WO2022033435A1 - Anticorps anti-coronavirus et son utilisation - Google Patents

Anticorps anti-coronavirus et son utilisation Download PDF

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Publication number
WO2022033435A1
WO2022033435A1 PCT/CN2021/111550 CN2021111550W WO2022033435A1 WO 2022033435 A1 WO2022033435 A1 WO 2022033435A1 CN 2021111550 W CN2021111550 W CN 2021111550W WO 2022033435 A1 WO2022033435 A1 WO 2022033435A1
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seq
amino acid
acid sequence
variable region
alternatively
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Chinese (zh)
Inventor
郭宇
饶子和
刘礼乐
邬俊
张宏恺
汪皛皛
付丹
聂翠
缴鹏
卢忠华
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Harbour Biomed Suzhou Co Ltd
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Harbour Biomed Suzhou Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • 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
    • C07K2317/70—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
    • 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 field of biomedicine, in particular to an anti-coronavirus antibody and application thereof, wherein the coronavirus is a novel coronavirus, in particular to an antibody that specifically binds to the novel coronavirus antigen S-RBD protein and its antigen binding Fragment.
  • the novel coronavirus infection (the disease caused by the novel coronavirus officially named by the World Health Organization as COVID-19, and the novel coronavirus as SARS-CoV-2) has caused enormous damage to public health and economies around the world. It spread quickly and became a global pandemic with unprecedented severity. It has wreaked havoc on the global economy and health care system, and the effects are likely to continue for a long time.
  • SARS-CoV-2 virus utilizes the self-produced spike protein (S protein) to bind to angiotensin-converting enzyme II (ACE2) on human cell membranes to invade and further infect target cells.
  • S protein self-produced spike protein
  • ACE2 angiotensin-converting enzyme II
  • the S protein of SARS-CoV-2 consists of S1 and S2 subunits, and its domain for binding to the ACE2 protein (also known as the receptor binding domain, RBD) is located in the S1 subunit (SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor, Hoffmann et al., 2020, Cell 181, 1–10).
  • small molecule drugs such as remdesivir and chloroquine have shown some positive results in some treatment cases, but have not proven effective in subsequent clinical trials.
  • the side effects of these small molecule drugs can be severe (Rethinking the role of hydroxychloroquine in the treatment of COVID-19, Meyerowitz et al., FASEB J. 2020 May;34(5):6027-6037;Hydroxychloroquine and chloroquine: a potential and controversial treatment for COVID-19, Zou et al., Arch Pharm Res. 2020 Aug 1; Remdesivir for the Treatment of COVID-19: A Systematic Review of the Literature, Musa et al., West J Emerg Med. 2020 May 20;21(4):737-741).
  • Vaccines are the most common treatment against viral infections, with large numbers of infections as expected. This includes traditional vaccines and novel vaccines, such as mRNA vaccines.
  • the large number of patient blood samples provides sufficient resources for vaccine development, and the estimated vaccine production timeline is relatively fast compared to the development of other new drugs.
  • Currently, dozens of vaccines are currently reported in clinical trials.
  • some recent studies have begun to question the tolerance of vaccine-stimulated neutralizing antibodies to SARS-CoV-2, which could seriously affect the effectiveness of this treatment (QX Long et al., Clinical and immunological assessment of asymptomatic SARS- CoV-2 infections, Nature Medicine 26, 1200–1204 (2020)).
  • antibody drugs which have become a major force in treating major diseases such as cancer.
  • antibody drugs have distinct advantages in specificity, frequency of administration, and toxic side effects.
  • the development of antibody drugs can take a long time.
  • most of the first round of research and development results of anti-SARS-CoV-2 antibodies focus on screening neutralizing antibodies from the blood of recovered patients, or screening neutralizing antibodies from previous coronaviruses (such as the SARS virus in 2003) that are related to SARS.
  • -CoV-2 has better cross-reactive neutralizing antibodies.
  • the second wave of anti-SARS-CoV-2 antibodies mostly uses SARS-CoV-2 S1 protein or SARS-CoV-2 S-RBD protein as a specific antigen for specific immune screening, resulting in better specificity and higher affinity. high antibody.
  • Antibodies generated in this way need to undergo more and longer screening development and are therefore expected to have better properties such as higher binding affinity, higher neutralizing activity, more prominent stability, and better clinical effect, etc.
  • the technical problem to be solved by the present invention is to overcome the lack of effective anti-coronavirus antibodies in the prior art, and to provide an anti-novel coronavirus antibody, especially an antibody that specifically binds to the novel coronavirus antigen S-RBD protein and the same. Antigen-binding fragments.
  • the present invention mainly solves the above technical problems through the following technical means.
  • a first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, Described light chain variable region comprises LCDR1, LCDR2 and LCDR3;
  • the HCDR1 comprises the sequence shown in SEQ ID NO.1 or variant 1 thereof, the sequence shown in SEQ ID NO.2 or variant 2 thereof, the sequence shown in SEQ ID NO.3 or a variant thereof 3.
  • the amino acid sequence in the group consisting of the sequences shown in SEQ ID NO.112, 52, 28 to 31, the HCDR2 comprises an amino acid sequence selected from the group consisting of: The sequence shown in SEQ ID NO.4 or its variant 4, the sequence shown in SEQ ID NO.5 or its variant 5, the sequence shown in SEQ ID NO.6 or its variant 6, the sequence shown in SEQ ID NO.6 or its variant 6
  • the sequence shown in NO.8 or its variant 8 the amino acid sequence in the group consisting of the sequences shown in SEQ ID NO.7, 32-34, 68 and 189
  • the HCDR3 comprises a sequence selected from the group consisting of SEQ ID NO. 10 or its variant 10, the sequence shown in SEQ ID NO.12 or its variant 12, the sequence shown in SEQ ID NO.69, 35, 73, 36, 37-42 and 74 of the group consisting of amino acid sequence;
  • the LCDR1 comprises a sequence selected from the group consisting of the sequence shown in SEQ ID NO.14 or its variant 14, the sequence shown in SEQ ID NO.15 or its variant 15, the sequence shown in SEQ ID NO.16 or its variant Variant 16, the sequence shown in SEQ ID NO. 17 or its variant 17, the sequence shown in SEQ ID NO. 18 or its variant 18, the sequence shown in SEQ ID NO.
