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WO2022041745A1 - Anticorps dirigé contre la proteine s du coronavirus sars-cov-2 et son utilisation - Google Patents

Anticorps dirigé contre la proteine s du coronavirus sars-cov-2 et son utilisation Download PDF

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WO2022041745A1
WO2022041745A1 PCT/CN2021/086477 CN2021086477W WO2022041745A1 WO 2022041745 A1 WO2022041745 A1 WO 2022041745A1 CN 2021086477 W CN2021086477 W CN 2021086477W WO 2022041745 A1 WO2022041745 A1 WO 2022041745A1
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amino acid
seq
substitutions
antibody
sequences
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Chinese (zh)
Inventor
李强
孙见宇
武翠
张晓峰
刁家升
周利
马心鲁
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Ampsource Biopharma Shanghai Inc
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Ampsource Biopharma Shanghai Inc
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Priority claimed from CN202010889404.6A external-priority patent/CN114106161A/zh
Priority claimed from CN202010887508.3A external-priority patent/CN114106159A/zh
Priority claimed from CN202010889425.8A external-priority patent/CN114106163A/zh
Priority claimed from CN202010889418.8A external-priority patent/CN114106162B/zh
Priority claimed from CN202010889400.8A external-priority patent/CN114106160A/zh
Application filed by Ampsource Biopharma Shanghai Inc filed Critical Ampsource Biopharma Shanghai Inc
Publication of WO2022041745A1 publication Critical patent/WO2022041745A1/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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to the field of therapeutic antibodies and molecular immunology, more particularly, to a recombinant monoclonal antibody of the S protein of SARS-CoV-2 coronavirus, and the use of this antibody, especially in the treatment, prevention and diagnosis of Use in COVID-19 disease caused by SARS-CoV-2.
  • the novel coronavirus SARS-CoV-2 can cause severe respiratory disease and COVID-19 pneumonia with fever, fatigue, and dry cough as the main manifestations.
  • the World Health Organization on August 19, 2020, there were 21,756,357 confirmed cases globally, resulting in 771,635 deaths.
  • the new pathogen turned out to be a novel member of the genus betacoronavirus.
  • the genome sequence similarity between SARS-CoV-2 and bat coronavirus RaTG13 is 96.2% (Zhou P et al, 2020, Nature, 579:270-273), which is similar to two bat SARS-like coronaviruses bat-SL-CoVZC45 (same as bat-SL-CoVZC45). 88% homology) and bat-SL-CoVZXC21 (87% homology) are closely related, but relatively distantly related to SARS-CoV (79% homology) and MERS-CoV (50% homology) ( Lu R et al, 2020, Lancet, 395:565-574). Compared with SARS-CoV, the SARS-CoV-2 coronavirus is more easily transmitted from person to person.
  • the WHO has declared the COVID-19 disease a global pandemic, and the new coronavirus has now spread all over the world.
  • the novel coronavirus SARS-CoV-2 is a positive-sense RNA virus that encodes several major proteins, S, M, N, and E, the RNA-dependent RNA polymerase RDRP, and more than a dozen nonstructural proteins.
  • S, M, N and E proteins are used to package the virus structure, and RDRP and more than a dozen non-structural proteins are used for the replication of viral genome RNA and the synthesis of each protein mRNA.
  • SARS-CoV-2 is very similar to SARS-CoV virus and has a high degree of amino acid sequence homology.
  • the number of amino acids of its S, M, N, E and RDRP proteins and the degree of homology to SARS-CoV are 1273 ( 76%), 222 (91%), 419 (91%), 75 (95%), 932 (96%).
  • the SARS-CoV-2 virus is spherical in shape with an envelope and crowned spikes lining its periphery.
  • the spike S protein of SARS-CoV-2 forms a trimer (Wrapp D et al, 2020, Science, 6483:1260-1263), shaped like a mushroom, embedded in the outer membrane of the virus.
  • the S protein is the main antigenic component of the virus and is responsible for the binding of the virus to the receptor ACE2 of the invaded host cell and the fusion of the virus and the cell. Similar to the SARS-CoV virus S protein (Yuan Y et al, 2017, Nat Commun, 8:15092), the SARS-CoV-2 coronavirus S protein is mainly divided into two domains, S1 (1-685) and S2 ( 686-1122), as well as a short transmembrane region and cytoplasmic tail. In the mushroom-like S protein trimer, three S1 domains form the "mushroom cap" and three S2 domains form the "mushroom stem".
  • the RBD domain (Receptor binding domain, amino acids 331-527) in S1 is responsible for binding to the invaded host cell receptor ACE2, and S2 is responsible for fusion with the host cell.
  • the S2 domain typically exists in a folded or coiled-compressed conformation in the overall S protein, and when the virus fuses with the host cell after S1 shedding, S2 displays an extended conformation for insertion into the host cell membrane (Walls AC et al, 2017, Proc Natl Acad Sci USA, 114:11157-11162).
  • the Furin site containing polybasic amino acids can also be used by other lysine or arginine as Targeted enzymes, such as cell surface enzyme TMPRSS2, endosomal cathepsin L enzyme or possibly trypsin (Trypsin), etc.
  • the SARS-CoV virus S1/S2 is only connected by a basic amino acid arginine, where the S protein is cleaved by the cell surface enzyme TMPRSS2 and cathepsin L in the endosome to infect host cells (Belouzard S et al. al, 2012, Viruses, 4:1011-1033; Belouzard S et al, 2009, Proc Natl Acad Sci USA, 106:5871-5876). Therefore, the above two differences, namely the existence of the Furin cleavage site and the high affinity with the human receptor ACE2, may be the reasons for the high infectivity of the SARS-CoV-2 coronavirus. Since the S protein is responsible for binding to human host cell receptors and fusion with host cells, the S protein is a major target for therapeutic neutralizing antibodies against SARS-CoV and SARS-CoV-2 coronaviruses.
  • Neutralizing antibodies prevent the spread of the virus by blocking the virus from invading the host cell, and achieve the purpose of treating the disease.
  • Regeneron Pharmaceuticals has developed a series of SARS-CoV-2 neutralizing antibodies against the RBD domain using transgenic mice and single B cell sequencing platforms (Hansen J et al, 2020, Science, 369:1010-1014).
  • LY-CoV555 is a potent neutralizing antibody against the SARS-CoV-2 spike protein S of the IgG1 subtype.
  • Regeneron Pharmaceuticals' double antibody cocktail REGN-COV2 entered the clinical research stage for the first time, and based on the good safety data of the Phase I clinical study, the study has now been directly entered into the Phase III clinical study.
  • JS016 is the first new coronavirus neutralizing antibody to enter the clinic in China.
  • SARS-CoV-2 coronavirus S protein neutralizing antibody should be developed as soon as possible, with higher specificity, better clinical efficacy and lower treatment cost, which will give SARS - CoV-2-infected patients provide more medication options.
  • the present invention provides an antibody that can specifically recognize and bind to the S protein of SARS-CoV-2 coronavirus with high affinity.
  • the antibody of the present invention can block the infection of host cells by SARS-CoV-2.
  • the S protein antibodies disclosed herein can be used (alone or in combination with other formulations or therapeutic methods) for the treatment, prevention and/or diagnosis of diseases caused by SARS-CoV-2, such as COVID-19.
  • the first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to the S protein of SARS-CoV-2 coronavirus, wherein the variable region (VH) of the heavy chain contained in the antibody or the antigen-binding fragment thereof comprises at least One, two or three complementarity determining regions (CDRs) selected from the group consisting of:
  • HCDR1 having the sequence set forth in SEQ ID NO: 1, 7, 16, 22, 31, 37, 46, 52, 61, 67, 76, 82, 111, 117, 126 or 132, or with A sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
  • HCDR2 having as SEQ ID NO: 2, 8, 17, 23, 32, 38, 47, 53, 62, 68, 77, 83, 101, 104, 112, 118, 127, 133, 165 or The sequence shown in 167, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or additions) compared to any of the above sequences; and
  • HCDR3 having as SEQ ID NO: 3, 9, 18, 24, 33, 39, 48, 54, 63, 69, 78, 84, 102, 105, 113, 119, 128, 134, 146, 151, 153, 158, 160, 166 or 168, or with one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or add) sequence;
  • the light chain variable region (VL) it comprises comprises at least one, two or three complementarity determining regions (CDRs) selected from the group consisting of:
  • LCDR1 having as SEQ ID NO: 4, 10, 19, 25, 34, 40, 49, 55, 64, 70, 79, 85, 91, 92, 103, 106, 114, 120, 129, A sequence shown in 135, 152 or 159, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
  • LCDR2 having the sequence set forth in SEQ ID NO: 5, 11, 20, 26, 35, 41, 50, 56, 65, 71, 80, 86, 115, 121, 130, 136 or 141, or a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions) compared to any of the above sequences; and
  • LCDR3 which has the sequence shown in SEQ ID NO: 6, 21, 36, 51, 66, 81, 116, 131, or has one or more amino acid substitutions, deletions compared to any of the above sequences or additions (eg 1, 2 or 3 substitutions, deletions or additions) of the sequence.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region, and/or the LCDR1, LCDR2 and LCDR3 contained in the light chain variable region are defined by the Kabat or IMGT numbering system .
  • Table 5 in Example 6 exemplifies the CDR amino acid sequences of murine antibodies as defined by the Kabat or IMGT numbering system.
  • the antibody or antigen-binding fragment thereof comprises 3 VH variable region CDRs and 3 VL variable region CDRs selected from the following 26 groups:
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 7, 8, 9, 10, 11 or 6, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 16, 17, 18, 19, 20 or 21, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 22, 23, 24, 25, 26 or 21, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 31, 32, 33, 34, 35 or 36, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 37, 38, 39, 40, 41 or 36, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 46, 47, 48, 49, 50 or 51, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 52, 53, 54, 55, 56 or 51, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 61, 62, 63, 64, 65 or 66, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 67, 68, 69, 70, 71 or 66, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 76, 77, 78, 79, 80 or 81, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 82, 83, 84, 85, 86 or 81, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 1, 2, 3, 91, 5 or 6, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 7, 8, 9, 92, 11 or 6, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 16, 101, 102, 103, 20 or 21, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 22, 104, 105, 106, 26 or 21, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 111, 112, 113, 114, 115 or 116, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 117, 118, 119, 120, 121 or 116, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 126, 127, 128, 129, 130 or 131, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 132, 133, 134, 135, 136 or 131, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 1, 2, 3, 4, 141 or 6, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 22, 23, 146, 25, 26 or 21, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 76, 77, 151, 152, 80 or 81, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 82, 83, 153, 85, 86 or 81, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 76, 77, 158, 159, 80 or 81, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 82, 83, 160, 85, 86 or 81, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 126, 165, 166, 129, 130 or 131, respectively, or have one or more in comparison with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequence shown in SEQ ID NO: 132, 167, 168, 135, 136 or 131, respectively, or have one or more in comparison with any of the above sequences
  • a sequence of several amino acid substitutions, deletions or additions eg 1, 2 or 3 substitutions, deletions or additions.
  • the antibody or antigen-binding fragment thereof is murine or chimeric, and its heavy chain variable region comprises the heavy chain FR region of a murine IgGl, IgG2, IgG3, or variant thereof; and
  • the light chain variable region comprises the light chain FR regions of murine kappa, lambda chains or variants thereof.
  • the variable region amino acid sequence numbers of some preferred murine antibodies are given in Table 6 in Example 6.
  • the murine antibody or antigen-binding fragment thereof comprises VH and VL domains selected from the following 11 groups:
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 12, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 13, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 27, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 28, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 42, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 43, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 57, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g. conservative substitutions); and its VL domain comprises the amino acid sequence shown in SEQ ID NO: 58, or is substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 72, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 73, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 87, or is substantially identical to the above sequence (for example at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g. conservative substitutions); and its VL domain comprises the amino acid sequence shown in SEQ ID NO: 88, or is substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 93, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 94, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 97, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g. conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 98, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 107, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g. conservative substitutions); and its VL domain comprises the amino acid sequence shown in SEQ ID NO: 108, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 122, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 123, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 137, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 138, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) .
  • the antibody or antigen-binding fragment thereof is humanized.
  • Example 6 gives the basic flow of the humanization strategy, and Table 6 gives the amino acid sequence numbers of the variable regions of some preferred humanized antibodies.
  • the humanized antibody or antigen-binding fragment thereof comprises VH and VL domains selected from the following 9 groups:
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 14, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 15, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 44, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g. conservative substitutions); and its VL domain comprises the amino acid sequence shown in SEQ ID NO: 45, or is substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 74, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 75, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions))
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 142, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 143, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 147, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 148, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 154, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 155, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 161, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 162, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 124, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 125, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) ;
  • the VH domain comprises the amino acid sequence shown in SEQ ID NO: 169, or is substantially identical to the above sequence (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, A sequence of 99% or higher identity or having one or more amino acid substitutions (e.g., conservative substitutions); and its VL domain comprising the amino acid sequence shown in SEQ ID NO: 170, or substantially the same as the above-mentioned sequence Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or have one or more amino acid substitutions (eg, conservative substitutions)) .
