WO2022041745A1 - Antibody against sars-cov-2 coronavirus s protein and application thereof - Google Patents

Antibody against sars-cov-2 coronavirus s protein and application thereof 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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PCT/CN2021/086477
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French (fr)
Chinese (zh)
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李强
孙见宇
武翠
张晓峰
刁家升
周利
马心鲁
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安源医药科技（上海）有限公司
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Priority claimed from CN202010889404.6A external-priority patent/CN114106161A/en
Priority claimed from CN202010887508.3A external-priority patent/CN114106159A/en
Priority claimed from CN202010889418.8A external-priority patent/CN114106162A/en
Priority claimed from CN202010889425.8A external-priority patent/CN114106163A/en
Priority claimed from CN202010889400.8A external-priority patent/CN114106160A/en
Application filed by 安源医药科技（上海）有限公司 filed Critical 安源医药科技（上海）有限公司
Publication of WO2022041745A1 publication Critical patent/WO2022041745A1/en

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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

Provided are an antibody against SARS-CoV-2 coronavirus S protein and an application thereof in preparation of a drug for treating novel coronavirus pneumonia COVID-19. The antibody can specifically recognize and bind to the SARS-CoV-2 coronavirus S protein with a high affinity. This ensures that the antibody can block the infection of human cells by SARS-CoV-2.