  • the LCDR2 comprising amino acids selected from the group consisting of amino acids shown in SEQ ID NO.20 or variants thereof 20, as shown in SEQ ID NO.21
  • the amino acid sequence shown in the group consisting of the amino acid or its variant 21, the amino acid shown in SEQ ID NO.22 or its variant 22, and the sequence shown in SEQ ID NO.44-47, the LCDR3 comprises selected Free as shown in SEQ ID NO.23 or its variant 9, as shown in SEQ ID NO.24 or its variant 11, as shown in SEQ ID NO.25 or its variant 25, as The sequence shown in SEQ ID NO.26 or its variant 26, the sequence shown in SEQ ID NO.27 or its variant 13, SEQ ID NO.48-50, 95, 104 and 101 in the group consisting of amino acid sequence;
  • the variant 1 contains one or more of the mutations F2Y, S5N/T, S6D/Y/E, Y7N/F and G8T
  • the variant 2 contains the mutations T6S and/or T10N
  • the variant 3 contains One or more of the mutations E6R, T8D, M9I and H10Y/L/N
  • the variant 7 contains the mutation G6S;
  • the variant 4 contains one or more of the mutations I2L, N6Q, G7D, T9N, S10T/G and F15L, the variant 5 contains the mutations T9S and/or K16M, and the variant 6 contains the mutations V1I, N8Y , one or more of K9R/T/Q, F10Y and Q16K, the variant 8 contains mutations V7A and/or K16Q;
  • the variant 10 contains one or more of the mutations A1G, D2G, G3T and E5D and the variant 12 contains one or more of the mutations D1E, Y7F and M9L;
  • the variant 14 contains the mutation G10S
  • the variant 15 contains one or more of the mutations S3T, E4K, D7S, S8T, Y9S, N11Y, F13Y and H15F
  • the variant 16 contains the mutations T3S and/or H10Y
  • the variant 17 contains the mutation G7D
  • the variant 18 contains one or more of the mutations I6V, W9Y/D and A11N
  • the variant 19 contains one or more of the mutations H8Q, N10D and F14Y;
  • the variant 20 contains the mutation Y4H/S
  • the variant 21 contains one or more of the mutations K1A, A2T and K6E/Q
  • the variant 22 contains one or more of the mutations S1R/L, T2A and A6E or Multiple
  • the variant 9 contains the mutation N5T
  • the variant 11 contains the mutation G3A
  • the variant 25 contains the mutation T5S and/or T7P
  • the variant 26 contains the mutation S7W
  • the variant 13 contains the Q1S , one or more of N4T, E5H, and G6V/D.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO.3 or its variant 3
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO. .4 or variant 4 thereof
  • the HCDR3 is shown in SEQ ID NO. 12 or variant 12 thereof;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.1 or its variant 1
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.6 or its variant 6
  • amino acid of the HCDR3 The sequence is shown in SEQ ID NO. 38, 40, 42 or 74.
  • the sequence of the variant 1 is shown in SEQ ID NO.51, 54, 55 or 108, and the sequence of the variant 2 is shown in SEQ ID NO.11 or 81
  • the sequence of the variant 3 is shown in SEQ ID NO.109, 110, 111 or 13
  • the sequence of the variant 7 is shown in SEQ ID NO.53
  • the sequence of the variant 4 is shown in SEQ ID NO. 56 to 59 and any one of SEQ ID NO.187
  • the sequence of the variant 5 is as shown in SEQ ID NO.60
  • the sequence of the variant 6 is as shown in any of SEQ ID NO.106 or 61 to 67.
  • the sequence of the variant 8 is shown in SEQ ID NO.107
  • the sequence of the variant 10 is shown in SEQ ID NO.70 or SEQ ID NO.186
  • the sequence of the variant 12 is shown in SEQ ID NO. 71, 72 or 188.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO.51
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO.56
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO.69;
  • amino acid sequence of HCDR1 is shown in SEQ ID NO.11
  • amino acid sequence of HCDR2 is shown in SEQ ID NO.60
  • amino acid sequence of HCDR3 is shown in SEQ ID NO.10;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.112
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.32
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.35;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.9
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.107
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.73;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.2
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.5
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.70;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.52
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.33
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.36;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.109
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.4
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.71;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.110
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.57
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.12;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.111
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.4
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.12;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.3
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.58
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.72;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.81
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.5
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.10;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.53
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.8
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.73;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.3
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.59
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.69;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.28
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.68
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.37;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.54
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.106
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.38;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.29
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.34
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.39;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.1
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.6
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.40;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.55
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.61
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.74;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.30
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.62
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.74;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.13
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.7
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.41;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.55
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.63
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.74;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.31
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.64
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.74;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.108
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.65
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.74;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.1
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.66
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.74;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.1
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.67
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.42;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.81
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.5
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.186;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.109
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.187
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.188;
  • amino acid sequence of the HCDR1 is shown in SEQ ID NO.9
  • amino acid sequence of the HCDR2 is shown in SEQ ID NO.189
  • amino acid sequence of the HCDR3 is shown in SEQ ID NO.73;
  • the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO. 113-137 or a variant thereof, and the variant has at least 90%, at least 95% or at least 99% of the sequence before the mutation. % sequence identity, while retaining at least the function of the pre-mutation sequence; the sequence of the variant is preferably shown in any one of SEQ ID NO. 164-166 and SEQ ID NO. 171-179.
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO.15 or its variant 15
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO.22 or its variant 22
  • the LCDR3 is shown in SEQ ID NO.27 or Its variant 13 is shown;
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO. 17 or its variant 17
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO. 20 or its variant 20
  • the LCDR3 is shown in SEQ ID NO. 23 or its variant 9;
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO. 18 or its variant
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO. 21 or its variant
  • the LCDR3 is shown in SEQ ID NO. 26 or its variant 26;
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO.19 or its variant 19
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO.47
  • the LCDR3 is shown in SEQ ID NO.24 or a variant thereof 11 shown.
