 and 108 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 109, 110, and 111 or variants with up to three amino acid mutations, respectively, from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 112, 113 and 114 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 115, 116 and 117 or variants with up to three amino acid mutations respectively from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 118, 119 and 120, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences set forth in SEQ ID NOs: 121, 122 and 123 or variants with up to three amino acid mutations, respectively, from the amino acid sequences set forth; or
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 124, 125 and 126 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 127, 128 and 129 or variants with up to three amino acid mutations respectively from the amino acid sequences shown.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the sequence:
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 37, 38 and 39;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 43, 44 and 45;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 49, 50 and 51;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 52, 53 and 54 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 61, 62 and 63;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 64, 65 and 66 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 67, 68 and 69;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 70, 71 and 72 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 73, 74 and 75:
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 76, 77 and 78 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 79, 80 and 81;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 82, 83 and 84 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 85, 86 and 87;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 88, 89 and 90 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 91, 92 and 93;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 94, 95 and 96 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 97, 98 and 99;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 100, 101 and 102 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 103, 104 and 105;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 106, 107 and 108 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 109, 110 and 111;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 112, 113 and 114 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 115, 116 and 117;
  • HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 118, 119 and 120, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 121, 122 and 123, respectively; or
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 124, 125 and 126, respectively; and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 127, 128 and 129, respectively.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the sequence:
  • amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NOs: 100, 101 and 102 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 103, 104 and 105;
  • amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NOs: 112, 113 and 114 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NOs: 115, 116 and 117;
  • the heavy chain variable region sequence comprises and SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or
  • the amino acid sequence shown in 32 has an amino acid sequence with at least 80% identity;
  • the amino acid sequences shown in 23, 25, 27, 29, 31 or 33 have amino acid sequences that are at least 80% identical.
  • the heavy chain variable region sequence comprises and SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or
  • the amino acid sequence shown in 32 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence;
  • the light chain variable region sequence comprises the same as SEQ ID NO: 3, 5, 7, 9, 11, 13, 15,
  • the amino acid sequence shown in 17, 19, 21, 23, 25, 27, 29, 31 or 33 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, Amino acid sequences that are 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.
  • the heavy chain variable region sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 The amino acid sequence shown; the light chain variable region sequence comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 show the amino acid sequence.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 2 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 3
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 4 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 5
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 6 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 7
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 8 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 9
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 10 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 11
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 12 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 13
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 14 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 15
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 16 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 17
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 18 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 19
  • the sequence shown has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 20 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 21
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 22 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 23
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 24 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 25
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 26 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 27
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 28 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 29
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 30 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 31
  • the sequence shown has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences; or
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 32 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 33
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:5;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 20
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:31;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 25;
  • the present invention provides isolated polynucleotides encoding the above-described monoclonal antibodies against the extramembrane region of the SARS-CoV-2 spike protein or functional fragments thereof.
  • the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the above-mentioned anti-SARS-CoV-2 spike protein outer-membrane monoclonal antibody or a functional fragment thereof, and encoding the The nucleotide sequence of the light chain variable region of a monoclonal antibody against the extramembrane region of the SARS-CoV-2 spike protein or a functional fragment thereof.
  • the present invention provides an expression vector comprising the polynucleotide.
  • the present invention provides a host cell or cell-free expression system comprising the expression vector.
  • the present invention provides a pharmaceutical composition comprising the anti-SARS-CoV-2 spike protein monoclonal antibody or its functional fragment and pharmaceutically acceptable vector.
  • the present invention provides the application of the monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane or its functional fragment in the preparation of a drug for the treatment of coronavirus.
  • the present invention provides a kit for detecting coronavirus, which comprises the monoclonal antibody or its functional fragment.
  • the coronavirus is selected from SARS-CoV, MERS-Cov or SARS-Cov-2, preferably SARS-Cov-2. In other embodiments, the coronavirus is SARS-Cov-2.
  • the present invention provides a method for preparing a monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane or a functional fragment thereof, comprising:
  • PBMC peripheral blood mononuclear Cell
  • variable region coding sequence for recombinant antibody production to obtain a functional monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane.