  • the antibody comprises a heavy chain constant region and a light chain constant region derived from human immunoglobulin.
  • the antibody comprises the human kappa chain constant region amino acid sequence (amino acid sequence shown in SEQ ID NO: 95).
  • the antibody comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; more preferably, comprises a heavy chain selected from human IgG1, IgG2 and IgG4 A constant region; and, the heavy chain constant region has the native sequence or a sequence with one or more amino acid substitutions, deletions or additions compared to the native sequence from which it is derived.
  • the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgGl (amino acid sequence set forth in SEQ ID NO: 96).
  • the humanized antibody molecule comprises the heavy chain constant region of human IgG1 containing the M252Y, S254T, T256E and M428L mutations according to EU numbering (amino acid sequence set forth in SEQ ID NO: 190). In another embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG2 (amino acid sequence set forth in SEQ ID NO: 99). In one embodiment, the humanized antibody molecule comprises human IgG2 modified in the hinge region according to EU numbering (e.g. deletion of ERKCC, amino acid sequence shown in SEQ ID NO: 100), see Chinese Patent No. CN104177496B.
  • the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG4 (amino acid sequence set forth in SEQ ID NO: 109). Or use a modified human IgG4 constant region sequence; in one embodiment, the humanized antibody molecule comprises a human IgG4 (amino acid sequence such as SEQ ID NO: 228) mutated (e.g., S to P) according to EU numbering. 110).
  • the heavy chain of the antibody has the amino acid sequence set forth in SEQ ID NO: 29, 59, 89, 139, 144, 149, 156, 163 or 171; Any sequence having one or more substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the sequences; or at least 80 compared to any of the above sequences %, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher identical and/or, the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 30, 60, 90, 140, 145, 150, 157, 164 or 172; compared to a sequence having one or several substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions); or at least 80% compared to any of the above
  • the aforementioned substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the present invention wherein the SARS-CoV-2 coronavirus S protein has:
  • (b) is an amino acid sequence obtained by replacing, deleting or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 189.
  • substitutions include K417 and/or L452 and/or E484 and/or N501.
  • the substituted K417 is K417N, and/or L452 is L452R, and/or E484 is E484K, and/or N501 is N501Y.
  • the antibody or antigen-binding fragment thereof of the present invention is capable of binding to the SARS-CoV-2 coronavirus S protein with a KD of 10 nM or lower, more preferably, with a KD of 1 nM or lower S protein; more preferably, with a KD of 100 pM or less; more preferably, with a KD of 10 pM or less; most preferably, with a KD of 1 pM or less . .
  • the second aspect of the present invention provides a DNA molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.
  • the DNA molecule encoding the heavy chain of the antibody has the nucleotide sequence shown in SEQ ID NO: 173, 175, 177, 179, 181, 183, 185 or 187, and encoding the The DNA molecule of the antibody light chain has the nucleotide sequence set forth in SEQ ID NO: 174, 176, 178, 180, 182, 184, 186 or 188.
  • a third aspect of the present invention provides a vector comprising the above DNA molecule.
  • the fourth aspect of the present invention provides a host cell comprising the above-mentioned vector;
  • the host cell comprises prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells;
  • the fifth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient, carrier or diluent.
  • the sixth aspect of the present invention also provides a method for preparing the antibody or its antigen-binding fragment of the present invention, comprising: (a) obtaining the gene of the antibody or its antigen-binding fragment, and constructing an expression vector for the antibody or its antigen-binding fragment (b) transfecting the above-mentioned expression vector into host cells by genetic engineering methods; (c) culturing the above-mentioned host cells under conditions that allow the production of the antibody or its antigen-binding fragment; (d) isolating and purifying the resulting the antibody or antigen-binding fragment thereof.
  • the expression vector in step (a) is selected from one or more of plasmids, bacteria and viruses, preferably, the expression vector is pcDNA3.1;
  • the constructed vector is transfected into host cells by genetic engineering method, and the host cells include prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells.
  • step (d) separates and purifies the antibody or its antigen-binding fragment by a conventional immunoglobulin purification method, including protein A affinity chromatography and ion exchange, hydrophobic chromatography or molecular sieve method.
  • a conventional immunoglobulin purification method including protein A affinity chromatography and ion exchange, hydrophobic chromatography or molecular sieve method.
  • the seventh aspect of the present invention provides the use of the antibody or its antigen-binding fragment in the preparation of a medicament for the treatment and prevention of diseases caused by SARS-CoV-2 coronavirus.
  • the disease is novel coronavirus pneumonia (COVID-19); for example, the disease is novel coronavirus pneumonia (COVID-19) caused by B.1.351 mutant strain and/or B.1.1.7 virus strain .
  • the eighth aspect of the present invention provides an immunoassay method for detecting or determining the presence or quantification of SARS-CoV-2 virus or its antigen in a biological sample by using the above-mentioned antibody; the method comprises combining the biological sample to be detected with the present invention.
  • the invented anti-SARS-CoV-2 virus S protein monoclonal antibody or its antigen-binding fragment is incubated to form an antigen-antibody complex, and qualitative detection and quantitative determination of the formed binding complex are carried out.
  • the existence or content of SARS-CoV-2 virus; specifically, the method includes the following steps:
  • Monoclonal antibodies or antigen-binding fragments thereof according to the present invention may be independent of the label used (eg, enzyme, fluorescence, etc.) and independent of the detection mode (eg, fluorescent immunoassay, ELISA, or chemical luminescence assay, etc.) or assay principles (eg, sandwich method, competition method, etc.) are used in the above-mentioned immunoassay methods; wherein, examples of the antigen-binding fragment include, but are not limited to, F(ab')2, Fab ', Fab and Fv.
  • the label used eg, enzyme, fluorescence, etc.
  • detection mode e.g, fluorescent immunoassay, ELISA, or chemical luminescence assay, etc.
  • assay principles eg, sandwich method, competition method, etc.
  • the above immunoassays include enzyme immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescence immunoassays, Western blotting, immunochromatography, latex agglutination assays, etc.;
  • the method uses a marker-labeled antigen or antibody to determine the target antigen in a biological sample.
  • the above competitive method is based on the quantitative competitive binding reaction of SARS-CoV-2 virus and a known amount of labeled SARS-CoV-2 virus S protein in the detection sample with the monoclonal antibody of the present invention or its antigen-binding fragment; specifically
  • the above competition method includes: embedding a predetermined amount of the monoclonal antibody of the present invention against the S protein of SARS-CoV-2 virus or its antigen-binding fragment on a solid-phase carrier, and then adding the SARS-CoV-2 virus containing SARS-CoV-2 virus to be detected.
  • the solid phase was sufficiently washed and detected or retained on the carrier. Determining the signal value of the label not retained on the support; then comparing the measured signal value with the signal value of a predetermined amount of control samples measured in parallel to determine the presence of SARS-CoV-2 virus in the sample and its Relative amounts; preferably, the labeled antigen and the biological sample to be detected are added almost simultaneously.
  • the above-mentioned sandwich method is based on the fact that the monoclonal antibody or its antigen-binding fragment of the present invention as a capture antibody (or solid-phase antibody) and the labeled antibody that can be used in combination can specifically bind to the SARS-CoV-2 virus in the biological sample.
  • the above sandwich method includes: the specific monoclonal antibody against the SARS-CoV-2 virus S protein of the present invention or its antigen-binding fragment Binding to the solid phase carrier to form a solid phase antibody (also known as capture antibody or primary antibody), then add the biological sample to be tested and the control sample to the coated solid phase carrier and incubate for a long enough time under appropriate conditions.
  • a solid phase antibody also known as capture antibody or primary antibody
  • the second antibody can also be other polyclonal antibodies; preferably, the second antibody is a monoclonal antibody.
  • the second antibody is selected from any monoclonal antibody or antigen-binding fragment thereof that can be used in conjunction with the first antibody of the present invention.
  • the label can be a radioisotope (eg, 125I), an enzyme, an enzyme substrate, a phosphorescent substance, a fluorescent substance, a biotin, and a coloring substance.
  • the labels used in the present invention include alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, urease and glucose oxidase; the labels can also be fluorescent substances, such as fluorescein derivatives and rhodamine derivatives; in addition, the label can also be a rare earth element or a rare earth element complex, such as europium or europium complex, which allows for time-resolved fluorescence determination; in addition, the label can be a phosphorescent substance, such as acridine esters and isorubic acid Minoan; or radioactive isotopes such as 125I, 3H, 14C and 32P; in addition, the label can be a colored substance such as latex particles and colloidal gold. That is, the present invention includes qualitatively or quantitatively determining the presence or content of SARS-CoV-2 virus in biological components by measuring color, fluorescence, time-resolved fluorescence, chemiluminescence
  • the solid phase needs to be washed sufficiently to measure the activity of binding to the label.
  • the label is a radioisotope
  • the measurement is performed with a pore counter or a liquid scintillation counter.
  • the label is an enzyme
  • the substrate is added and the enzyme activity is measured colorimetrically or fluorometrically after color development.
  • the label is a fluorescent substance, a phosphorescent substance, or a coloring substance, it can be measured by methods known in the art, respectively.
  • the biological samples mentioned above are selected from plasma, whole blood, mouthwash, throat swabs, urine, feces and bronchial perfusate.
  • solid supports mentioned above include, but are not limited to, nitrocellulose membranes, latex particles, magnetic particles, colloidal gold, beads or sensors such as glass, fiberglass or polymers such as polystyrene or polyvinyl chloride or fiber optic sensors.
  • the ninth aspect of the present invention provides the use of the above-mentioned monoclonal antibody in the preparation of a SARS-CoV-2 virus detection kit.
  • the tenth aspect of the present invention provides a detection kit for SARS-CoV-2 virus, which comprises at least one monoclonal antibody or an antigen-binding fragment thereof of the present invention; the monoclonal antibody used for preparing the detection reagent does not Subject to special limitation, any of the above-mentioned monoclonal antibodies of the present invention or their antigen-binding fragments (such as F(ab')2, Fab', Fab and scFv) can be used as one of solid-phase antibodies or labeled antibodies. It can also be used in combination with two monoclonal antibodies or antigen-binding fragments thereof directed against different antigenic epitopes in the present invention as solid-phase antibodies or labeled antibodies, respectively.
  • any of the above-mentioned monoclonal antibodies of the present invention or their antigen-binding fragments such as F(ab')2, Fab', Fab and scFv
  • the detection kit includes:
  • the first antibody is any monoclonal antibody or antigen-binding fragment thereof selected from the present invention.
  • the second antibody is optionally labeled appropriately, and the second antibody is selected from the monoclonal antibodies or antigen-binding fragments thereof described in the present invention that can be used in combination with the first antibody in (1).
  • the monoclonal antibody or its antigen-binding fragment of the present invention contained in the above detection reagent can be pre-immobilized on a solid-phase carrier to form a solid-phase antibody
  • the solid-phase carrier includes but is not limited to nitrocellulose membrane, latex particles, magnetic particles , colloidal gold, beads or sensors such as glass, fiberglass or polymers (such as polystyrene or polyvinyl chloride) or fiber optics; in a preferred embodiment of the present invention, the solid support is a microtiter plate.
  • the monoclonal antibody of the present invention or its antigen-binding fragment contained in the above-mentioned immunoassay reagent can be labeled with a label in advance to form a labeled antibody, and the label includes but is not limited to radioisotopes (such as 125I), enzymes, enzyme substrates , phosphorescent substances, fluorescent substances, biotin and coloring substances; preferably, the enzymes include, for example, alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, urease and glucose oxidase; the fluorescent substances Including such as fluorescein derivatives and rhodamine derivatives and rare earth elements or rare earth element complexes, such as europium or europium complexes; the phosphorescent substances include such as acridine esters and isoluminol; the radioisotopes include such as 125I, 3H, 14C and 32P; the coloring substances include, for example, late
  • the eleventh aspect of the present invention provides the use of the above immunoassay reagent in diagnosing diseases caused by SARS-CoV-2 virus infection.
  • the disease is novel coronavirus pneumonia (COVID-19); for example, the disease is novel coronavirus pneumonia (COVID-19) caused by B.1.351 mutant strain and/or B.1.1.7 virus strain .
  • mice Using the mouse hybridoma platform to immunize mice with S protein (amino acids 326-685) and S trimer (amino acids 16-1213) as immunizing antigens, a series of murine sources of SARS-CoV-2 coronavirus S protein were obtained Antibodies, these antibodies can specifically recognize and bind to S protein with high affinity, and the KD value reaches pM level.