  • sequence of the variant 14 is preferably shown in SEQ ID NO. 75
  • sequence of the variant 15 is preferably shown in SEQ ID NO. 76 or 77
  • sequence of the variant 16 is preferably shown in SEQ ID NO. ID NO.78 or 79
  • sequence of the variant 17 is preferably as shown in SEQ ID NO.80
  • sequence of the variant 18 is preferably as shown in SEQ ID NO.82 or 83
  • the variant 19 The sequence of the variant 20 is preferably shown in any one of SEQ ID NO.84-86
  • sequence of the variant 20 is preferably shown in SEQ ID NO.89 or 90
  • sequence of the variant 21 is preferably shown in SEQ ID NO.93 Or shown in 94
  • sequence of the variant 22 is preferably shown in SEQ ID NO.
  • the sequence of the variant 9 is preferably shown in SEQ ID NO. 96
  • the sequence of the variant 11 is preferably shown It is preferably shown in SEQ ID NO.103
  • the sequence of the variant 25 is preferably shown in SEQ ID NO.100
  • the sequence of the variant 26 is preferably shown in SEQ ID NO.105
  • the sequence of the variant 13 is preferably shown in SEQ ID NO.105.
  • the sequence is preferably shown in SEQ ID NO.98 or 99, preferably:
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO.14
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO.44
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO.95;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.15
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.87
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.98;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.77
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.88
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.48;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.78
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.22
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.25;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.76
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.87
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.27;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.43
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.45
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.99;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.80
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.89
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.96;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.17
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.20
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.23;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.80
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.20
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.96;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.17
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.90
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.23;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.16
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.22
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.100;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.75
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.44
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.101;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.79
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.92
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.49;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.82
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.93
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.50;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.83
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.46
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.102;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.18
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.94
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.26;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.84
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.47
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.24;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.85
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.47
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.103;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.18
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.21
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.105;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.86
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.47
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.103;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.19
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.47
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.103;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.84
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.47
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.104;
  • amino acid sequence of LCDR1 is shown in SEQ ID NO.80
  • amino acid sequence of LCDR2 is shown in SEQ ID NO.90
  • amino acid sequence of LCDR3 is shown in SEQ ID NO.96;
  • the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.
  • the sequence has at least 90%, at least 95% or at least 99% sequence identity, while at least retaining the function of the sequence before the mutation; the sequence of the variant is preferably such as SEQ ID NO. 158-163, SEQ ID NO. 167-170 and any one of SEQ ID NO. 180-183.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.113, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.138;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.114, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.139;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.115
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.140;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.116
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.141;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.117
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.142;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.118, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.143;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.119
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.144;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.120, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.145;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.121
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.146;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.122, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.147;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.123, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.139;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.124, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.148;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.125
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.149;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.126
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.150;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.127
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.151;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.128, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.152;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.129, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.153;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.130, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.154;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.131, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.155;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.132
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.156;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.133
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.154;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.134
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.157;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.135, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.155;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.136
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.91;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.137
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.97;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.174
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.150;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.173, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.169;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.173, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.168;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.173, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.167;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.172
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.169;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.172
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.168;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.172
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.167;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.171, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.169;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.171, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.168;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.171, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.167;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.179
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.183;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.178
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.182;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.178
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.181;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.178
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.180;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.177
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.182;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.177
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.181;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.177
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.180;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.176
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.182;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.176
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.181;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.176
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.180;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.175, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.182;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.175, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.181;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.175, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.180.
  • the numbers in the table refer to SEQ ID NO.
  • the present invention uses the Chothia numbering rule, and the CDR region division refers to the Chothia and KABAT mixing rule.
  • the antibody or antigen-binding fragment thereof as described above which can be a full-length antibody, Fab, Fab', F(ab') 2 , Fv, bispecific antibody, multispecific antibody, heavy chain antibody, single domain antibody or In the form of single domain antibodies, either monoclonal or polyclonal antibodies prepared from the above antibodies.
  • its light chain constant region can comprise human ⁇ , ⁇ chain or its variants
  • the amino acid sequence of the light chain constant region used in a preferred embodiment of the present invention is as SEQ ID NO. 184; its heavy chain constant region may comprise human IgG1, 2, 3, 4 or variants thereof.
  • the amino acid sequence of the heavy chain constant region used in a preferred embodiment of the present invention is shown in SEQ ID NO. 185 in the sequence listing.
  • antibodies or antigen-binding fragments thereof of the present invention can specifically bind to, but are not limited to, the novel coronavirus antigen S-RBD protein.
  • a second aspect of the present invention provides an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to the first aspect of the present invention.
  • a third aspect of the present invention provides an expression vector comprising the isolated nucleic acid according to the second aspect of the present invention.
  • the fourth aspect of the present invention provides a host cell comprising the expression vector according to the third aspect of the present invention; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.
  • the fifth aspect of the present invention provides a method for preparing an anti-novel coronavirus antigen S-RBD protein or an antigen-binding fragment thereof, which comprises culturing the host cell according to the fourth aspect of the present invention.
  • a sixth aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.
  • the seventh aspect of the present invention provides the use of the antibody or its antigen-binding fragment according to the first aspect of the present invention in the preparation of a medicament for treating novel coronavirus-related diseases.
  • An eighth aspect of the present invention provides a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof as described above.
  • a ninth aspect of the present invention provides an antibody drug conjugate comprising a cytotoxic agent, and the antibody or antigen-binding fragment thereof as described above.
  • a tenth aspect of the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described above, a pharmaceutical composition as described above, a chimeric antigen receptor as described above and/or as described above Antibody Drug Conjugates.
  • An eleventh aspect of the present invention provides a set of medicine kits, which comprises a medicine box A and a medicine box B, wherein:
  • the kit A contains the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned pharmaceutical composition, the above-mentioned chimeric antigen receptor and/or the above-mentioned antibody-drug conjugate;
  • the kit B contains other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies, and/or is composed of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, One or more of the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • An eleventh aspect of the present invention provides a method of diagnosing, treating and/or preventing a coronavirus-mediated disease or disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen thereof as described above A binding fragment, a chimeric antigen receptor as described above, an antibody drug conjugate as described above, or a pharmaceutical composition as described above, or a kit as described above is used to treat a patient in need thereof.