  • the monoclonal antibody is a rabbit-derived, chimeric, humanized or human antibody. In some preferred embodiments, the monoclonal antibody is of rabbit origin. In other preferred embodiments, the monoclonal antibody is humanized.
  • the monoclonal antibody against the membrane outer region of the SARS-CoV-2 spike protein developed by the present invention can specifically bind to the membrane outer region of the S protein, and can specifically recognize S1 or S2.
  • the monoclonal antibodies provided can recognize 11 epitopes of antigens, and the diversity of antibodies facilitates the development of detection kits.
  • Fig. 1 is the result figure of the rabbit serum titer detection result after immunization
  • Fig. 2 shows the result of SDS-PAGE identification after purification of some monoclonal antibodies
  • Figure 3 is a graph showing the results of affinity detection between monoclonal antibody and S-ECD recombinant protein
  • Fig. 4 is a paired detection diagram of monoclonal antibody 3C4 and antibody 39G6 on recombinant protein S-ECD;
  • Figure 5 is a graph showing the paired detection of recombinant protein S-ECD by monoclonal antibody 2F8 and antibody 39G6.
  • the present invention relates to a functional monoclonal antibody of the S protein membrane outer region of the virus SARS-CoV-2.
  • the embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • novel coronavirus SARS-CoV-2
  • 2019-nCoV refers to a new type of viral infection that began to appear and spread in 2019. It belongs to the genus beta coronavirus, with an envelope and round or Oval, often pleomorphic, 60-140 nm in diameter. Its genetic characteristics are significantly different from SARSr-Cov and MERSr-CoV. Studies have shown that it shares more than 85% homology with bat SARS-like coronavirus (bat-SL-CoVZC45). When isolated and cultured in vitro, 2019-nCov can be found in human respiratory epithelial cells in about 96 hours, while it takes about 6 days to isolate and culture in Vero E6 and Huh-7 cell lines.
  • antibody is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains (wherein two heavy (H) and two light (L) chains are connected to each other by disulfide bonds (ie, "complete antibody molecule")) , and multimers thereof (eg, IgM) or antigen-binding fragments thereof.
  • Each heavy chain consists of a heavy chain variable region ("HCVR” or “VH”) and a heavy chain constant region (consisting of the domains CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (“LCVR or "VL”) and a light chain constant region (CL).
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), Intervening are more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to hydroxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3 , CDR3, FR4.
  • the FRs of the antibody may be identical to human germline sequences or may be naturally or artificially modified.
  • the term "monoclonal antibody” refers to a homogeneous antibody directed against only a particular epitope. Each monoclonal antibody is directed against a single epitope on an antigen, in contrast to typical polyclonal antibody preparations that include different antibodies directed against different antigenic determinants (epitopes).
  • the modifier "monoclonal” denotes a uniform characteristic of an antibody and is not to be construed as requiring that the antibody be produced by any particular method.
  • the monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods, or obtained by screening methods as described elsewhere herein.
  • mutation refers to a monoclonal antibody or functional fragment thereof comprising an alteration of one or more (several) amino acid residues at one or more (several) positions, ie, a substitution, insertion and/or deletion of a polypeptide.
  • isolated polynucleotide refers to a polynucleotide that is not naturally found in nature, including polynucleotides isolated from nature (including in vivo) by biological techniques, as well as artificially synthesized polynucleotides.
  • An isolated polynucleotide can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof. It should be pointed out that those skilled in the art can design the provided nucleotide sequences with different nucleotide sequences according to the amino acid sequences of the heavy chain variable region and light chain variable region provided herein and based on the degeneracy of codons. nucleotide sequences, but both encode the same amino acid sequence. These altered nucleotide sequences are also included within the scope of the present invention.
  • vector when referring to a polynucleotide refers to any molecule (eg, nucleic acid, plasmid or virus, etc.) used to transfer information encoded by a nucleotide into a host cell.