  • S protein amino acids 326-685
  • S trimer amino acids 16-1213
  • the mouse-derived antibody has been humanized to reduce the immunogenicity.
  • the humanized antibody retains the affinity and pseudovirus inhibitory activity of the murine antibody, the binding affinity KD value reaches pM level, and the pseudovirus inhibitory activity is equivalent to nM level.
  • the above characteristics lay the foundation for the clinical application of antibodies.
  • the antibody provided by the present invention can also be used to detect the presence of SARS-CoV-2 virus or its corresponding antigen in a sample, and the detection sensitivity of the antibody is lower than 100 pg/ml; more preferably, lower than 10 pg/ml.
  • CDR Complementarity-Determining Regions complementarity determining regions in immunoglobulin variable regions, defined by the Kabat, IMGT, Chothia or AbM numbering system (see term “hypervariable region” or “CDR region” or “complementarity determining region”).
  • V region A segment of an IgG chain whose sequence varies between different antibodies. It extends to Kabat residue 109 of the light chain and residue 113 of the heavy chain.
  • EC50 refers to the concentration of an antibody or antigen-binding fragment thereof that induces a 50% response in an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof, ie, the concentration halfway between the maximal response and baseline.
  • EU refers to the first human IgG1 immunoglobulin isolated and purified by Gerald M Edelman et al in the late 1960s (1968-1969), named EU, Its amino acid sequence was determined and numbered (Edelman GM et al, 1969, Proc Natl Acad USA, 63:78-85). The amino acid sequences of the heavy chain constant regions of other immunoglobulins are aligned with EU, and the corresponding amino acid positions are EU numbering.
  • the EU numbering system mainly targets the constant regions of immunoglobulin heavy chains, including CH1, CH2, CH3 and hinge regions.
  • Lambda light chains do not contain residue at position 10, whereas Lambda and Kappa light chains are encoded by two different genes, located on different chromosomes. Lambda and Kappa light chains can be distinguished by differences in their constant region amino acid sequences. Unlike the EU numbering system, which addresses only heavy chain constant regions, the Kabat numbering system covers the full-length immunoglobulin sequence, including the variable and constant regions of immunoglobulin light and heavy chains.
  • binding defines the affinity interaction between a specific epitope on an antigen and its corresponding antibody, generally also understood as “specific recognition”.
  • Specific recognition means that the antibody of the invention does not, or substantially does not cross-react with, any polypeptide other than the target antigen.
  • the degree of its specificity can be judged by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry and the like.
  • the specific identification is preferably determined by flow cytometry, and the standard of the specific identification in a specific case can be judged by a person of ordinary skill in the art according to the common knowledge in the art.
  • the term "antigen" is a foreign substance that can trigger an organism's own or human to produce antibodies, and is any substance that can induce an immune response, such as bacteria, viruses, and the like.
  • Foreign antigen molecules are recognized and processed by B cells or antigen-presenting cells (such as macrophages, dendritic cells, endothelial cells, and B cells, etc.), and combined with major histocompatibility complexes (such as MHC II molecules) The complex reactivates T cells and triggers a continuous immune response.
  • antigenic epitope or “antigenic determinant” refers to a specific chemical group or peptide sequence on a molecule that is antigenic (ie, elicits a specific immune response), and is an antigen to which an immunoglobulin or antibody specifically binds (such as site on the S protein of SARS-CoV-2.
  • Epitope-determining regions usually consist of chemically active surface groups of molecules (eg, amino acids or glycosyl side chains) and usually have specific three-dimensional structural properties as well as specific charge properties.
  • Antigens have two types of epitopes or epitopes, B cell epitopes and T cell epitopes, which are recognized by B cells and T cells, respectively.
  • B cell antigenic epitopes are located on the surface of antigen molecules and are antigenic sites that bind to B cell receptors (BCR, an antibody located on the B cell membrane). B cell epitopes can be directly recognized by B cells without processing. Then B cells engulf antigen molecules, process them into small peptides (about 15 amino acids in size, antigen T cell epitopes), and present them to Th cells (helper T cells). At the same time, antigen molecules can also be processed into small peptides and presented to Th cells through another pathway, such as phagocytosis by macrophages.
  • BCR B cell receptors
  • Th is co-stimulated by B cells and macrophages, the three cells interact together, and Th cells send feedback signals to B cells, instructing B cells to proliferate and differentiate into plasma cells and memory cells.
  • Plasma cells have the function of secreting antibodies and mediate humoral adaptive immunity.
  • Antibody binds antigen molecules through its variable region Fv part, and binds to receptor FcR on various immune cells through its constant region Fc part, thereby directing various immune cells to kill antigen molecules, using ADCC (through NK cells), CDC (via complement) and ADCP (via macrophages) functions.
  • ADCC through NK cells
  • CDC via complement
  • ADCP via macrophages
  • B cell epitopes can be divided into continuous epitopes and conformational epitopes (or discontinuous epitopes) according to their continuity in the protein amino acid sequence.
  • B cell epitopes vary in size, ranging from 5 to 20 amino acids in size.
  • T-cell epitopes are recognized by T cells. Unlike B-cell epitopes, T-cell epitopes can be located anywhere in the antigen molecule (such as viral proteins), so T-cell epitopes run through the entire protein sequence. .
  • T cell epitopes are continuous determinants, typically 10-20 amino acids in size.
  • T cell epitopes bind to MHC class I (MHC I) or class II (MHC II) MHC molecules and are presented on the cell surface, where they are captured by two distinct subsets of T cells, CD8 + T cells (killer T cells) and CD4 + , respectively. T cell (helper Th cell) recognition. Therefore, there are two types of T cell epitopes, CD8 + and CD4 + T cell epitopes.
  • MHC I molecules are expressed by almost all cells and can provide some conditions in the cells. For example, if the cell is infected by a virus, the small peptide molecules of virus fragments will be displayed on the cell surface through MHC I, which can be recognized by killer CD8 + T cells. , for culling.
  • MHC II molecules are mostly located on antigen-presenting cells, such as macrophages. This type of MHC II molecule provides the situation outside the cell (such as in body fluids), such as the invasion of bacteria in the tissue. After the macrophage engulfs it, the bacterial debris is prompted by MHC II to the helper Th cells to initiate an immune response.
  • B cells and T cells can only recognize and bind to the antigenic epitopes of foreign antigen molecules, but have no binding ability to antigen fragments derived from the organism itself, such as protein molecules and their fragments, because B cells and T cells have no binding ability.
  • B cells and T cells with high affinity for self-protein molecules or fragments are inhibited from maturation or undergo apoptosis.
  • antibody generally refers to protein-binding molecules that have immunoglobulin-like functions. Typical examples of antibodies are immunoglobulins, and derivatives or functional fragments thereof, so long as they exhibit the desired binding specificity. Techniques for preparing antibodies are well known in the art. "Antibody” includes different classes of native immunoglobulins (eg, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgGl, IgG2, IgAl, IgA2, etc.).
  • native immunoglobulins eg, IgA, IgG, IgM, IgD, and IgE
  • subclasses eg, IgGl, IgG2, IgAl, IgA2, etc.
  • Antibody also includes unnatural immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (eg, humanized murine antibodies), and heteroconjugated antibodies (eg, bispecific antibodies), and antigen-binding fragments thereof (for example, Fab', F(ab') 2 , Fab, Fv and rIgG). See also, eg, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co, Rockford, Ill); Kuby J, Immunology, 3rd Ed, WH Freeman & Co, New York, 1997. Antibodies can bind to one antigen, called “monospecific”; or to two different antigens, called “bispecific”; or to more than one different antigen, called “multispecific””.
  • Antibodies can be monovalent, bivalent or multivalent, ie antibodies can bind to one, two or more antigenic molecules at a time. Antibodies bind "monovalently" to a particular protein, ie a molecule of antibody binds to only one molecule of protein, but the antibody may also bind to a different protein. An antibody binds "monovalently” to each protein when it binds only to each molecule of two different proteins, and the antibody is “bispecific” and binds "monovalently” to two different proteins each type of protein. An antibody may be “monomeric", that is, it comprises a single polypeptide chain.
  • Antibodies may comprise multiple polypeptide chains ("multimeric") or may comprise two ("dimeric"), three (“trimeric") or four ("tetrameric") polypeptide chain. If the antibody is multimeric, the antibody may be a homomultimer, ie the antibody contains more than one molecule of only one type of polypeptide chain, including homodimers, homotrimers or homodimers source tetramer. Alternatively, a multimeric antibody may be a heteromultimer, ie the antibody comprises more than one different polypeptide chain, including heterodimers, heterotrimers or heterotetramers.
  • mAb refers to an antibody obtained from a population of substantially homogeneous antibodies, eg, the population comprising individual antibodies that are identical except for mutations that may be present in minor amounts, such as naturally occurring mutations.
  • the attribute "monoclonal” means that the antibody is characterized as not being a mixture of discrete antibodies.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, eg, by fusing myeloma cells with immune splenocytes to prepare hybrid antibody-producing cells. It is synthesized by hybridoma culture and will not be contaminated by other immunoglobulins.
  • Monoclonal antibodies can also be obtained using, for example, recombinant techniques, phage display techniques, synthetic techniques, or other available techniques.
  • single-chain Fv antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, the variable heavy (VH) and light chain regions being joined by a linker (VL), the linker allows the two domains to cross-link to form the antigen binding site, and the linker sequence generally consists of a flexible peptide, such as but not limited to G2(GGGGS) 3 .
  • the size of scFv is generally 1/6 of that of a complete antibody.
  • Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain.
  • an “intact antibody” refers to an antibody consisting of two antibody heavy chains and two antibody light chains.
  • An “intact antibody heavy chain” is composed in the N-terminal to C-terminal direction of the antibody heavy chain variable domain (VH), the antibody constant heavy chain domain 1 (CH1), the antibody hinge region (HR), the antibody heavy chain Consists of constant domain 2 (CH2) and antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and in the case of antibodies of the IgE subclass, optionally also antibody heavy Chain constant domain 4 (CH4).
  • VH antibody heavy chain variable domain
  • CH1 constant heavy chain domain 1
  • HR antibody hinge region
  • CH2 antibody heavy chain Consists of constant domain 2
  • CH3 antibody heavy chain constant domain 3
  • an “intact antibody heavy chain” is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
  • An “intact antibody light chain” is a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.
  • the antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda).
  • Intact antibody chains are linked together by interpolypeptide disulfide bonds between the CL and CH1 domains (ie, between the light and heavy chains) and between the hinge regions of the intact antibody heavy chains. Examples of typical intact antibodies are native antibodies such as IgG (eg, IgGl and IgG2), IgM, IgA, IgD and IgE.
  • antibody fragment refers to antigen-binding fragments and antibody analogs of antibodies that retain the ability to specifically bind to an antigen (eg, the S protein of SARS-CoV-2 coronavirus), which generally includes at least a portion of The antigen binding or variable region of the parent antibody (Parental Antibody).
  • Antibody fragments retain at least some of the binding specificity of the parent antibody.
  • antibody fragments retain at least 10% of the parent binding activity when the activity is expressed in molar units ( KD ).
  • the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the binding affinity of the parent antibody for the target.
  • Antibody fragments include, but are not limited to: Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, Fd fragments, complementarity determining region (CDR) fragments, disulfide stabilizing proteins (dsFv), etc.; linear antibodies ( Linear Antibody), single chain antibody (such as scFv single antibody), single antibody (Unibody, technology from Genmab), bivalent single chain antibody, single chain phage antibody, single domain antibody (Single Domain Antibody) (such as VH domain antibody) , domain antibodies (Domantis, technology from Domantis), nanobodies (nanobodies, technology from Ablynx); multispecific antibodies formed from antibody fragments (eg, tribodies, tetrabodies, etc.); and engineered antibodies such as chimeric Antibody (Chimeric Antibody) (eg, humanized murine antibody), Heteroconjugate Antibody, etc. These antibody fragments are obtained using conventional techniques known to those of skill in the
  • VL domain refers to the amino-terminal variable region domain of an immunoglobulin light chain.
  • VH domain refers to the amino-terminal variable region domain of an immunoglobulin heavy chain.
  • hinge region includes that portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain.
  • the hinge region comprises about 25 residues and is flexible, allowing the two N-terminal antigen binding regions to move independently.
  • the hinge region can be divided into three distinct domains: upper, middle, and lower hinge domains (Roux KH et al, 1998, J Immunol, 161:4083-4090).
  • domain refers to a three-dimensional structure capable of specifically recognizing and/or binding to an epitope, such as an antibody or antibody fragment, including native intact antibodies, single-chain antibodies (scFv), Fd fragments, Fab fragments, F( ab') 2 fragments, single domain antibody fragments, isolated CDR fragments and derivatives thereof.
  • single-stranded means that the first and second functional domains are covalently linked, and can be represented by a co-linear amino acid sequence encoded by one nucleic acid molecule.