  • the twelfth aspect of the present invention provides a method for immunodetection or determination of coronavirus, comprising using the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned chimeric antigen receptor, the above-mentioned antibody drug conjugate A conjugate or a pharmaceutical composition as described above; preferably, the detection is for non-diagnostic and/or therapeutic purposes.
  • a thirteenth aspect of the present invention provides a combination therapy comprising administering to a patient in need thereof the antibody or antigen-binding fragment thereof as described above, the chimeric antigen receptor as described above, and the antibody drug conjugate as described above, respectively.
  • the combination or the pharmaceutical composition as described above, and a second therapeutic agent; the second therapeutic agent preferably comprises other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies, and/or is composed of hormone preparations , targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines. one or more of the group.
  • a fourteenth aspect of the present invention provides a drug delivery device, characterized in that the drug delivery device comprises the above-described antibody or antigen-binding fragment thereof, the above-described chimeric antigen receptor, and the above-described antibody A drug conjugate or a pharmaceutical composition as described above;
  • the drug delivery device further comprises means for administering the antibody or antigen-binding fragment thereof, the chimeric antigen receptor, the antibody drug conjugate or the pharmaceutical composition to a subject, eg Syringe or infusion set.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the antibody or antigen-binding fragment thereof of the present invention has higher binding ability to the new coronavirus, and has better neutralizing activity (the IC50 values of neutralizing activity are all lower than ⁇ g/mL level), and better coverage of mutant virus , diverse binding epitopes.
  • the antibody or antigen-binding fragment thereof of the present invention has better blocking effect on the new crown S protein and human ACE2 protein, as well as better stability and druggability.
  • Figure 1 shows the neutralizing activity of humanized antibodies.
  • Figure 2 shows the neutralizing activity of murine antibodies.
  • Figure 3 shows an alignment of the variants of PR300902 VH and VL with the original sequence.
  • Figure 4 shows an alignment of the variants of PR300928 VH and VL to the original sequence.
  • Figure 5 shows an alignment of the variants of PR300961 VH and VL to the original sequence.
  • Figure 6 shows PR301077 mice virus prevention and treatment experiments.
  • the term “antibody” generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone portion that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antibody to the antigen.
  • An antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both may typically be included.
  • VL variable region
  • VH antibody heavy chain variable region
  • the "heavy chain antibody” in this application does not contain a VL region, but only contains a VH region.
  • the VH or VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH or VL can consist of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • antibodies include, but are not limited to, full-length antibodies, heavy chain antibodies (HCAb), antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and/or or dAb), immunoconjugates, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • HCAb heavy chain antibodies
  • Fab antigen-binding fragments
  • Fab' antigen-binding fragments
  • F(ab)2 fragment fragments
  • F(ab')2 fragments
  • scFv di-scFv and/or or dAb
  • immunoconjugates eg, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • variable generally refers to the fact that some portion of the sequence of the variable domains of an antibody varies strongly which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the variable domains of the light and heavy chains, called the CDRs or hypervariable regions (HVRs), with the FRs being the more highly conserved parts of the variable domains.
  • the variable domains of native heavy and light chains each comprise four FR regions, mostly in a ⁇ -sheet configuration, connected by three CDRs, forming loops connecting, and in some cases forming part of, a ⁇ -sheet structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions, and together with the CDRs from the other chain form the antigen-binding site of the antibody, the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions , eg involved in antibody-dependent cytotoxicity of antibodies.
  • nucleic acid refers to DNA molecules and RNA molecules. It may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • the term “specifically binds” generally refers to the binding of an antibody to an epitope through its antigen binding domain, and that binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an antigen when it is more likely to bind to an epitope through its antigen-binding domain than to a random, unrelated epitope than to a random, unrelated epitope.
  • Fab generally refers to the antigen-binding portion of a conventional antibody (eg, IgG), including the heavy chain variable region VH, light chain variable region VL and heavy chain constant region domain CH1 and light chain variable region of the antibody.
  • Chain constant region CL In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is further linked to the hinge region of the heavy chain and other constant The domains are linked to form the heavy chain.
  • Fab also refers to variant structures of Fab.
  • the C-terminus of VH is linked to the N-terminus of CL to form a polypeptide chain
  • the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming a Fab (cross VH/VL) structure
  • the CH1 of the Fab is not linked to the hinge region, but the C-terminus of the CL is linked to the hinge region of the heavy chain to form a Fab (cross Fd/LC) structure.
  • VH generally refers to the heavy chain variable region VH domain of an antibody, that is, it can be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of humans or other animals, or it can be Camelidae, etc.
  • the heavy chain variable region VHH of an animal heavy chain antibody (HCAb structure) can also be the heavy chain variable region VH of a fully human heavy chain antibody (HCAb structure) produced by using Harbour HCAb transgenic mice.
  • the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997).
  • the present application may also determine amino acid residues in variable domain sequences and full-length antibody sequences using the Combined definition rule comprising the Kabat definition and the Chothia definition. In the present invention, each sequence is determined according to the Chothia definition.
  • the various anti-SARS-CoV-2 antibodies involved in the present invention are related to R&D personnel using SARS-CoV-2-S-RBD as a specific antigen to immunize wild-type or humanized mice (H2L2 https://harbourantibodies.com /science-technology/h2l2/#.Xxj_oZ4zbIU), and further through the Beacon platform (Ref. https://www.berkeleylights.com/systems/beacon/, Rapid single B cell antibody discovery using nanoopens and structured light, mAbs, https ://doi.org/10.1080/19420862.2019.1624126) for single B cell screening. They have high affinity for S-RBD and excellent neutralizing effect against SARS-CoV-2 virus. These antibodies have the potential to be developed into effective drugs and therapies that could provide an effective treatment for COVID-19.
  • selected murine neutralizing antibodies are humanized, and several selected neutralizing antibodies are subjected to post-translational modification sequence optimization.
  • the BLI (or ELISA) method was used to investigate the change of their affinity for the new crown S protein
  • the competition ELISA method was used to investigate the mutual blocking of the new crown S protein and human ACE2 protein. Activity changes.