  • expression vector or “expression cassette” refers to a vector suitable for expressing a gene of interest (nucleotide sequence to be expressed) in a host cell, usually including parts of the gene of interest, promoter, terminator, marker gene and the like.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest.
  • the term includes progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent cell, so long as the progeny has the selected gene of interest.
  • Commonly used host cells include bacteria, yeast, mammalian cells, and the like.
  • antibody functional fragment means antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody.
  • antibody fragments capable of binding the coronavirus spike (S) protein or a portion thereof include, but are not limited to, sdAb (single domain antibodies), Fab (eg, antibodies obtained by papain digestion), F(ab')2 ( For example, by pepsin digestion), Fv or scFv (eg by molecular biology techniques).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and sustained release agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the Standard References in the latest edition of Remington's Pharmaceutical Sciences, which is hereby incorporated by reference in its entirety. Examples of suitable carriers or diluents include, but are not limited to, water, saline solution, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and hydrophobic-hydrophobic vehicles such as fixed oils can also be used.
  • the use of pharmaceutically active media and agents is well known in the art. Except for those conventional media or agents that are incompatible with the active ingredient, its use in the ingredients can achieve the desired effect.
  • amino acid substitution refers to the replacement of existing amino acid residues with different amino acid residues in a predetermined (original) amino acid sequence.
  • amino acid substitutions are preferably made in accordance with the substitutions shown below:
  • Percent (%) amino acid sequence identity with respect to a peptide or polypeptide sequence is defined as after comparing the sequences and introducing gaps where necessary to obtain maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity, a candidate The percentage of amino acid residues in a sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed in a variety of ways that are within the skill in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • administer and “treat” in reference to an animal, human, subject, cell, tissue, organ or biological fluid, it refers to combining an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, or biological fluid. Person, cell, tissue, organ or biological fluid contact.
  • administering and “treatment” can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, eg, by agents, diagnostic agents, binding compositions, or by other cells.
  • subject refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention and/or treatment of a disease or disorder such as a viral infection.
  • the term includes human subjects with or at risk of infection with a coronavirus such as SARS-CoV-2.
  • the term “effective amount” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • “Pharmaceutically acceptable carrier” refers to a carrier for administration, including various excipients, diluents and buffers, etc., which are suitable for human and/or animal administration without excessive adverse side effects, and at the same time Suitable for maintaining the viability of a drug or active agent located therein.
  • S-ECD spike protein extramembrane domain
  • the animal immunization antigen adopts recombinant protein S-ECD-His (the sequence is shown in SEQ ID NO: 1).
  • New Zealand white rabbits were immunized subcutaneously with 200 ⁇ g of S-ECD fusion protein. Subsequently, the immunization was repeated every other week, so that the experimental rabbits were boosted 3 times in total.
  • the serum titers of the two rabbits reached more than 10 5 after three immunizations (Fig. 1).
  • the experimental rabbit numbered 7967 was selected to collect sterile blood 7 days after the last immunization for subsequent antibody discovery.
  • the enriched cells were plated into a 96-well cell culture plate plated with feeder cells in advance at a density of 10 cells per well. Incubate the plate at 37 °C in 5% CO . After 6 days of incubation, fresh medium was exchanged, and on day 7 the overnight culture supernatants were collected and tested for the presence of antibodies against the extramembrane region of the S protein using ELISA binding as described below.
  • Indirect ELISA was used to assess the binding ability of the antibodies in the supernatant to the S-ECD protein.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-ECD protein at 1 ⁇ g/ml in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 150 ⁇ l/well of PBST containing 1% BSA for 14 hours at 37°C. The blocking solution was then discarded and 100 ⁇ l of B cell culture supernatant was added to each plate, followed by incubation at 37°C for 1 hour.
  • RNA from total cells in wells with an OD value greater than 1.0 was extracted using TRIzol (Life Technology, 15596-026) and reverse transcribed into cDNA using universal primers (Prime Script TM 1 st Strand cDNA Synthesis Kit, Takara).