  • Fab fragment consists of the variable and CH1 regions of a heavy chain and a light chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a "Fab antibody” is 1/3 the size of an intact antibody, which contains only one antigen-binding site.
  • Fab' fragment contains a light chain, the VH and CH1 domains of a heavy chain, and the constant region portion between the CH1 and CH2 domains.
  • F(ab') 2 fragment contains the VH and CH1 domains of two light and two heavy chains and a portion of the constant region between the CH1 and CH2 domains, thereby forming between the two heavy chains Interchain disulfide bonds.
  • an F(ab') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • Fd fragment consists of the variable region of a heavy chain and CH1, and is the portion of the heavy chain remaining after the light chain has been removed from the Fab fragment.
  • Fv region comprises variable regions from both heavy and light chains, but lacks the constant regions, and is the smallest fragment that contains a complete antigen recognition and binding site.
  • disulfide stabilizing protein introduces a cysteine mutation point in the VH and VL regions, respectively, thereby forming a disulfide bond between VH and VL to achieve structural stability.
  • disulfide bond includes a covalent bond formed between two sulfur atoms.
  • the amino acid cysteine contains a sulfhydryl group that can form a disulfide bond or bridge with a second sulfhydryl group.
  • the CH1 and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds, at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system) connection.
  • heavy chain constant region includes amino acid sequences from immunoglobulin heavy chains.
  • a polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (eg, upper hinge region, middle hinge region, and/or lower hinge region) domain, CH2 domain, CH3 domain, or variants thereof. body or fragment.
  • an antigen-binding polypeptide used in the present application may comprise a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a portion of a hinge domain and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; A polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain having a CH1 domain, at least a portion of a hinge structure, a CH2 domain, and a CH3 domain.
  • the polypeptide of the present application includes a polypeptide chain having a CH3 domain.
  • the antibodies used in the present application may lack at least a portion of the CH2 domain (eg, all or a portion of the CH2 domain).
  • CH2 domain e.g, all or a portion of the CH2 domain.
  • heavy chain constant regions may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.
  • light chain constant region includes amino acid sequences from antibody light chains.
  • the light chain constant region comprises at least one of a constant kappa domain and a constant lambda domain.
  • Fc region or “Fc fragment” refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the hinge region, the CH2 domain, and the CH3 domain, which mediate the interaction of the immunoglobulin with host tissues or factors. Binding includes binding to Fc receptors located on various cells of the immune system (eg, effector cells) or to the first component (Clq) of the classical complement system. Fc regions include native sequence Fc regions and variant Fc regions.
  • the Fc region of a human IgG heavy chain is the stretch from its amino acid residue at position Cys 226 or Pro 230 to the carboxy terminus, although the boundaries may vary.
  • the C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may or may not be present.
  • Fc can also refer to this region that exists independently, or in the case of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein", also referred to as an "Fc fusion protein" (eg, an antibody or immunoadhesin). ).
  • the native sequence Fc regions in the antibodies of the invention are derived from IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4 including mammalian (eg, human).
  • the aforementioned Fc region amino acid differences may be Fc alterations that prolong half-life, alterations that increase FcRn binding, alterations that enhance Fc ⁇ receptor (FcyR) binding, and/or alterations that enhance ADCC, ADCP, and/or CDC.
  • the Fc region comprises the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the IgM and IgE Fc regions comprise three heavy chain constants in each polypeptide chain domain (CH2-4 domain).
  • Fc receptor refers to a receptor that binds the Fc region of an immunoglobulin.
  • FcRs can be native sequence human FcRs, eg, can be FcRs that bind IgG antibodies (gamma receptors), as well as allelic variants and alternatively spliced forms of these receptors.
  • the FcyR family consists of three activating receptors (FcyRI, FcyRIII and FcyRIV in mice; FcyRIA, FcyRIIA and FcyRIIIA in humans) and one inhibitory receptor (FcyRIIb in mice or the equivalent FcyRIIB in humans) .
  • FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibiting receptor”), which have similar amino acid sequences.
  • the cytoplasmic domain of Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM).
  • the cytoplasmic domain of FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see Daeron M, 1997, Annu Rev Immunol, 15:203-234).
  • Fc ⁇ RIII activating Fc receptor
  • Fc ⁇ RIIIA activating Fc receptor
  • Fc ⁇ RIIIA activating Fc receptor
  • FcyRIIB Inhibitory FcyRIIB in mice and humans.
  • Human IgGl binds to most human Fc receptors and is considered equivalent to murine IgG2a in the type of activating Fc receptor it binds.
  • FcR herein encompasses other FcRs, including those to be identified in the future.
  • Fc receptor or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer RL et al, 1976, J Immunol, 117:587-593). Methods for measuring binding to FcRn are known (see, eg, Ghetie V and Ward ES, 1998, Immunol Today, 18:592-598; Ghetie V et al, 1997, Nat Biotechnol, 15:637-640). In vivo binding and serum half-life of human FcRn high affinity binding polypeptides to FcRn can be determined, eg, in transgenic mice or transfected human cell lines expressing human FcRn.
  • chimeric antibody means that a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequence derived from another antibody class or subclass.
  • Corresponding sequences in antibodies of one species or belonging to another antibody class or subclass are identical or homologous, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US Pat. No. 4,816,567; Morrison SL et al, 1984, Proc Natl Acad Sci USA, 81:6851-6855).
  • chimeric antibody can include antibodies (eg, human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a primary antibody (eg, a murine antibody) and the heavy and The light chain constant region is derived from a second antibody (eg, a human antibody).
  • a primary antibody eg, a murine antibody
  • a second antibody eg, a human antibody
  • human antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences.
  • Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody. Most or all of the amino acids outside the CDR domains of a non-human antibody, eg, a mouse antibody, are replaced with corresponding amino acids from a human immunoglobulin, while most or all of the amino acids within one or more of the CDR regions are unchanged. Additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not eliminate the ability of the antibody to bind to a specific antigen. "Humanized" antibodies retain similar antigenic specificity as the original antibody.
  • the source of the CDR is not particularly limited, and can be derived from any animal.
  • CDR regions derived from mouse antibodies, rat antibodies, rabbit antibodies, or non-human primate (eg, cynomolgus monkey) antibodies can be utilized.
  • the framework region can be obtained by searching the IMGT antibody germline database (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) to obtain the human antibody germline sequence, generally selecting the homology with the modified non-human antibody High human germline antibody sequences serve as framework regions for humanized antibodies.
  • variable region or “CDR region” or “complementarity determining region” refers to the amino acid residues of an antibody responsible for antigen binding and is a non-contiguous sequence of amino acids.
  • CDR region sequences can be determined by the methods of Kabat, Chothia, IMGT (Lefranc et al, 2003, Dev Comparat Immunol, 27:55-77) and AbM (Martin ACR et al, 1989, Proc Natl Acad Sci USA, 86:9268-9272) The amino acid residues within the variable region that are defined or identified by any CDR region sequence determination method well known in the art.
  • a hypervariable region comprises the following amino acid residues: amino acid residues from a "complementarity determining region" or "CDR" (Kabat numbering system) defined by a sequence alignment, e.g., 24-34 of the light chain variable domain ( LCDR1), residues 50-56 (LCDR2) and 89-97 (LCDR3) and residues 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) of the heavy chain variable domain , see Kabat et al, 1991, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md.; and/or from "hypervariable loops" (HVL) defined by structure Residues (Chothia numbering system), eg, residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) of the light chain variable domain and 26- Residues 32 (HCDR1), 53-
  • “Framework” residues or "FR” residues are variable domain residues other than hypervariable region residues as defined herein.
  • the CDRs contained by an antibody or antigen-binding fragment thereof of the invention are preferably determined by the Kabat, IMGT or Chothia numbering systems.
  • the Kabat residue numbering for a given antibody can be determined by comparing the antibody sequence to each "standard” numbered sequence for regions of homology. Based on the sequence numbering scheme provided herein, it is well within the routine skill of those skilled in the art to determine the numbering of any variable region sequence in the Sequence Listing.
  • polypeptide or polynucleotide refers to a form of the polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which can be achieved by combining polynucleotides or polypeptides that do not normally occur together combined together to achieve.
  • isolated antibody molecule refers to an antibody molecule that has been identified and separated and/or recovered from components of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • nucleic acid refers to a molecule that is separated from other DNA or RNA, respectively, which occurs as a macromolecule of natural origin.
  • isolated as used herein also refers to a nucleic acid or polypeptide that is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when prepared by chemical synthesis.
  • isolated nucleic acid is meant to include nucleic acid fragments that are not naturally occurring fragments and are not found in their natural state.
  • isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. An isolated polypeptide is meant to include purified and recombinant polypeptides.
  • cross-reactivity refers to the ability of the antibodies described herein to bind antigens from different species.
  • the antibodies described herein that bind the SARS-CoV-2 coronavirus S protein may also bind to the S protein from other species (eg, the S protein of SARS-CoV).
  • Cross-reactivity can be detected by detecting specific reactivity with purified antigen in binding assays (eg, SPR, ELISA), or binding to, or otherwise functional interaction with, cells that express the physiological antigen. Measurement. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (eg, Biacore) or similar techniques (eg, Kinexa or Octet).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as NK cells, neutrophils, or macrophages.
  • the FcR present on phagocytes binds these cytotoxic effector cells specifically to antibody-attached target cells, which then kill the target cells by secreting cytotoxins.
  • Methods for detecting ADCC activity of antibodies are known in the art and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an FcR (eg, CD16a).
  • ADCP antibody-dependent cell-mediated phagocytosis
  • complement system refers to the large number of small proteins found in the blood called complement factors, which normally circulate as inactive precursors (preproteins). This term refers to the ability of this system to "complement” the ability of antibodies and phagocytes to clear pathogens such as bacteria and antigen-antibody complexes from an organism.
  • An example of a complement factor is complex C1, which contains C1q and two serine proteases, C1r and C1s.
  • Complex C1 is a component of the CDC pathway.
  • C1q is a hexavalent molecule with a molecular weight of approximately 460,000 and has a structure resembling a tulip bouquet, with six collagen "stems" attached to six spherical head regions.
  • C1q In order to activate the complement cascade, C1q must bind to at least two molecules of IgG1, IgG2 or IgG3.
  • complement-dependent cytotoxicity refers to a form of cytotoxicity that activates the complement cascade by binding complement component C1q to an antibody Fc.
  • Methods for detecting the CDC activity of an antibody are known in the art, and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an Fc receptor (eg, C1q).
  • immunobinding and “immunobinding properties” refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength or affinity of an immunobinding interaction can be expressed in terms of the equilibrium dissociation constant (K D ) of the interaction, where a smaller K D value indicates a higher affinity.
  • K D equilibrium dissociation constant
  • the immunobinding properties of selected polypeptides can be determined using methods well known in the art. One assay involves measuring the rate of antigen/antibody complex formation and dissociation.
  • association rate constants K a or K on
  • dissociation rate constants K d or K off
  • immune cell includes cells of hematopoietic origin and that play a role in the immune response, including lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils cells, mast cells, basophils and granulocytes.
  • lymphocytes such as B cells and T cells
  • natural killer cells such as myeloid cells, such as monocytes, macrophages, eosinophils cells, mast cells, basophils and granulocytes.
  • immune response refers to cells of the immune system such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils. ) and the action of soluble macromolecules (including antibodies, cytokines and complement) produced by either of these cells or the liver that result in selective targeting, binding, injury, destruction and/or removal from vertebrates Removal of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.
  • An immune response includes, for example, activation or suppression of T cells (eg, effector T cells or Th cells, such as CD4 + or CD8 + T cells), or suppression of Treg cells.
  • immunogenicity refers to the ability of a particular substance to elicit an immune response.
  • host cell refers to a cell in which a vector can be propagated and its DNA can be expressed, which cell can be a prokaryotic cell or a eukaryotic cell.
  • the term also includes any progeny of the subject host cell. It should be understood that not all progeny are identical to the parental cell, and such progeny are included due to the possibility of mutation during replication.
  • Host cells include prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by an adenine, or both A position in each of the polypeptides is occupied by a lysine)
  • the molecules are identical at that position.
  • the "percent identity” between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6).
  • comparisons are made when two sequences are aligned for maximum identity.
  • Align program DNAstar, Inc.
  • Needleman SB and Wunsch CD 1970, J Mol Biol, 48:443-453.
  • mutant refers to the substitution, deletion or insertion of one or more nucleotides or amino acids as compared to the native nucleic acid or polypeptide (ie, a reference sequence that can be used to define wild-type) .
  • effector functions refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), and which vary among antibody isotypes.
  • antibody effector functions include, but are not limited to: Fc receptor binding affinity, ADCC, ADCP, CDC, downregulation of cell surface receptors (eg, B cell receptors), B cell activation, cytokine secretion, antibodies and antigens /half-life/clearance of antibody complexes, etc.