  • using 293 transient expression, protein purity (SEC and SDS-PAGE), and Uncle (Unchained Labs) to detect Tm, Tagg, molecular aggregation and other indicators, to investigate and compare its druggability. The results showed that these candidate proteins showed good druggability, and their humanization and post-translational modification improvements were successful.
  • Example 1 Preliminary acquisition of candidate antibodies that can efficiently bind to the new crown RBD protein and the new crown S protein
  • RBD domain receptor binding domain
  • SARS-CoV2-2019 S protein of the 2019 novel coronavirus
  • BALB/c wild-type
  • humanized H2L2, 10 in total
  • mice Using the RBD domain (receptor binding domain) in the S protein of the 2019 novel coronavirus (SARS-CoV2-2019) to immunize wild-type (BALB/c, 5 in total) or humanized (H2L2, 10 in total) Mice, and the spleen cells and bone marrow cells of the mice whose plasma was positive for the antigen were selected and enriched with Miltenyi (Miltenyi Biotec, #130-092-530) and Stem Cell's kit (Stemcell, #18957) Antibody-expressing plasma cells (plasma B cells).
  • Miltenyi Miltenyi Biotec, #130-092-530
  • Stem Cell's kit Stem Cell's kit
  • Beacon (Berkeley Lights, Optofluidic System) preliminarily investigated the binding of single B cell culture supernatant to the new crown RBD protein, and derived the positive cells that the culture supernatant can effectively bind to the new crown RBD, and then amplified the antibody variable region in a single B cell by molecular cloning sequence and sequenced.
  • the antibody with completely identical CDR region sequences was regarded as an independent sequence, and a total of 191 independent mouse antibody heavy and light chain pairs were obtained (an independent mouse antibody pair included a VH, a VL two sequences), and 105 independent human antibody heavy and light chain pairs.
  • the obtained independent sequences were obtained by gene synthesis and molecular cloning to obtain mammalian expression vectors of the corresponding antibodies, which were transfected into HEK293T cells using PEI (SIGMA, #24885) and cultured in 24-well plates for 3 days. Culture conditions: 37°C, 5% carbon dioxide. Take 0.1 mL of the culture supernatant to detect its binding activity to the new crown S protein (hereinafter referred to as S protein) by ELISA.
  • S protein new crown S protein
  • S protein SARS-CoV-2 (2019-nCoV) Spike Protein (S1 Subunit, His Tag), (Sino Biological, #40591-V08H) coated immune plate overnight at 4°C, after blocking, added 100 ⁇ L supernatant, room temperature After 1 hour of reaction, secondary antibody anti-Human IgG(Fc) HRP (Jackson Immuno Research Labs, #109-035-098) was added to react for 1 hour.
  • Use TMB color development kit (Chaozhou Yingchuang Biotechnology Co., Ltd., TMB-S-003) to develop color, read its OD405 on SpectraMax PLUS 384 microplate reader (Molecular Devices) after the reaction is terminated. OD405>0.25 was marked as positive.
  • the screened candidate antibody strains will be transiently cultured in the HEK293 suspension cell system, and purified antibody proteins will be obtained for further investigation.
  • the mammalian expression vector of the positive antibody strain obtained by screening in Example 1 was transfected into HEK293F suspension cells (Gibco, #R79007) using PEI (SIGMA, #24885), and cultured in the culture conditions for 7 days. Culture conditions: 37°C, 5% carbon dioxide, 125rpm. After the culture supernatant was harvested, it was purified by affinity chromatography with a protein A (AmMag Protein A Magnetic Beads, Genscript, L00695) purification filler. The purity of the obtained protein was preliminarily investigated by SDS-PAGE (SurePAGE, Bis-Tris, 10 ⁇ 8, 4-12%, 12 wells, Genscript, M00653).
  • the expression of all candidate proteins in the HEK293 transient expression system was between about 10 mg/L and 100 mg/L. And the SDS-PAGE results of these candidate antibodies all showed that the purity of the obtained antibodies was >90%.
  • the positive candidate antibody obtained in the previous step was tested for its binding activity to the new crown S protein by ELISA.
  • S protein SARS-CoV-2 2019,-nCoV
  • S1 Subunit, His Tag (Sino Biological, #40591-V08H) and 2019-nCoV Spike Protein (S1 Subunit, His Tag) (Genscript, Batch No.: P9FE001)
  • coat the immunoplate overnight at 4°C after blocking, add the candidate antibody of gradient dilution (the concentration selected by gradient dilution, such as 15 ⁇ g/mL initial, 10-fold gradient dilution, to 0.000015 ⁇ g/mL, a total of 7 concentrations ), react at room temperature for 1 hour, and then add secondary antibody anti-Human IgG(Fc)HRP (Jackson Immuno Research Labs, #109-035-098) to react for 1 hour.
  • the candidate antibody of gradient dilution the concentration selected by gradient dilution, such as 15 ⁇ g/mL initial, 10-fold gradient dilution, to 0.000015 ⁇ g/mL, a total of 7 concentrations
  • TMB color development kit Chozhou Yingchuang Biotechnology Co., Ltd., TMB-S-003
  • TMB-S-003 Use TMB color development kit (Chaozhou Yingchuang Biotechnology Co., Ltd., TMB-S-003) for color development, and read its OD405 on SpectraMax PLUS 384 microplate reader (Molecular Devices) after the reaction is terminated.
  • the OD405 of each antibody was plotted against the logarithmic value of its concentration with Graphpad, and the EC50 value of the antibody binding to the new coronavirus S protein was obtained by four-parameter fitting.
  • the EC50 values of the above antibodies and the new crown S protein ELISA detection basically reached the level of ⁇ 10pM or even lower. This result is superior to existing antibodies reported in the literature (D.Wrapp et al., Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies, 2020, Cell 181, 1–12; Z.Lv et al ., Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody, Science 10.1126/science.abc5881(2020)), indicating that the candidate antibody of the present invention has stronger binding to the new coronavirus S protein ability.
  • VH and VL sequences of the above antibodies were numbered using the Chothia rules, and the CDR regions were determined comprehensively considering the rules of Chothia and KABAT. For details, see Tables 1-2 and 1-3.