  • the rabbit immunoglobulin heavy and light chain V-region fragments were subsequently amplified by antibody signal peptide and constant region-specific primers, and the resulting PCR fragments were homologously recombined into the pCDNA3.4 vector using vector-specific primer pairs Inserts were sequenced.
  • strains of rabbit IgG antibodies were obtained, which are the unique V-region protein amino acids of 3B4, 2F6, 5D10, 3D5, 3C4, 2F8, 2G4, 5F7, 5E4, 5H7, 2F12, 3F4, 5A10, 3E5, 5C9, and 2E12 clones. sequences and plasmids.
  • 2F6 heavy chain variable region amino acid sequence (SEQ ID NO: 4):
  • 5D10 heavy chain variable region amino acid sequence (SEQ ID NO: 6):
  • 5D10 light chain variable region amino acid sequence (SEQ ID NO: 7):
  • 3D5 heavy chain variable region amino acid sequence (SEQ ID NO: 8):
  • 3C4 heavy chain variable region amino acid sequence (SEQ ID NO: 10):
  • Plasmids containing antibody heavy chain and light chain, respectively, were co-transfected into HEK293-6E (NRC, 11565) cells, and after culturing in shake flasks at 37°C for 6 days, the supernatant was collected for antibody purification.
  • the Protein A column was equilibrated with buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2x the above buffer and filter sterilized. The filtered supernatant was incubated with the protein A column for 2 hours at room temperature.
  • the IgG was eluted with sterile 0.1 M sodium citrate (pH 3.5), and the eluate was collected and washed with 1/9. Neutralize with one volume of sterile 1M Tris-HCl (pH 9.0). Under sterile conditions, the product was buffer exchanged to PBS (pH 7.4) to remove residual elution buffer, and antibodies were quantified by OD280nm using an extinction coefficient Ec (0.1%) of 1.43.
  • Purified antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system using 10% precast gels (GenScript, M42012C). The gel was stained with Estain2.0 (GenScript, L00687R) and the molecular size and purity were estimated by comparing the stained bands with Protein Ladder (Takara, 3452), as shown in Figure 2 for the detection results of 3B4, 3C4, and 5B10 antibodies .
  • Example 5 Binding of monoclonal antibody recombinant supernatant to viral S-ECD protein, S-ECD trimer protein, S-RBD protein, S1 protein and S2 protein
  • Indirect ELISA was used to evaluate antibodies in the supernatant after reconstitution against S-ECD protein (Genscript, Z03481), S-ECD trimer protein (produced by Genscript), S-RBD protein (Genscript, T80302), S1 protein (Genscript, T80302) , Z03485) and S2 protein (produced by Genscript).
  • ELISA plates were coated with 100 ⁇ l/well of 1 ⁇ g/ml of recombinant protein to be tested in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 150 ⁇ l/well of PBST containing 1% BSA for 1 hour at 37°C.
  • the negative judgment standard is that the OD value is less than 2.1 times of the negative control well, and the positive judgment standard is that the OD value is greater than 1.0.
  • the test results are shown in Table 2 ("+” means positive; "-” means negative).
  • S-RBD (SEQ ID NO: 130, NCBI Accession No: QNA38155.1):
  • Example 6 EC50 test of monoclonal antibody binding to viral S-ECD protein
  • Indirect ELISA was used to assess the binding ability of purified antibodies to S-ECD protein.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-ECD protein at 0.5 ⁇ g/ml in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 250 ⁇ l/well of PBST containing 1% BSA for 2 hours at 37°C. Subsequently, the blocking solution was discarded, and 100 ⁇ l of 1 ⁇ g/ml purified antibody was added to the first well, and diluted in a 3-fold gradient, for a total of 11 test concentration gradients plus a blank well. It was then incubated at 37°C for 1 hour.