  • Methods of altering the effector function of antibodies are known in the art, eg, by introducing mutations in the Fc region.
  • pharmaceutically acceptable carrier and/or excipient and/or stabilizer refers to a carrier and/or excipient and/or that is pharmacologically and/or physiologically compatible with the subject and the active ingredient or stabilizers, which are not toxic to the cells or mammals to which they are exposed at the doses and concentrations employed. Including but not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate salts and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, glucan, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate) and the like.
  • sugars such as sorbitol, mannitol, starch, sucrose , lactose, glucan, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dry whey, albumin or casein
  • degradation products such as lactalbumin hydrolyzate
  • prevention refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (eg, tumor or infection) in a subject or to minimize its effects if it occurs.
  • a disease or disorder or symptom eg, tumor or infection
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell, and thereby replicate together with the host genome.
  • vectors are capable of directing the expression of the genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
  • expression vectors useful in recombinant DNA technology typically exist in the form of plasmids.
  • viral vectors eg, replication-defective retroviruses, adenoviruses and adeno-associated viruses
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical outcomes include, but are not limited to, reduced rate of disease progression, improved or lessened disease state, and regression or improved prognosis, whether detectable or undetectable.
  • the amount of therapeutic agent effective to relieve symptoms of any particular disease may vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to elicit the desired response in the subject. Relief of disease symptoms can be assessed by any clinical measure commonly used by physicians or other skilled health care providers to assess the severity or progressive state of the symptoms.
  • the antibodies of the invention can be used as therapeutic agents. These agents can generally be used to treat or prevent the novel coronavirus pneumonia (COVID-19) in a subject, increase vaccine efficacy, or improve innate immune responses.
  • An antibody preparation preferably one with high specificity and high affinity for its target antigen S protein, is administered to a subject and generally has an effect due to its binding to the target. Administration of antibodies can eliminate or inhibit or interfere with the activity of the SARS-CoV-2 coronavirus S protein. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • variable region sequences of antibodies which retain the ability to bind target protein sequences.
  • Such peptides can be chemically synthesized and/or prepared by recombinant DNA techniques (see, eg, Marasco WA et al, 1993, Proc Natl Acad Sci USA, 90:7889-7893).
  • the antibody or fragment thereof that specifically binds to the SARS-CoV-2 coronavirus S protein of the present invention can be administered in the form of a pharmaceutical composition.
  • a formulation may contain more than one active compound, preferably those having complementary activities that do not adversely affect each other, as desired for the particular indication being treated.
  • the composition may contain an agent that enhances its function.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention can be used in an immunoassay for detection or quantification of SARS-CoV-2 virus.
  • the immunoassay method itself is known, and any known immunoassay method can be used. That is, if it is classified by the measurement format, there are sandwich methods, competition methods, aggregation methods, Western blotting methods, etc., and if they are classified by the labels used, there are fluorescence methods, enzymatic methods, radioactive methods, biotin methods, etc. can be used. Diagnosis can also be made by immunohistostaining.
  • the method for labeling the antibody itself is known, and any known method can be used.
  • the sandwich method is to fix the antibody or antigen-binding fragment of the present invention as the first antibody on a solid phase, react with the biological sample to be tested, rinse After that, it was reacted with the secondary antibody, and after washing, the secondary antibody bound to the solid phase was measured.
  • the second antibody bound to the solid phase can be measured by labeling the second antibody with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like.
  • the first antibody and the second antibody can also be substituted in the above description.
  • the antibody or antigen-binding fragment thereof of the present invention is immobilized on particles such as latex, reacted with a sample, and the absorbance is measured.
  • a plurality of standard substances of known concentration are measured, and a standard curve is prepared according to the relationship between the measured labeled amount and the content of the standard substance. Quantification of SARS-CoV-2 viral antigens in samples.
  • the biological sample to be supplied to the immunoassay method is not particularly limited as long as it contains the S protein of the SARS-CoV-2 virus.
  • it can be derived from human and animal serum, plasma, and whole blood, as well as nasal cavity.
  • Swabs nasal swabs
  • nasal aspiration fluids nasal aspiration fluids
  • throat swabs pharyngeal swabs
  • other body fluid extracts saliva, respiratory secretions, urine, feces, cell or tissue homogenate, etc.
  • the antibody can be used as at least one of a solid-phase antibody and a labeled antibody to prepare a SARS-CoV-2 virus immunoassay reagent.
  • solid phases used in conventional immunoassays such as ELISA plates, latex, gelatin particles, magnetic particles, polystyrene, glass, etc., can be used as the solid phase bound to the above-mentioned monoclonal antibody, beads, etc.
  • Insoluble carriers such as substrates for transporting liquids, etc.
  • labeled antibodies can be prepared by labeling antibodies with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances, and the like.
  • reagents for use in enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, and the like can be prepared.
  • assay reagents are reagents for measuring the target antigen in a sample by a sandwich method or a competitive binding assay.
  • the reagent for immunoassay by the sandwich method for example, two kinds of monoclonal antibodies of the present invention are prepared, one of which is the labeled antibody and the other is the solid-phase antibody bound to the solid phase.
  • a sample containing an antigen to be measured is reacted with the solid-phase antibody, and then a labeled antibody (secondary antibody) is reacted with the antigen captured on the solid-phase antibody to detect the presence of the label bound to the insoluble carrier. or activity, immunoassays can be performed.
  • a sample containing the antigen to be assayed is reacted with a solid-phase antibody, and then a labeled antibody (secondary antibody) is reacted with the antigen captured on the solid-phase antibody, and the presence of the label bound to the insoluble carrier or the Activity, ie quantification of the amount of antigen to be assayed by the amount of labeled antibody, allows immunometric measurements to be performed.
  • a monoclonal antibody can be used as the solid-phase antibody and the labeled antibody (for example, when the antigen is a polymer), but it is generally preferable to use two different epitopes that can respectively recognize the antigen to be assayed. 2 or more antibodies.
  • any solid-phase antibody and labeled antibody can be selected and used in combination from two or more monoclonal antibodies.
  • an immunoassay reagent using a competitive binding assay for example, a certain amount of viral antigens labeled with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances and the like can be prepared.
  • a competitive reaction can be performed with, for example, a sample containing a certain amount of the monoclonal antibody of the present invention, the labeled viral antigen described above, and the antigen to be assayed, and the amount of labeled viral antigen bound or unbound to the antibody to be assayed.
  • the amount of antigen in the sample is quantified to perform an immunoassay.
  • the above-mentioned labeled anti-SARS-CoV-2 virus monoclonal antibody can be prepared by binding the anti-SARS-CoV-2 virus monoclonal antibody to a labeled substance.
  • Labels can be enzymes, colloidal metal particles, colored latex particles, fluorescent latex particles, luminescent substances, fluorescent substances, and the like.
  • Enzymes can be various enzymes used in enzyme-linked immunoassays (EIA), such as alkaline phosphatase, peroxidase, ⁇ -D-galactosidase, etc.; colloidal metal particles such as colloidal gold can be used Granules, colloidal selenium granules, etc.
  • the method of binding the label to the anti-SARS-CoV-2 virus monoclonal antibody can utilize a known method for generating a covalent bond or a non-covalent bond.
  • the methods of combining are, for example, the glutaraldehyde method, the periodate method, the maleimide method, the dithiodipyridine method, the method using various cross-linking agents, etc. 31, 37-45 (1985)).
  • a cross-linking agent for example, N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidohexanoic acid can be used as the cross-linking agent.
  • the functional groups present in the antibody can be used depending on the use of functional groups.
  • functional groups such as thiol group, amino group, carboxyl group, and hydroxyl group can be introduced into the antibody according to conventional methods, and then the above-mentioned binding method can be used.
  • the functional group is combined with the label, thereby preparing a labeled anti-SARS-CoV-2 virus monoclonal antibody.
  • the substrate various chromogenic substrates, fluorescent substrates, luminescent substrates, and the like can be used corresponding to the enzymes of the labels and shown below.
  • Chromogenic substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3',5, in combination with hydrogen peroxide 5'-Tetramethylbenzidine (TMB), Diaminobenzidine (DAB) for peroxidase; 5-Bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolyl sodium phosphate (BCIP ⁇ Na) was used for alkaline phosphatase.
  • Fluorescent substrates 4-methylumbelliferyl phenyl phosphate (4-MUP) for alkaline phosphatase; 4-methylumbelliferyl phenyl- ⁇ -D-galactoside (4MUG) for in ⁇ -D-galactosidase.
  • 4-MUP 4-methylumbelliferyl phenyl phosphate
  • 4-MUG 4-methylumbelliferyl phenyl- ⁇ -D-galactoside
  • Luminescent substrate 3-(2'-Spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane ⁇ 2 Sodium salt (AMPPD) for alkaline phosphatase; 3-(2'-spiroadamantane)-4-methoxy-4-(3"- ⁇ -D-galactopyranosyl)phenyl-1, 2-Dioxetane (AMGPD) was used for ⁇ -D-galactosidase; luminol, isoluminol obtained in combination with hydrogen peroxide were used for peroxidase.
  • AMPPD Sodium salt
  • AMGPD 2-Dioxetane
  • Diagnosis of SARS-CoV-2 virus infection can be performed by assaying various biological samples from humans or animals using the monoclonal antibody of the present invention against the S protein of SARS-CoV-2 virus.
  • conservative modification is intended to mean that an amino acid modification does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions refer to the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are well described in the art.
  • These families include those with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine) , proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g.
  • basic side chains eg lysine, arginine, histidine
  • acidic side chains eg aspartic acid, glutamic acid
  • uncharged polar side chains eg glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryp
  • one or more amino acid residues in the CDR regions of the antibodies of the invention can be replaced with other amino acid residues from the same family of side chains.
  • the antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application can be applied to prepare a pharmaceutical composition or a sterile composition, for example, the antibody is mixed with a pharmaceutically acceptable carrier, excipient or stabilizer.
  • a pharmaceutical composition may include one or a combination (eg, two or more different) antibodies of the invention.
  • a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on a target antigen.
  • Formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions.
  • pharmaceutically acceptable means that the molecular entity, molecular fragment or composition does not produce an adverse, allergic or other untoward reaction when properly administered to an animal or human.
  • Specific examples of some substances that may be pharmaceutically acceptable carriers or components thereof include sugars (eg, lactose), starch, cellulose and derivatives thereof, vegetable oils, gelatin, polyols (eg, propylene glycol), alginic acid, and the like.
  • the antibodies of the present invention or nucleic acids or polynucleotides encoding the antibodies of the present application may be used alone or in combination with one or more other therapeutic agents, such as vaccines.
  • Figure 3-1 Determination of the binding ability of purified murine antibodies S1B-73-3, S1B-34-4 and S1B-8-2 to SARS-CoV-2 S1.
  • Figure 3-2 Determination of the binding ability of purified murine antibodies S1B-48-2 and S1B-64-2 to SARS-CoV-2 S1.
  • Figure 3-3 Determination of the binding ability of purified murine antibody S1B-91-3 to SARS-CoV-2 S1.
  • Figure 3-4 Determination of the binding ability of purified murine antibody S1B-82-5 to SARS-CoV-2 S1.
  • Figure 3-5 Determination of the binding ability of purified murine antibody S1B-30-3 to SARS-CoV-2 S1.
  • Figure 3-6 Determination of the binding ability of purified murine antibody S1B-14-3 to SARS-CoV-2 S1.
  • Figure 3-7 Determination of the binding ability of purified murine antibodies ST-10-4 and ST-35-4 to S-CoV-2 ST.
  • Figure 4-1 Cross-reactivity determination of purified murine antibodies S1B-73-3, S1B-34-4 and S1B-8-2 with SARS-CoV S.
  • Figure 4- Cross-reactivity determination of purified murine antibodies S1B-48-2 and S1B-64-2 with SARS-CoV S.
  • Figure 4-3 Cross-reactivity determination of purified murine antibody S1B-91-3 with SARS-CoV S.
  • Figure 4-4 Cross-reactivity determination of purified murine antibody S1B-82-5 with SARS-CoV S.
  • Figure 4-5 Cross-reactivity determination of purified murine antibody S1B-30-3 with SARS-CoV S.
  • Figure 5-1 The ability of murine antibodies S1B-73-3, S1B-34-4 and S1B-8-2 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
  • Figure 5-2 The ability of murine antibodies S1B-48-2 and S1B-64-2 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
  • Figure 5-3 The ability of mouse monoclonal antibody S1B-91-3 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
  • Figure 5-4 The ability of mouse monoclonal antibody S1B-82-5 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
  • Figure 5-5 The ability of mouse monoclonal antibody S1B-30-3 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
  • Figure 5-6 The ability of mouse monoclonal antibody S1B-14-3 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
  • FIG. 6-1 Murine antibodies S1B-73-3, S1B-48-2, S1B-91-3, S1B-82-5, S1B-30-3 and blocking spike S protein and 293T-ACE2 cells combine.