  • Example 2 The positive candidate antibodies obtained in Example 2 were tested by ELISA for their blocking activity on the interaction of the new coronavirus S protein-human ACE2 protein
  • ACE2 protein Human ACE2 protein, His tag (Acro, #AC2-H52H8) and Human ACE2 protein, hFc tag (Genscript, #T80801 (no catalog product), Lot: T2006002) were coated on the immunoplate overnight at 4°C and blocked. Then add the candidate antibody to be detected (concentration selected by gradient dilution, such as 15 ⁇ g/mL starting, 10-fold gradient dilution, to 0.000015 ⁇ g/mL, a total of 7 concentrations), and human RBD protein (mFc-tag) (Sino Biological, #40592-V05H), react at 37°C for 1 hour.
  • secondary antibody anti-Mouse IgG(Fc)HRP (Sigma, #A0168) was added to react for 1 time.
  • Use TMB color development kit Chozhou Yingchuang Biotechnology Co., Ltd., #TMB-S-003 for color development, and read its OD405 on SpectraMax PLUS 384 microplate reader (Molecular Devices) after the reaction is terminated.
  • the OD405 of each antibody was plotted against the logarithmic value of its concentration with Graphpad, and four-parameter fitting was used to obtain the IC50 value of the antibody blocking SARS-CoV-2 S protein and human ACE2 protein.
  • IC50 values of some positive antibodies are shown in Table 2 below. For incomplete blocking antibodies, IC50 values are not presented, only the inhibition rate at the highest concentration is written, and antibodies that do not block completely are denoted by "NA".
  • the candidate antibodies with a blocking effect have a low IC50 value of blocking the interaction between the new crown S protein and human ACE2 protein, basically reaching the level of nM or sub-nM, and some of them have reached the level of nM or sub-nM. ⁇ 10pM level.
  • the positive candidate antibody obtained in Example 2 was tested for the binding kinetics of the new crown S protein and the new crown RBD protein by the method of Biolayer Interferometry (BLI). Immobilized on the AHC biosensor, and for kinetic measurement, the antigenic protein (new crown S protein, or new crown RBD protein, see above for relevant information) was diluted with 10X Kinetics buffer to 3 of 800nM, 400nM, 200nM, 100nM, 50nM, and 25nM, respectively. Each concentration (depending on the strength of the binding signal) was injected for 100 s, the dissociation time was 400-800 s, and the regeneration was performed with 10 mM glycine HCl (pH 1.5) for 15 s.
  • BBI Biolayer Interferometry
  • Example 6 The neutralizing activity of candidate positive antibodies was investigated by in vitro virus infection
  • a mixture of SARS-CoV-2 virus (100 TCID 50 per well) and diluted antibody was incubated at 37°C for 1 hour, then seeded into VeroE6 cells pre-passaged into 96-well plates and incubated at 37°C for an additional 48 hours.
  • the IC50 value information of each molecule is shown in Table 3-2 below (the IC50 unit in the table is ⁇ g/mL).
  • Antibody name IC50 Antibody name IC50 Antibody name IC50 Antibody name IC50 PR301052 0.6482 PR301078 0.5416 PR300902 1.093 PR301056 0.4059 PR301100 ⁇ 0.6077 PR300911 0.8193 PR301077 0.02791 PR301103 0.04720 PR300914 ⁇ 0.1048 PR301086 0.4353 PR301261 1.302 PR300928 0.04681 PR301099 ⁇ 0.05034 PR300872 0.6417 PR300992 0.1798 PR301105 0.4044 PR300874 0.1155 PR300961 0.1723 PR301042 1.752 PR300886 0.1488 PR300953 0.01659 PR300929 0.03002 PR300950 0.06778 PR300985 0.1598 PR300965 1.489
  • Example 7 Neutralizing antibodies examine their binding epitope differences between each other by competitive ELISA
  • Example 6 The candidate antibodies with neutralizing activity confirmed in Example 6 were examined for their binding epitope differences by competitive ELISA
  • Method description At a concentration of 1 ⁇ g/mL of the antibody to be detected, the immunoplate was coated overnight at 4°C, and after blocking, a competing antibody mixed with the new crown S protein (GenScript, batch number P9FE001, for Biotin labeling) was added. The concentration of competing antibody was 10 ⁇ g/mL or 25 ⁇ g/mL. The control group without the addition of the competing antibody was reacted at 37°C for 1 hour. Then Streptavidin HRP (sigma, #S2438) was added to react for 1 time.
  • a competing antibody mixed with the new crown S protein GeneScript, batch number P9FE001, for Biotin labeling
  • TMB color development kit Chozhou Yingchuang Biotechnology Co., Ltd., #TMB-S-003 to develop color, and read its OD405 on SpectraMax PLUS 384 microplate reader (Molecular Devices) after the reaction is terminated. Using this formula, the percentage blocking of the two antibodies (OD405 of the control group - OD405 of the detection group)/OD405 of the control group x 100% was obtained.
  • candidate antibodies were grouped by epitope according to the mutual blocking of these antibodies. Among them, the percentage of ELISA blocking between pairs of antibodies in the same epitope group is >40%.
  • These candidate antibodies can be divided into several epitope groups as follows. Where there is no pairwise competition between groups 1, 2, and 3 (ie, the percentage of blocking between the antibodies in epitope group 1 and the antibodies in epitope groups 2 and 3 is less than 20%), group 1.5 There is partial competition with Groups 1 and 2, and Group 2.5 with partial competition with Groups 2 and 3. Group 4 competes completely with Group 1, partially competes with Group 2, 2.5, 1.5, and basically does not compete with Group 3. See Table 4 for details.
  • the antigenic epitope grouping results obtained by competitive ELISA show the diversity of the binding epitopes of the novel coronavirus candidate antibodies obtained by the present invention.
  • Example 6 The candidate antibody with neutralizing activity confirmed in Example 6 was examined for its purity by HPLC-SEC
  • Example 9 The neutralizing activity of candidate neutralizing antibodies in vitro was investigated by pseudovirus method
  • Example 6 According to physicochemical properties, antigenic epitope groups, and other activity results, some candidate antibodies with neutralizing activity confirmed in Example 6 were selected, and their neutralizing activity was investigated by in vitro pseudovirus infection.