  • Antibody EC50 (ng/ml)
  • Antibody EC50 (ng/ml) 3B4 1.145 5E4 6.144 2F6 1.354 5H7 6.727 5D10 2.198 2F12 7.182 3D5 2.402 3F4 7.723 3C4 2.796 5A10 8.130 2F8 3.029 3E5 8.327 2G4 4.523 5C9 11.873 5F7 4.630 2E12 12.253
  • ELISA Competition ELISA was used to assess epitopes of cell culture supernatants.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-ECD protein at 1 ⁇ g/ml in PBS for 2 hours at 37°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 250 ⁇ l/well of PBST containing 1% BSA for 1 hour at 37°C. The blocking solution was then discarded, and 50 ⁇ l of each cell supernatant premixed with horseradish peroxidase-conjugated goat anti-rabbit IgG (Fc-specific) (GenScript, A01856) secondary antibody was added to each well and each cell 50 ⁇ l of supernatant stock solution.
  • Fc-specific horseradish peroxidase-conjugated goat anti-rabbit IgG
  • the difference of OD value is close to 1.0, and there is obvious competition effect.
  • the difference between the OD values of the supernatant B and the supernatant A-HRP mixture was about 0.9, there was an obvious competitive effect. Therefore, it can be determined that supernatant A and supernatant B recognize the same antigenic determinant.
  • the difference between the OD values of the supernatant C and the supernatant A-HRP mixture was not significantly different from the value of well 0, and there was no competition effect. Therefore, it can be determined and analyzed that supernatant C and supernatant A recognize different antigenic determinants of the antigen.
  • ELISA plates were coated with 100 ⁇ l/well of 2.5 ⁇ g/ml of unlabeled purified antibody (eg 3C4 or 2F8) in PBS overnight at 4°C. Plates were washed with PBST (0.05% Tween) and blocked with 250 ⁇ l/well of PBST containing 1% BSA for 2 hours at 37°C. Subsequently, the blocking solution was discarded, and 100 ⁇ l of recombinant S-ECD protein with a concentration of 20 ng/ml, 2-fold dilution and 0 ng/ml in the first well was added, and incubated at 37° C. for 1 hour.
  • PBST 0.05% Tween
  • the plate was washed 4 times with PBST, and a biotin-labeled antibody (such as 39G6-biotin) was added to the wells of the plate, 100 ⁇ l per well, and incubated at 37° C. for 1 hour. Plates were washed 4 times with PBST and incubated with 100 ⁇ l/well of streptavidin HRP (SA-HRP, GenScript) for 15 minutes at 37°C. Plates were finally washed 4 times with PBST, then TMB developer solution (GenScript) was added and incubated at 25°C for 15 minutes in the dark. The reaction was stopped by adding 50 ⁇ l of 1 M HCl stop solution (GenScript).
  • a biotin-labeled antibody such as 39G6-biotin
  • antibodies 3C4 and 39G6 are relatively good paired antibodies, and their sensitivity can reach 0-40pg/ml, which can be used as the development of ELISA kits for double-antibody sandwich detection of antigens.
  • antibodies 2F8 and 39G6 are also good paired antibodies, and their sensitivity can reach 0-40pg/ml.
  • the antibody 39G6 is derived from the rabbit monoclonal antibody specific for RBD in the PCT patent (PCT/CN2021/095228), and the variable region amino acid sequence of the antibody is shown in SEQ ID NO: 135 and SEQ ID NO: 136.

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Abstract

La présente invention relève du domaine de la détection et du diagnostic de virus, et concerne un anticorps monoclonal dirigé contre un domaine extracellulaire de la protéine de spicule du SARS-CoV-2, et des séquences d'acides aminés d'une région variable de chaîne lourde et d'une région variable de chaîne légère de celui-ci. L'anticorps monoclonal dirigé contre un domaine extracellulaire de la protéine de spicule du SARS-CoV-2 peut se lier de manière spécifique à un domaine extracellulaire de protéine S1 ou S2, peut être utilisé pour détecter un antigène viral du SARS-CoV-2, et offre une possibilité et une commodité pour la détection du virus du SARS-CoV-2.
PCT/CN2021/121803 2020-09-30 2021-09-29 Anticorps monoclonal dirigé contre le domaine extracellulaire de la protéine de spicule du sars-cov-2, et son utilisation WO2022068895A1 (fr)

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