  • FIG. 6-2 Murine antibody S1B-14-3 blocks the binding of spike S protein to 293T-ACE2 cells.
  • FIG. 6-3 Murine antibodies ST-10-4 and ST-35-4 block the binding of spike S protein to 293T-ACE2 cells.
  • FIG. 7 Murine antibodies S1B-73-3, S1B-48-2, S1B-82-5, S1B-91-3/RBD molecular docking model, ACE2/RBD structure (PDB 6M0J) and antibody CR3022/RBD structure ( PDB 6W41) three structures superimposed.
  • FIG. 1 Murine antibodies S1B-30-3, S1B-14-3, ST-10-4, ST-35-4/RBD molecular docking model, ACE2/RBD structure (PDB 6M0J) and antibody CR3022/RBD structure ( PDB 6W41) three structural overlays.
  • Figure 9-1 The results of the amino acid sequence alignment of the heavy chain variable region of the humanized antibody hS1B-73-3 and its parent murine antibody.
  • Figure 9-2 The comparison result of the amino acid sequence of the light chain variable region of the humanized antibody hS1B-73-3 and its parental murine antibody.
  • Figure 10-1 The results of the alignment of the amino acid sequences of the heavy chain variable region of the humanized antibody hS1B-48-2 and its parental murine antibody.
  • Figure 10-2 The comparison results of the amino acid sequence of the light chain variable region of the humanized antibody hS1B-48-2 and its parent murine antibody.
  • Figure 11-1 The results of the alignment of the amino acid sequences of the heavy chain variable region of the humanized antibody hS1B-91-3 and its parent murine antibody.
  • Figure 11-2 The comparison results of the amino acid sequence of the light chain variable region of the humanized antibody hS1B-91-3 and its parental murine antibody.
  • Figure 12-1 The results of the alignment of the amino acid sequences of the heavy chain variable region of the humanized antibody hS1B-82-5 and its parent murine antibody.
  • Figure 12-2 The comparison results of the amino acid sequence of the light chain variable region of the humanized antibody hS1B-82-5 and its parental murine antibody.
  • Figure 13-1 The results of the alignment of the amino acid sequences of the heavy chain variable region of the humanized antibody hS1B-30-3 and its parent murine antibody.
  • Figure 13-2 The comparison results of the amino acid sequence of the light chain variable region of the humanized antibody hS1B-30-3 and its parental murine antibody.
  • Figure 14-1 The results of the amino acid sequence alignment of the heavy chain variable regions of the humanized antibodies hS1B-14-3-1 and hS1B-14-3-2 and their parental murine antibodies.
  • Figure 14-2 The amino acid sequence alignment results of the light chain variable regions of the humanized antibodies hS1B-14-3-1 and hS1B-14-3-2 and their parental murine antibodies.
  • Figure 15-1 Indirect ELISA method to determine the binding ability of humanized antibody hS1B-73-3 to SARS-CoV-2 S trimer antigen.
  • Figure 15-2 Indirect ELISA method to determine the binding ability of humanized antibody hS1B-48-2 to SARS-CoV-2 S trimer antigen.
  • Figure 15-3 Indirect ELISA method to determine the binding ability of humanized antibody hS1B-91-3 to SARS-CoV-2 S trimer antigen.
  • Figure 15-4 Indirect ELISA method to determine the binding ability of humanized antibody hS1B-82-5 to SARS-CoV-2 S trimer antigen.
  • Figure 15-5 Indirect ELISA method to determine the binding ability of humanized antibody hS1B-30-3 to SARS-CoV-2 S trimer antigen.
  • Figure 16-1 Competitive ELISA assay to determine the ability of humanized antibody hS1B-73-3 to block the binding of SARS-CoV-2 S trimer to human ACE2.
  • Figure 16-2 Competitive ELISA assay to determine the ability of humanized antibody hS1B-48-2 to block the binding of SARS-CoV-2 S trimer to human ACE2.
  • Figure 16-3 Competitive ELISA assay to determine the ability of humanized antibody hS1B-91-3 to block the binding of SARS-CoV-2 S trimer to human ACE2.
  • Figure 16-4 Competitive ELISA assay to determine the ability of humanized antibody hS1B-82-5 to block the binding of SARS-CoV-2 S trimer to human ACE2.
  • Figure 16-5 Competitive ELISA assay to determine the ability of humanized antibody hS1B-30-3 to block the binding of SARS-CoV-2 S trimer to human ACE2.
  • FIG. 17-1 Humanized antibodies hS1B-73-3, hS1B-48-2, hS1B-91-3, hS1B-82-5, hS1B-30-3 block the interaction of spike S protein with 293T-ACE2 cells combine.
  • FIG 17-2 Humanized antibody hS1B-14-3 blocks the binding of spike S protein to 293T-ACE2 cells.
  • Figure 18-1 Measurement of the activity of humanized antibody hS1B-91-3 for inhibiting pseudovirus in vitro.
  • Figure 18-2 Measurement of the activity of humanized antibody hS1B-30-3 for inhibiting pseudovirus in vitro.
  • Figure 18-3 In vitro activity assay of humanized antibodies hS1B-48-2, hS1B-82-5 and hS1B-14-3 to inhibit pseudovirus mutants.
  • FIG. 19-1 Map of published antibody binding to RBD structures, and the spatial positions of RBD residues K417, E484 and N501.
  • 1A The structure of LY-CoV555/RBD is taken from PDB 7KMG;
  • 1B The structure of LY-CoV016/RBD is taken from PDB 7C01;
  • 1C The structure of REGN-10933/RBD is taken from PDB 6XDG;
  • 1D The structure of COV2-2196/RBD is taken from COV2 -2196 structure modeling, and computational simulation of molecular docking with the RBD structure (PDB 6M0J) by ZDOCK software.
  • FIG. 19-2 Binding diagram of ACE2, antibody hS1B-48-2 or ST-35-4 and RBD structure.
  • 2A ACE2/RBD structure was taken from PDB 6M0J;
  • 2B Antibody hS1B-48-2/RBD structure was derived from hS1B-48-2 structural modeling and molecular docking computational simulations with the RBD structure (PDB 6M0J) by ZDOCK software ;
  • 2C Antibody ST-35-4/RBD structure is derived from ST-35-4 structural modeling and computational simulation of molecular docking with the RBD structure (PDB 6MOJ) by ZDOCK software.
  • Antigen preparation SARS-CoV-2 antigen preparation process: According to the full-length amino acid sequence of the new coronavirus S protein published in Uniprot (Uniprot Entry P0DTC2 ), select the 326-685aa segment (S1 protein, marked as S1B), and among them The 16-1213aa segment (S trimer, labeled ST) was used as the antigen for screening antibodies in this example.
  • the coding genes of S1B and ST were artificially modified and optimized, and the eukaryotic expression vectors pcDNA3.1-S1B and pcDNA3.1-ST of the target gene were constructed according to conventional molecular biology methods.
  • the correctly sequenced recombinant expression plasmid was transfected into CHO cells, and expressed and purified according to conventional methods, and the purified antigen was obtained for immunization.
  • mice with the highest anti-SARS-CoV-2 antibody titers and the highest levels of neutralizing antibodies were then boosted 3 days before fusion. After 3 days, the mice were sacrificed and the spleens of the mice were removed to fuse with the mouse myeloma Sp2/0 cell line.
  • high-throughput ELISA was used to detect the ability of antibodies in the supernatant to compete with HRP-labeled human ACE2 for binding to SARS-CoV-2, so as to screen out the positive wells that compete with human ACE2 (see Example 2.3 for the method).
  • the above-mentioned fusion cells in the well containing the monoclonal antibody that can inhibit the binding of HRP-labeled ACE2 to SARS-CoV-2 were then subcloned, and the hybridoma cell line expressing the high-affinity mouse monoclonal antibody was also screened by competitive ELISA method.
  • Antibodies were purified on a protein A column and the monoclonal antibody eluate was dialyzed against 150 mM NaCl. Filter-sterilize the dialyzed solution through a 0.2 ⁇ m filter to obtain the purified murine monoclonal antibodies to be tested S1B-73-3, S1B-48-2, S1B-91-3, S1B-82-5, S1B-30- 3. S1B-14-3, S1B-34-4, S1B-8-2, S1B-64-2, ST-10-4, ST-35-4.
  • ELISA plates were separately coated with SARS-CoV-2 S1 and S trimer (ACRO Biosystems) overnight at room temperature. The coating solution was discarded, blocked with skim milk powder dissolved in PBS buffer for 1 h, and the plate was washed 3-4 times with PBST (PBS containing 0.05% Tween-20, pH 7.4). Then, 100 ⁇ L of purified murine antibody against SARS-CoV-2 to be tested and human antibody CR3022 against SARS-CoV and SARS-CoV-2 S1 (the variable region of the heavy chain and the variable region of the light chain) were added to each well.
  • PBST PBS containing 0.05% Tween-20, pH 7.4
  • the binding ability of the obtained murine monoclonal antibodies to SARS-CoV S was determined.
  • SARS-CoV S protein (ACRO Biosystems) was diluted to 0.1 ⁇ g/mL with PBS buffer, added to a 96-well plate in a volume of 100 ⁇ L/well, and placed at 4°C for 16-20 h. Aspirate the supernatant, wash the plate once with PBST buffer, add 200 ⁇ L of PBST containing 1% nonfat dry milk (PBST/1% nonfat dry milk) to each well, and incubate at room temperature for 1 h to block. Remove the blocking solution, wash the plate three times with PBST buffer, add the above anti-SARS-CoV-2 mouse antibody, 100 ⁇ L/well, and incubate at room temperature for 1.5 h.
  • PBST/1% nonfat dry milk 1% nonfat dry milk
  • the reaction system was removed, and after washing the plate three times with PBST, 50 ⁇ L/well of HRP-labeled goat anti-mouse IgG secondary antibody (The Jackson Laboratory) diluted at 1:4000 was added, and incubated at room temperature for 1 h. After washing the plate three times with PBST, 100 ⁇ L of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50 ⁇ L of 0.2 M H 2 SO 4 was added to each well to stop the reaction, and the OD value was read at dual wavelengths 450/620 nm with a microplate reader.
  • the mouse-derived antibody S1B-82-5 has strong cross-reaction with SARS-CoV, while S1B-73-3, S1B-48-2, S1B-91-3 , S1B-30-3, S1B-34-4, S1B-8-2, S1B-64-2 and SARS-CoV S do not cross.
  • SARS-CoV-2 S1 protein (ACRO Biosystems) was diluted to 0.1 ⁇ g/mL with PBS buffer and added to 96-well plates at 100 ⁇ L/well, overnight at room temperature. The coating solution was discarded, 200 ⁇ L of PBST/1% nonfat dry milk was added to each well, and the cells were incubated for 1 h at room temperature for blocking. The blocking solution was removed, and the plate was washed three times with PBST buffer, and then 100 ⁇ L of the mixture of horseradish peroxidase (HRP)-labeled ACE2 and the mouse antibody to be tested was added to each well. PBST was used as blank control.
  • HRP horseradish peroxidase
  • S1B-34-4, S1B-8-2, and S1B-64-2 can compete with ACE2 for binding to SARS-CoV-2 S1, that is, by blocking the combination of SARS-CoV-2 S1 and ACE2.
  • the Luciferase luminescence value RLU was detected by chemiluminescence, and the pseudovirus inhibition rate of the antibody to be tested was calculated according to the RLU reading value, and the neutralization of the antibody to be tested was evaluated. Effect.
  • the genome of the new coronavirus Spike pseudovirus encodes firefly luciferase. When the viral genome is integrated into cells, the expression and activity of firefly luciferase is proportional to the number of transduced cells. In contrast to true viruses, pseudoviruses can only infect cells once.
  • the specific steps are as follows for the inhibition experiment of murine antibody on the infection of wild-type (WT) pseudovirus or mutant pseudovirus (B.1.1.7, B.1.351 or E484K).
  • WT wild-type
  • B.1.351 or E484K mutant pseudovirus
  • 293T-ACE2 cells (Shanghai Yisheng Biotechnology) were cultured to logarithmic growth phase with medium DMEM+10% FBS, seeded in 384-well white plates at 3000 cells/well, and cultured at 37°C, 5% CO 2 Incubate overnight.
  • Antibody samples to be tested S1B-14-3, ST-10-4 or ST-35-4 were diluted with DMEM containing 10% FBS, the initial concentration was 10 ⁇ g/mL, 5 times dilution, 9 gradients; wild type (WT ) Pseudovirus or mutant pseudovirus (B.1.1.7, B.1.351 or E484K) (Jiman Bio) was taken out from -80°C, thawed at 4°C, and the reconstituted pseudovirus was diluted 125 times as a work The diluted test antibody and pseudovirus working solution were added to the 96-well U-bottom plate at 50 ⁇ L/well and mixed well, and pre-incubated at 37°C for 30 minutes; then 20 ⁇ L/well was added to the 384-well white plate where the cells were plated on the previous day.