  • the pseudovirus neutralization experiment used the new coronavirus pseudovirus constructed by the conventional murine leukemia virus (MLV) system.
  • MLV new coronavirus pseudovirus The preparation method of MLV new coronavirus pseudovirus is in the relevant literature (Millet, JK & Whittaker, GRMurine Leukemia Virus(MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection.Bio Protoc 6, https://doi.org/10.21769/BioProtoc.2035 10.21769/BioProtoc.2035.(2016) and Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody, Nature, 583, 290–295 (2020)) are described in detail.
  • Antibodies S309 and REGN10987 in Table 6 are respectively derived from the prior art document "Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody Nature.2020 Jul; 583(7815):290-295.doi:10.1038/ s41586-020-2349-y.
  • Example 6 According to physicochemical properties, antigenic epitope groups and other activity results, some candidate antibodies with neutralizing activity confirmed in Example 6 were selected and optimized according to their amino acid sequence properties.
  • the obtained optimized antibody variable region sequences are shown in Table 7 below.
  • the optimized antibody was obtained, the antibody protein was obtained by the method of Example 2, and its drugability was preliminarily investigated and compared with the original sequence.
  • Antibody name parent antibody variable heavy chain variable region light chain EC50 binds S1,nM PR300961 SEQ ID NO. 174 SEQ ID NO. 170 0.005 PR301441 PR300961 SEQ ID NO. 173 SEQ ID NO. 169 0.0154 PR301555 PR300961 SEQ ID NO. 173 SEQ ID NO. 168 0.02315/0.01639 PR301556 PR300961 SEQ ID NO. 173 SEQ ID NO. 167 0.02719 PR301557 PR300961 SEQ ID NO. 172 SEQ ID NO. 169 0.04382 PR301442 PR300961 SEQ ID NO. 172 SEQ ID NO. 168 0.02938 PR301558 PR300961 SEQ ID NO.
  • Example 6 According to physicochemical properties, antigenic epitope groups, and other activity results, some candidate antibodies with neutralizing activity confirmed in Example 6 were selected, and their binding activities to the new coronavirus RBD proteins with different mutations were detected by ELISA. At the same time, using a pseudovirus with a mutated S protein, the degree of coverage of the mutant by the candidate antibody was further verified using the pseudovirus neutralization experiment as described in the above example.
  • the mutants examined include: F342L, N354D, N354D+D364Y, V367F, R408I, A435S, W436R, K458R, G476S, V483A, D364Y, V341I, D364Y, (Emergence of RBD mutations in circulating SARS-CoV-2 strains enhancing the structural stability and human ACE2 receptor affinity of the spike protein, https://doi.org/10.1101/2020.03.15.991844); Q493N (Structural basis of receptor recognition by SARS-CoV-2.Nature.2020 May;581(7807):221 -224.); K444Q, V445A, Y453F, L455F, F486V, Q493K (Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies.
  • the above mutant RBD protein was coated overnight at 4°C on the immune plate, and after blocking, the candidate neutralizing antibody (the concentration selected by gradient dilution, such as 15 ⁇ g/mL starting, 10-fold gradient dilution, to 0.000015 ⁇ g/mL) was added. mL, a total of 7 concentrations), react at room temperature for 1 hour, and then add secondary antibody anti-Human IgG(Fc)HRP (Jackson Immuno Research Labs, Cat. No. 109-035-098) to react for 1 hour.
  • the candidate neutralizing antibody the concentration selected by gradient dilution, such as 15 ⁇ g/mL starting, 10-fold gradient dilution, to 0.000015 ⁇ g/mL
  • TMB color development kit Chozhou Yingchuang Biotechnology Co., Ltd., TMB-S-003
  • TMB-S-003 Use TMB color development kit (Chaozhou Yingchuang Biotechnology Co., Ltd., TMB-S-003) for color development, and read its OD405 on SpectraMax PLUS microplate reader (Molecular Devices, model) after the reaction is terminated.
  • the OD405 of each antibody was plotted against the logarithmic value of its concentration using Graphpad, and by four-parameter fitting, the EC50 value (nM) of the antibody binding to the mutant RBD protein was obtained. According to the EC50 value, its binding situation was investigated, and some results are shown in Table 9-1 to Table 9-4 below. It can be seen that the candidate antibodies of the present invention can cover most of the mutants.
  • the above antibodies bind to the novel coronavirus RBD protein mutants reported in the literature, and the binding ability is at the same level as that of the wild-type novel coronavirus RBD protein. This result shows that the candidate antibody of the present invention has a better coverage of mutant virus, indicating that it will have better coverage and curative effect on mutant virus strains during clinical use.
  • inhibition rate [1-(mean luminescence intensity of sample group-mean value of blank control)/(mean luminescence intensity of negative group-mean value of blank control)]*100%.
  • IC50 was calculated by Reed-Muench method according to the results of neutralization inhibition rate. Its IC50 ( ⁇ g/ml) is shown in Table 10 below.
  • Example 6 According to physicochemical properties, antigenic epitope groups, and other activity results, some candidate antibodies with neutralizing activity confirmed in Example 6 were selected, and Uncle was used to preliminarily investigate the druggability.
  • Example 13 In vivo efficacy of candidate neutralizing antibodies was investigated by mouse model
  • the candidate antibody PR301077 with neutralizing activity confirmed in Example 6 was selected, and its neutralizing effect in vivo was investigated using a mouse model.
  • mice aged about 8 weeks were selected, firstly injected with neutralizing antibody against IFNaR1 to block the activity of IFNaR1, and then injected with Adv-hACE2 to make their in vivo cells express human ACE2 protein.
  • mice were infected with the SARS-CoV-2 virus.
  • mice were injected with a certain dose (5 mg/kg, 20 mg/kg two dose groups) of the antibody to be investigated, and the negative control group used an equal volume of PBS solution instead of the antibody. Changes in body weight of mice were observed over the following 4 days. The mice were sacrificed on the 4th to 5th day after infection, and the virus load in the lungs of the mice, the secretion of various cytokines in the mice, and the histopathological staining results of the lungs were investigated.