  • WT wild type Pseudovirus or mutant pseudovirus
  • positive control group pseudovirus working solution and DMEM medium containing 10% FBS were added to 384-well white plate at 10 ⁇ L/well, respectively.
  • Negative control group DMEM medium containing 10% FBS was added to 384-well white plate at 20 ⁇ L/well, and 4 wells were set up. After 24 hours, add DMEM medium containing 10% FBS at 30 ⁇ L/well, and continue to culture in the cell incubator for 24 hours; carefully remove the supernatant with a pipette, and add freshly prepared luciferase at 30 ⁇ L/well.
  • Inhibition rate (%) [1-(sample RLU signal value-negative control RLU signal value)/(positive control RLU signal value-negative control RLU signal value)] ⁇ 100.
  • the software GraphPad Prism 6 was used for analysis to obtain the antibody dose-response curve, and the non-linear regression curve was used to calculate the median inhibitory concentration (IC 50 ).
  • Table 2-1 to Table 2-4 show the experimental results of anti-SARS-CoV-2 murine antibodies inhibiting pseudovirus infection in vitro.
  • S1B-14-3 can well inhibit the new coronavirus pseudovirus
  • ST-10-4 or ST-35-4 can well inhibit wild type, B.1.1.7, B.1.351 or E484K SARS-CoV-2 infection
  • the neutralization effect of ST-10-4 or ST-35-4 on B.1.1.7 or B.1.351 mutants was significantly better than that of wild type, ST-35-4 in B. Better neutralization on .1.351.
  • the antibody to be tested was determined by FACS to compete to block the binding of spike S-mouse Fc fusion protein (S-mFc) to 293T-ACE2 cells, and the binding on the cells was detected by fluorescently labeled goat anti-mouse secondary antibody. The average fluorescence intensity of S-mFc was calculated, IC 50 of the antibody to be tested blocking the binding of S protein to ACE2 on the cell surface was calculated, and the blocking effect of the antibody to be tested was evaluated.
  • S-mFc spike S-mouse Fc fusion protein
  • S-mFc antigen preparation process According to the full-length amino acid sequence of the new coronavirus S protein published in Uniprot (Uniprot Entry P0DTC2), select the full-length segment of the S protein, and connect the mouse IgG2a Fc fragment (Uniprot Entry P01863 (107-330aa)) to construct the antigen S-mFc fusion protein used as the neutralizing antibody evaluated in this example.
  • the encoding gene of S-mFc was artificially modified and optimized, and the eukaryotic expression vector pcDNA3.1-S-mFc of the target gene was constructed according to conventional molecular biology methods.
  • the expression plasmid was transfected into CHO cells, and expressed and purified according to conventional methods.
  • the software GraphPad Prism 6 was used for analysis, and the IC50 value of the anti-SARS-CoV-2 mouse antibody blocking the binding of S-mFc protein to 293T-ACE2 cells was calculated.
  • anti-SARS-CoV-2 murine antibodies S1B-73-3, S1B-91-3, S1B-82-5, S1B-30 -3, S1B-48-2, S1B-14-3, ST-10-4 and ST-35-4 can compete well to block the binding of S protein to ACE2, the IC 50 is between 5-20ng/mL Among them, the blocking effect of mouse anti-S1B-73-3 was the best, followed by mouse anti-S1B-91-3, S1B-30-3, S1B-48-2, ST-35-4, and The blocking effect of S1B-82-5, S1B-14-3 and ST-10-4 was weak.
  • Example 4 Using computer molecular docking technology to evaluate the effect of anti-SARS-CoV-2 murine antibody on the binding ability between RBD/ACE2
  • the computer software Discovery Studio was used to model the structures of mouse antibodies S1B-73-3, S1B-15-5, S1B-30-3, S1B-48-2, S1B-82-5 and S1B-91-3, and The molecular docking spatial conformation of these murine antibodies and their antigen RBD domains was simulated to predict the binding site of the S protein murine antibody on the RBD domain, and to evaluate the effect of the antibodies on the binding ability between RBD/ACE2.
  • the three-dimensional structural model of the mouse antibody was constructed.
  • the modeling is carried out in three steps: 1. Searching for three-dimensional structural templates with high amino acid sequence similarity to the variable regions of the light chain and heavy chain of murine antibodies, respectively. Search for a three-dimensional structural template with high amino acid sequence similarity with the overall murine antibody variable region (light and heavy chains together), so as to determine the relative orientation of the light and heavy chains in the murine antibody variable region; 2. Apply step 1 The obtained 3 structural model templates and the amino acid sequences of the light and heavy chains of the variable region of the murine antibody are used to construct the structure model of the framework region of the murine antibody; 3. On the basis of step 2, the structure model of the six CDR loop regions is constructed.
  • the RBD structure model in the molecular docking simulation calculation adopts the protein database High-resolution RBD structure (PDB ID 6MOJ).
  • PBD ID 6MOJ protein database High-resolution RBD structure
  • the side chain conformation of the F486 residue in 6M0J was adjusted to be consistent with that in 6W41, the rotamer1 conformation. This conformation has the highest occupancy, and the side chain of F486 does not exist in this conformation due to the binding of ACE2 in RBD in 6M0J.
  • the software for molecular docking was ZDOCK software in the Discovery Studio software package. The parameters used in the molecular docking simulation experiment are all default values.
  • the murine antibody was used as the molecular docking receptor, and the RBD was used as the molecular docking ligand.
  • the receptor blocked residues are selected from the variable region amino acids which are far from the CDR region and whose spatial position is in the opposite direction of the CDR region.
  • the receptor binding site residues were selected as the top amino acid of the HCDR3loop exposed on the surface of the protein.
  • Murine Antibodies S1B-73-3, S1B-48-2, S1B-82-5, S1B-91-3, S1B-30-3, S1B-14-3, ST-10-4 and ST-35-4 See Figure 7 and Figure 8 for the results of molecular docking with RBD.
  • ACE2 (PDB 6MOJ, Lan J et al, 2020, Nature, 581:215-220) and antibody CR3022 structure (PDB 6W41, Yuan M et al, 2020, Science, 368:630-633) were introduced by superposition of RBD structures.
  • Molecular docking results showed that murine antibodies S1B-73-3, S1B-48-2, S1B-82-5, S1B-91-3, S1B-30-3, S1B-14-3, ST-10-4 and ST-35-4 competes with ACE2 for binding to RBD, and can block the binding between RBD and ACE2, providing a rational explanation at the molecular level for these antibodies to inhibit the infection of host cells by SARS-CoV-2 virus.
  • CDR grafting We use the CDR grafting method to humanize the mouse antibody.
  • the basic principle of CDR grafting is to graft the CDR region of mouse anti-antibody onto human antibody template, and at the same time introduce several or some key mouse anti-FR region residues which are important for stable CDR conformation and antigen-antibody binding.
  • human antibody template backmutations
  • PI isoelectric point
  • aggregation hydrophobic aggregation
  • PTM post-translational modification
  • the specific process of antibody humanization is as follows. Search the human antibody germline database of the IMGT website (IMGT human antibody germline database, http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) to obtain human antibody templates with high similarity to mouse antibodies (Table 4).
  • the mouse anti and human antibody templates were annotated with CDR regions using Discovery Studio, and the CDR regions were defined according to the Kabat or IMGT protocols (Table 5).
  • the six CDR regions of the human antibody template were replaced with the six CDR regions of the mouse antibody, respectively.
  • Each individual CDR region of the six CDR regions grafted can be an amino acid region as defined by Kabat, or an amino acid region as defined by IMGT.
  • the key mouse anti-FR region amino acids that stabilize antibody CDR region conformation and are important for antigen-antibody binding include four types of amino acid residues: 1) CDR region Amino acids buried under the surface of the antibody; 2) CDR regions 3) the interfacial amino acids between the antibody light and heavy chain domains; and 4) the vernier zone residues that stabilize the conformation of the antibody CDR regions (Foote J and Winter G, 1992, J Mol Biol, 224 :487-499).
  • the above four key murine anti-FR region residues were determined by establishing a three-dimensional structural model of murine anti-FR.
  • amino acids important for maintaining CDR conformation and antigen-antibody binding are selected through three-dimensional structural analysis, and amino acid transplantation or substitution from the mouse antibody to the human template is performed. Then, the isoelectric point, hydrophobic aggregation, post-translational modification and immunogenicity were further calculated for the humanized antibody generated after the transplantation of 4 types of amino acids, and the problematic amino acid was mutated to obtain the final humanized antibody sequence (Table 6) .
  • Figures 9-1 to 14-2 show the alignment results of the amino acid sequences of the heavy chain variable region and the light chain variable region of the above-mentioned humanized antibody and its parental murine antibody, respectively.
  • the VH and VL sequences shown in Table 6 were combined with the antibody heavy chain constant region (preferably from human IgG1, IgG2 or IgG4) and light chain constant
  • the sequence of the region preferably from the human kappa light chain, the amino acid sequence is shown in SEQ ID NO: 95
  • the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgGl (amino acid sequence set forth in SEQ ID NO: 96).
  • the humanized antibody molecule comprises the heavy chain constant region of human IgG1 containing the M252Y, S254T, T256E and M428L mutations according to EU numbering (amino acid sequence set forth in SEQ ID NO: 190).
  • the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG2 (amino acid sequence set forth in SEQ ID NO: 99).
  • use a modified human IgG2 constant region sequence in one embodiment, the humanized antibody molecule comprises a human IgG2 modified in the hinge region according to EU numbering (e.g. deletion of ERKCC, amino acid sequence shown in SEQ ID NO: 100) ).
  • the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG4 (amino acid sequence set forth in SEQ ID NO: 109). Or use a modified human IgG4 constant region sequence; in one embodiment, the humanized antibody molecule comprises a human IgG4 (amino acid sequence such as SEQ ID NO: 228) mutated (e.g., S to P) according to EU numbering. 110).
  • the cDNA encoding the heavy chain and light chain of the humanized antibody obtained in the above method is inserted into PcDNA3.1 or its derivative plasmid, or other eukaryotic expression vector to construct a humanized antibody expression vector.
  • the vector plasmid used should contain the cytomegalovirus early gene promoter-enhancer required for high-level expression in mammalian cells.
  • the vector plasmid contains a selectable marker gene that confers ampicillin resistance in bacteria and G418 resistance in mammalian cells.
  • the vector plasmid contains the DHFR gene, and in a suitable host cell, the humanized antibody gene and the DHFR gene can be co-amplified with methotrexate (Methotrexate, MTX, Sigma) (for example, see patent CN103333917B).
  • methotrexate Metalhotrexate, MTX, Sigma
  • the recombinant expression vector plasmids constructed above are transfected into mammalian host cell lines to express humanized antibodies.
  • the preferred host cell line is dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells (see, eg, US Pat. No. 4,818,679 to Chasin, L. et al.).
  • DHFR dihydrofolate reductase
  • CHO Chinese hamster ovary
  • the secretion rate of each cell line was measured by the methods of limiting dilution subcloning transfectants and ELISA, and the cell line expressing the humanized antibody at a high level was selected. Conditioned media of humanized antibodies are collected for determination of their in vitro and in vivo biological activities.
  • nucleotide sequences encoding the heavy chain and light chain of the humanized antibody shown in Table 8 are inserted into the expression vector constructed above. Cultivate and purify each target antibody.
  • Antibody number HC amino acid sequence LC amino acid sequence HC nucleotide sequence LC nucleotide sequence hS1B-73-3 SEQ ID NO: 144 SEQ ID NO: 145 SEQ ID NO: 173 SEQ ID NO: 174 hS1B-48-2 SEQ ID NO: 149 SEQ ID NO: 150 SEQ ID NO: 175 SEQ ID NO: 176 hS1B-91-3 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 177 SEQ ID NO: 178 hS1B-82-5 SEQ ID NO: 59 SEQ ID NO: 60 SEQ ID NO: 179 SEQ ID NO: 180 hS1B-30-3 SEQ ID NO: 89 SEQ ID NO: 90 SEQ ID NO: 181 SEQ ID NO: 182 hS1B-14-3-1 SEQ ID NO: 156 SEQ ID NO: 157 SEQ ID NO: 183 SEQ ID NO: 184 hST-10-4 SEQ ID NO: 139 SEQ ID NO:
  • SARS-CoV-2 S trimer protein (ACRO BioSystem) was diluted to 0.1 ⁇ g/ml with PBS buffer and added to 96-well plates at 100 ⁇ l/well, overnight at room temperature. The coating solution was discarded, 200 ⁇ l of PBST/1% nonfat dry milk was added to each well, and the cells were incubated for 1 h at room temperature for blocking. Remove the blocking solution, wash the plate three times with PBST buffer, and then add 100 ⁇ l of horseradish peroxidase (HRP)-labeled hACE2-Fc and humanized antibody hS1B-73-3 and anti-SARS- Mixture of receptor hACE2-Fc for CoV-2 S1 RBD. PBST was used as blank control.