  • candidate antibodies PR301077, PR300953 and PR300961 with neutralizing activity confirmed in Example 6 were selected for subsequent crystal structure analysis.
  • the light and heavy chain variable regions of the candidate antibodies are linked by (GGGGS)3 polypeptide linkers to form single chain variable region fragments.
  • the cDNA of this fragment was synthesized using the pAcGP67 vector (BD BaculoGold TM , 554756) containing BamHI and NotI restriction sites and expressed in Sf9 cells (Gibco TM , 11496015) after transformation with DH10Bac competent cells (Thermo Fisher, 10361012).
  • the expressed virus was de-transfected into Hi5 cells (Thermo Fisher, B85502), and the single-chain variable region fragment of the antibody was purified from the culture supernatant.
  • the SARS-CoV-2 RBD protein and purified single-chain variable region fragments were mixed at a molar ratio of 1.2:1, incubated for two hours, and then purified using a Superdex 75 16/60 size exclusion chromatography column (GE Healthcare).
  • the SARS-CoV-2 RBD/single-chain variable region fragment complexes at 4 mg/mL and 8 mg/mL were screened for crystallization at 16°C by vapor-diffusion sedimentation.
  • composition of crystallization mother liquor is 1%w/v tryptone, 0.001M sodium azide, 0.05M HEPES sodium salt (pH7.0), 12%w/v polyethylene glycol 3,350, or containing 0.8M sodium potassium tartrate tetrahydrate , 0.1M Tris pH 8.5, 0.5% w/v polyethylene glycol monomethyl ether 5,000, or 0.2M sodium chloride, 0.1M Tris base pH 8.5, 29% w/v polyethylene glycol 3,350. Columnar after 7 days Crystals start to appear. Crystals were cryopreserved with dry nitrogen vapor using 4M sodium formate as a protective agent.
  • Crystal diffraction data were collected at Beamline PX06SA of the Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland (wavelength, ) (PR301077), and Shanghai Synchrotron Radiation Facility (SSRF) BL17U1 (wavelength, ) (PR300953 and PR300961) completed.
  • Data processing uses MOSSLM or HKL3000 software package.
  • Structural elucidation was done in PHASER software using the molecular replacement method and SARS-CoV-2 RBD structural data (PDB ID: 6M0J).
  • the original model was built in COOT software and further optimized in PHENIX software. The geometry of the model was verified with the MolProbity program. Then use PyMOL to draw the structure diagram. Antibody epitopes, complementary sites and their interactions were confirmed by the European Bioinformatics Institute's PISA (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html).
  • the structures of antibody Fab and antigen Spike-RBD complexes were obtained by X-ray crystallography, respectively and Resolution of resolution results.
  • the structural results showed that all three mAbs recognized a novel epitope of the SARS-CoV-2 spike protein RBD (Spike-RBD).
  • Spike-RBD novel epitope of the SARS-CoV-2 spike protein RBD
  • RBM receptor body binding motif
  • PR300961 is adjacent to the receptor binding motif (RBM), making PR300961 a non-blocking neutralizing antibody against SARS-CoV-2.
  • the PR301077 binding epitope is directly located in the receptor binding motif (RBM) region of the SARS-CoV-2 Spike RBD; the buried surface area (BSA) of the heavy and light chains are and
  • RBM receptor binding motif
  • BSA buried surface area
  • All heavy chain complementarity determining regions (HCDRs) and light chain complementarity determining regions (LCDRs) are involved in interactions with the SARS-CoV-2 RBD, and the main interactions are polar interactions and hydrogen bonding interactions.
  • Y31, Y32, W52, Y53, D54, S56, N57, R58, G101, G102, R104, R106 of HCDR and H31, N33, Y35, Y54, Y37, T99 of LCDR also form hydrogen bonds with RBD, Further strengthen the combination between the two.
  • PR300953 The situation with PR300953 is slightly different. Although this antibody also has potent binding, blocking and neutralizing activities, the buried surface area between it and the RBD is much smaller than that of PR301077, where the heavy chain is The light chain is The complementarity determining regions (CDRs) of PR300953 formed a deep concave binding pocket and tightly covered the flexible tip of the RBM region (I472 to F490 on the RBD), partially overlapping the binding sites of RBD and hACE2. The interaction between PR300953 and RBD is mainly stabilized by a large network of hydrogen bonds.
  • CDRs complementarity determining regions
  • T33, H35, N52, N55, D57, T59, D99, Y101 and Y105 of the HCDR identified a raised region composed of Q474, A475, G476, S477, N487 and Y489 on the RBD.
  • the electrostatic interaction between Y91, N92, N93, Y94, W96 of LCDR and S477, S478, N481 and F486 on RBD further stabilized the binding of antibody to antigen.
  • Example 15 Part of candidate neutralizing antibodies were investigated for their neutralizing activity against new mutant strains by in vitro virus infection
  • PR301077 has a strong neutralizing activity against the Wuhan strain, but is ineffective against the South African strain.
  • PR300953 Wuhan strain and South Africa strain have the same neutralizing activity.
  • PR301446 obtained by humanization of PR300928 has strong neutralizing activity against Wuhan strain and South Africa strain.

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Abstract

L'invention concerne un anticorps anti-coronavirus et son utilisation, le coronavirus étant un nouveau coronavirus, et en particulier, un anticorps qui se lie de manière spécifique à une nouvelle protéine S-RBD du coronavirus et un fragment de liaison à l'antigène de celui-ci. L'anticorps ou le fragment de liaison à l'antigène de celui-ci comprend une région variable de chaîne lourde et/ou une région variable de chaîne légère, et la région variable de chaîne lourde comprenant HCDR1, HCDR2 et HCDR3, et la région variable de chaîne légère comprend LCDR1, LCDR2 et LCDR3. L'anticorps ou le fragment de liaison à l'antigène de celui-ci a une capacité de liaison plus élevée au nouveau coronavirus, et a une meilleure activité de neutralisation (les valeurs IC50 de l'activité de neutralisation sont toutes inférieures au niveau de μg/mL), un meilleur profil de couverture de virus mutant et divers épitopes de liaison.
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