  • HRP horseradish peroxidase
  • the results are shown in Table 9.
  • the above humanized antibody can compete with hACE2 for binding to SARS-CoV-2 S1, that is, it functions by blocking the binding of SARS-CoV-2 S1 and hACE2.
  • Example 2.4 The experimental methods for the determination of kinetic constants and affinity equilibrium dissociation constants of humanized antibodies are shown in Example 2.4. The experimental results are shown in Table 10. The affinity equilibrium dissociation constant of the humanized antibody remains at the same pM level compared to the murine antibody.
  • Anti-SARS-CoV-2 S1 humanized antibody blocks the binding of spike S protein to 293T-ACE2 cells
  • the Luciferase luminescence value RLU was detected by chemiluminescence, and the pseudovirus inhibition rate of the antibody to be tested was calculated according to the RLU reading value, and the neutralization of the antibody to be tested was evaluated. and effects.
  • the genome of the new coronavirus S protein pseudovirus encodes firefly luciferase. When the viral genome is integrated into cells, the expression and activity of firefly luciferase is proportional to the number of transduced cells. In contrast to true viruses, pseudoviruses can only infect cells once.
  • 1.351 or E484K) (Jiman Bio) was taken out from -80°C, reconstituted at 4°C, and the reconstituted pseudovirus was diluted 125 times as a working solution; the diluted antibody to be tested and pseudovirus working solution were mixed with 50 ⁇ L/ The wells were added to a 96-well U-bottom plate and mixed, and pre-incubated at 37°C for 30 minutes; then 20 ⁇ L/well was added to the 384-well white plate where the cells were plated on the previous day. Positive control group: pseudovirus working solution and DMEM containing 10% FBS The medium was added to 384-well white plates at 10 ⁇ L/well.
  • Negative control group DMEM medium containing 10% FBS was added to 384-well white plate at 20 ⁇ L/well, and 4 wells were set up. After 24 hours, add DMEM medium containing 10% FBS at 30 ⁇ L/well, and continue to culture in the cell incubator for 24 hours; carefully remove the supernatant with a pipette, and add freshly prepared luciferase at 30 ⁇ L/well. The chromogenic solution was incubated at room temperature for 5 minutes, and the 384-well plate was placed in a microplate reader to read the chemiluminescence signal of each well.
  • Inhibition rate (%) [1-(sample RLU signal value-negative control RLU signal value)/(positive control RLU signal value-negative control RLU signal value)] ⁇ 100.
  • the software GraphPad Prism 6 was used for analysis to obtain the antibody dose-response curve, and the non-linear regression curve was used to calculate the median inhibitory concentration (IC 50 ).
  • Table 12-1 to Table 12-5 show the experimental results of the anti-SARS-CoV-2 S1 humanized monoclonal antibody inhibiting pseudovirus in vitro.
  • the humanized monoclonal antibodies hS1B-91-3 and hS1B-30-3 can significantly and dose-dependently inhibit the wild-type
  • the new coronavirus pseudovirus infects 293T-ACE2 cells, which can block the early invasion of host cells by SARS-CoV-2 infection and play a protective role.
  • hS1B-82-5, hS1B-48-2 and hS1B-14-3 can well inhibit wild-type and B.1.1. 7 of the new coronavirus pseudovirus infection, while hS1B-82-5, hS1B-48-2 and hS1B-14-3 can also well inhibit B.1.351 or E484K new coronavirus pseudovirus mutant infection, and hS1B-82-5 or The neutralization effect of hS1B-48-2 on the B.1.351 mutant was significantly better than that of the wild type, and the performance of hS1B-48-2 was better.
  • the B.1.351 South African mutant strain contains 3 amino acid mutations in the RBD domain of the S protein, namely K417N, E484K and N501Y. These three amino acids K417, E484 and N501 are basically located on the interface between the S protein RBD domain and these antibodies ( Figure 19-1), which is also the interface between the S protein RBD domain and human ACE2 molecules ( Figure 19). -2A).
  • SARS-CoV-2 coronavirus antibodies against the South African mutant B.1.351 of SARS-CoV-2 coronavirus have inactivated or attenuated neutralizing activity, for example, Eli Lilly's antibodies LY-CoV55, LY - CoV016 and Regeneron's REGN-10933 antibodies were inactivated, and AstraZeneca's COV2-2196 neutralizing activity was attenuated by 14.6-fold (Wang et al, 2021, Nature, DOI: 10.1038/s41586-021-03398-2; Chen RE et al, 2021, Nat Med, DOI: 10.1038/s41591-021-01294-w).
  • the major amino acid mutation E484K at the LY-CoV555 antibody/RBD binding interface resulted in a free energy change of 6.54 Kcal/mol
  • the major amino acid mutation K417N at the LY-CoV016 antibody/RBD binding interface resulted in a 0.75 Kcal/mol free energy change
  • REGN- The major amino acid mutations K417N and E484K at the 10933 antibody/RBD binding interface resulted in free energy changes of 0.68 Kcal/mol and 2.0 Kcal/mol, respectively.
  • a mutation with a free energy change greater than 0.5Kcal/mol has a significant impact on the antigen/antibody binding capacity; the free energy change caused by the mutation is a positive value, indicating that this mutation reduces the antigen/antibody binding capacity. Since these antibodies are no longer able to bind to the S protein RBD domain of the B.1.351 virus strain or have weakened binding due to the above mutations, these antibodies lose neutralizing activity or have reduced neutralizing activity against the B.1.351 mutant strain.
  • the B.1.1.7 mutant strain contains one amino acid mutation N501Y in the S protein RBD domain, while the B.1.351 mutant contains three amino acid mutations (K417N, E484K, N501Y).
  • the K417N mutation results in enhanced hS1B-48-2 antibody/RBD binding, which allows the hS1B-48-2 antibody to more effectively neutralize the SARS-CoV-2 pseudovirus mutant strain B.1.351.
  • the E484K mutation had no significant effect on the neutralizing activity of the antibody compared to the wild-type virus, while the N501Y mutation resulted in enhanced neutralizing activity and a lower IC50 value, and the K417N mutation led to further enhanced neutralizing activity of the antibody , the IC50 is further reduced.

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Abstract

L'invention concerne un anticorps dirigé contre la protéine S du coronavirus SARS-CoV-2 et son utilisation dans la préparation d'un médicament pour le traitement de la nouvelle pneumonie à coronavirus COVID-19. L'anticorps peut reconnaître de manière spécifique et se lier à la protéine S du coronavirus SARS-CoV-2 avec une affinité élevée. Ceci garantit que l'anticorps peut bloquer l'infection des cellules humaines par le SARS-CoV-2.
PCT/CN2021/086477 2020-08-28 2021-04-12 Anticorps dirigé contre la proteine s du coronavirus sars-cov-2 et son utilisation Ceased WO2022041745A1 (fr)

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CN202010887508.3A CN114106159A (zh) 2020-08-28 2020-08-28 一种SARS-CoV-2刺突蛋白抗体及其应用
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114397452A (zh) * 2022-03-24 2022-04-26 江苏美克医学技术有限公司 新型冠状病毒Delta突变株或原型株检测试剂盒及其应用
CN116082500A (zh) * 2022-12-09 2023-05-09 珠海重链生物科技有限公司 抗SARS-CoV-2抗体nCoV1和nCoV2及其应用
CN116554314A (zh) * 2023-03-06 2023-08-08 优睿赛思(武汉)生物科技有限公司 抗新型冠状病毒s蛋白受体结合域的纳米抗体及其应用
WO2024094154A1 (fr) * 2022-11-04 2024-05-10 百奥泰生物制药股份有限公司 Préparation d'anticorps ciblant le coronavirus et son utilisation
WO2024131774A1 (fr) * 2022-12-20 2024-06-27 北京昌平实验室 Anticorps neutralisants à large spectre dirigés contre une nouvelle souche mutante de coronavirus omicron et utilisation
WO2024131775A1 (fr) * 2022-12-20 2024-06-27 北京昌平实验室 Anticorps neutralisant à large spectre dirigé contre le nouveau coronavirus xbb.1.5, variants associés et utilisation correspondante

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013054320A1 (fr) * 2011-10-11 2013-04-18 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anticorps dirigés contre la molécule d'adhésion cellulaire associée à l'antigène carcino-embryonnaire (ceacam)
CN103298833A (zh) * 2010-08-14 2013-09-11 Abbvie公司 β淀粉样蛋白结合蛋白
CN103665155A (zh) * 2012-09-14 2014-03-26 中国科学院上海生命科学研究院 一种抗流感病毒广谱中和性的中和分子1f2
CN109776677A (zh) * 2017-11-15 2019-05-21 上海开拓者生物医药有限公司 一种人源化抗il-13抗体及其制备方法和应用
CN110382544A (zh) * 2017-03-16 2019-10-25 先天制药公司 用于治疗癌症的组合物和方法
CN111333722A (zh) * 2020-03-03 2020-06-26 江苏省疾病预防控制中心(江苏省公共卫生研究院) SARS-CoV-2抑制剂及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103298833A (zh) * 2010-08-14 2013-09-11 Abbvie公司 β淀粉样蛋白结合蛋白
WO2013054320A1 (fr) * 2011-10-11 2013-04-18 Tel Hashomer Medical Research Infrastructure And Services Ltd. Anticorps dirigés contre la molécule d'adhésion cellulaire associée à l'antigène carcino-embryonnaire (ceacam)
CN103665155A (zh) * 2012-09-14 2014-03-26 中国科学院上海生命科学研究院 一种抗流感病毒广谱中和性的中和分子1f2
CN110382544A (zh) * 2017-03-16 2019-10-25 先天制药公司 用于治疗癌症的组合物和方法
CN109776677A (zh) * 2017-11-15 2019-05-21 上海开拓者生物医药有限公司 一种人源化抗il-13抗体及其制备方法和应用
CN111333722A (zh) * 2020-03-03 2020-06-26 江苏省疾病预防控制中心(江苏省公共卫生研究院) SARS-CoV-2抑制剂及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Genpept NCBI; . . : "Chain A, Spike glycoprotein", XP055906075, Database accession no. 6XR8_A *
WANG, C. Y. ET AL.: "A Human Monoclonal Antibody Blocking SARS-CoV-2 Infection.", NATURE COMMUNICATIONS, vol. 11, 4 May 2020 (2020-05-04), XP055737066, DOI: 10.1038/s41467-020-16256-y *
YAN GE, DUAN-HUA LI, CHEN ZHAO, JIN-JUN LI, LU WANG: "Advances in Biotechnology Drug Development for COVID-19", GUOWAI-YIYAO-KANGSHENGSU-FENCE : SHUANGYUEKAN = WORLD NOTES ON ANTIBIOTICS, ZHONGGUO-KANSHENGSU ZAZHISHE, CHENGDU, CHINA, vol. 41, no. 4, 31 July 2020 (2020-07-31), Chengdu, China , XP055906067, ISSN: 1001-8751, DOI: 10.13461/j.cnki.wna.005319 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114397452A (zh) * 2022-03-24 2022-04-26 江苏美克医学技术有限公司 新型冠状病毒Delta突变株或原型株检测试剂盒及其应用
WO2024094154A1 (fr) * 2022-11-04 2024-05-10 百奥泰生物制药股份有限公司 Préparation d'anticorps ciblant le coronavirus et son utilisation
CN116082500A (zh) * 2022-12-09 2023-05-09 珠海重链生物科技有限公司 抗SARS-CoV-2抗体nCoV1和nCoV2及其应用
CN116082500B (zh) * 2022-12-09 2023-08-22 珠海重链生物科技有限公司 抗SARS-CoV-2抗体nCoV1和nCoV2及其应用
WO2024131774A1 (fr) * 2022-12-20 2024-06-27 北京昌平实验室 Anticorps neutralisants à large spectre dirigés contre une nouvelle souche mutante de coronavirus omicron et utilisation
WO2024131775A1 (fr) * 2022-12-20 2024-06-27 北京昌平实验室 Anticorps neutralisant à large spectre dirigé contre le nouveau coronavirus xbb.1.5, variants associés et utilisation correspondante
CN116554314A (zh) * 2023-03-06 2023-08-08 优睿赛思(武汉)生物科技有限公司 抗新型冠状病毒s蛋白受体结合域的纳米抗体及其应用
CN116554314B (zh) * 2023-03-06 2023-11-24 优睿赛思(武汉)生物科技有限公司 抗新型冠状病毒s蛋白受体结合域的纳米抗体及其应用

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