Description

针对SARS-CoV-2冠状病毒S蛋白的抗体及其用途Antibodies against SARS-CoV-2 coronavirus S protein and uses thereof 技术领域technical field
本发明涉及治疗性抗体及分子免疫学领域，更具体地，涉及一种SARS-CoV-2冠状病毒S蛋白的重组单克隆抗体，以及这种抗体的用途，特别是在治疗、预防和诊断由SARS-CoV-2引起的COVID-19疾病中的用途。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.
背景技术Background technique
新型冠状病毒SARS-CoV-2作为一种新近出现的人类病原体，可引起发烧、乏力、干咳为主要表现的严重的呼吸系统疾病和COVID-19肺炎。根据世界卫生组织2020年8月19日的数据，全球已确诊病例为21,756,357人，导致771,635人死亡。依据基因组核酸序列，新的病原体被证明是β冠状病毒属的一种新型成员。SARS-CoV-2与蝙蝠冠状病毒RaTG13的基因组序列相似性达96.2％(Zhou P et al,2020,Nature,579:270-273)，与两种蝙蝠SARS样冠状病毒bat-SL-CoVZC45(同源性88％)和bat-SL-CoVZXC21(同源性87％)密切相关，但与SARS-CoV(同源性79％)和MERS-CoV(同源性50％)亲缘关系相对较远(Lu R et al，2020,Lancet,395:565-574)。与SARS-CoV相比，SARS-CoV-2冠状病毒更容易在人与人之间传播，WHO已宣布COVID-19疾病为全球大流行疾病，目前新冠病毒已传播至全球各地。As a newly emerged human pathogen, 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. According to data from the World Health Organization on August 19, 2020, there were 21,756,357 confirmed cases globally, resulting in 771,635 deaths. Based on the genomic nucleic acid sequence, 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.
与其它冠状病毒一样，新型冠状病毒SARS-CoV-2是一种正义RNA病毒，编码几种主要蛋白，S、M、N和E，依赖RNA的RNA聚合酶RDRP以及十几种非结构蛋白。其中S、M、N和E蛋白用于包装病毒结构，RDRP和十几种非结构蛋白用于病毒基因组RNA复制和各个蛋白mRNA的合成。SARS-CoV-2与SARS-CoV病毒很相似，氨基酸序列同源度较高，它的S、M、N、E和RDRP蛋白的氨基酸个数和与SARS-CoV的同源度分别为1273(76％)、222(91％)、419(91％)、75(95％)、932(96％)。类似于SARS-CoV病毒，SARS-CoV-2病毒呈球形，有包膜，外周排列有冠状刺突。SARS-CoV-2的刺突S蛋白形成三聚体(Wrapp D et al,2020,Science,6483:1260-1263)，型如蘑菇状，镶嵌在病毒外表面膜上。S蛋白是该病毒的主要抗原成分，负责病毒与被入侵宿主细胞受体ACE2结合以及病毒与细胞的融合。类似于SARS-CoV病毒S蛋白(Yuan Y et al,2017,Nat Commun,8:15092)，SARS-CoV-2冠状病毒S蛋白主要分为两个结构域，S1(1-685)和S2(686-1122)，以及一个短跨膜区和细胞质尾端。在型如蘑菇的S蛋白三聚体中，三个S1结构域形成“蘑菇帽”，三个S2结构域形成“蘑菇茎”。其中，S1中的RBD结构域(Receptor binding domain，氨基酸331-527)负责与被入侵宿主细胞受体ACE2结合，S2负责与宿主细胞融合。S2结构域在整体S蛋白中通常以折叠或蜷曲压缩的构象存在，当病毒在S1脱落后与宿主细胞融合时，S2展示伸展构象以便插入宿主细胞膜(Walls AC et al,2017,Proc Natl Acad Sci USA,114:11157-11162)。据报道，SARS-CoV-2病毒的S蛋白与人细胞受体的结合亲和力远高于SARS-CoV病毒的S蛋白(Wrapp D et al,2020,Science,6483:1260-1263；Walls AC et al,2020,Cell,181:281-292)。另一个与SARS-CoV病毒S蛋白的不同之处在于，SARS-CoV-2 S蛋白有一个Furin酶切位点RRAR(氨基酸682-685)，这个酶切位点把S蛋白分为S1和S2两部分，酶切后的S1和S2以非共价键的形式联系在一起。由于S1/S2之间存在Furin酶切位点以及Furin酶在真核组织及细胞中广泛 表达，同时，包含多碱性氨基酸的Furin位点，还可以被其它以赖氨酸或精氨酸为靶点的酶，如细胞表面酶TMPRSS2、核内体cathepsin L酶或可能的胰蛋白酶(Trypsin)等所降解(Hoffmann M et al,2020,Cell,181(2):271-280.e8；Shang J et al,2020,Proc Natl Acad Sci USA,117:11727-11734；Belouzard S et al,2012,Viruses,4:1011-1033)，因此，SARS-CoV-2的S1/S2之间更容易被切割，导致S1结构域在病毒与人细胞融合时更容易脱落，从而增加S2的融合能力和病毒侵染力。而SARS-CoV病毒S1/S2之间仅通过一个碱性氨基酸精氨酸连接，S蛋白在此处被细胞表面酶TMPRSS2和核内体中的cathepsin L酶切后而感染宿主细胞(Belouzard S et al,2012,Viruses,4:1011-1033；Belouzard S et al,2009,Proc Natl Acad Sci USA,106:5871-5876)。所以，以上两个不同之处，即Furin酶切位点的存在和与人受体ACE2的高亲和力，可能是造成SARS-CoV-2冠状病毒高传染力的原因。由于S蛋白负责与人体宿主细胞受体结合以及与宿主细胞的融合，S蛋白是SARS-CoV和SARS-CoV-2冠状病毒治疗性中和抗体的主要靶点。Like other coronaviruses, 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. Among them, 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%). Similar to the SARS-CoV virus, 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". Among them, 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). It has been reported that the binding affinity of the S protein of SARS-CoV-2 virus to human cell receptors is much higher than that of the S protein of SARS-CoV virus (Wrapp D et al, 2020, Science, 6483:1260-1263; Walls AC et al , 2020, Cell, 181:281-292). Another difference from the SARS-CoV virus S protein is that the SARS-CoV-2 S protein has a Furin cleavage site RRAR (amino acids 682-685), which divides the S protein into S1 and S2 The two parts, the digested S1 and S2, are linked together by non-covalent bonds. Since there is a Furin cleavage site between S1/S2 and Furin enzyme is widely expressed in eukaryotic tissues and cells, at the same time, 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. are degraded (Hoffmann M et al, 2020, Cell, 181(2): 271-280.e8; Shang J et al, 2020, Proc Natl Acad Sci USA, 117: 11727-11734; Belouzard S et al, 2012, Viruses, 4: 1011-1033), therefore, the S1/S2 of SARS-CoV-2 is more susceptible to The cleavage causes the S1 domain to be more easily shed when the virus is fused with human cells, thereby increasing the fusion ability and viral infectivity of S2. 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.
目前还没有治疗SARS-CoV-2冠状病毒的特效药物，疫苗和中和抗体似乎是目前最有希望成功的药物。中和抗体通过阻断病毒入侵宿主细胞而防止病毒传播，达到治疗疾病的目的。关于SARS-CoV-2的中和抗体，最近有许多文献报道。例如，再生元制药公司通过转基因鼠和单个B细胞测序平台开发了一系列针对RBD结构域的SARS-CoV-2中和抗体(Hansen J et al,2020,Science,369:1010-1014)。另有研究者们也利用单个B细胞测序技术平台开发了一系列针对SARS-CoV-2的S蛋白RBD结构域的中和抗体(Wu Y et al,2020,Science,368,1274-1278；Cao Y et al,2020,Cell,182:73-84；Pinto D et al,2020,Nature,583:290-295；Ju B et al,2020,Nature,584:115-119；Chi X et al,2020,Science,369:650-655)。。There is currently no specific drug for the treatment of SARS-CoV-2 coronavirus, and vaccines and neutralizing antibodies seem to be the most promising drugs for success. 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. There have been many recent literature reports on neutralizing antibodies against SARS-CoV-2. For example, 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). Other researchers have also used a single B cell sequencing technology platform to develop a series of neutralizing antibodies against the RBD domain of the S protein of SARS-CoV-2 (Wu Y et al, 2020, Science, 368, 1274-1278; Cao Y et al, 2020, Cell, 182:73-84; Pinto D et al, 2020, Nature, 583:290-295; Ju B et al, 2020, Nature, 584:115-119; Chi X et al, 2020 , Science, 369:650-655). .
目前已有三种针对SARS-CoV-2冠状病毒S蛋白的中和抗体进入临床研究。2020年6月1日，礼来公司与AbCellera合作研发的中和抗体LY-CoV555完成首批患者给药，进入I期临床研究。LY-CoV555是一种IgG1亚型的针对SARS-CoV-2刺突蛋白S的有效中和抗体。6月11日，再生元制药公司的双抗体鸡尾酒REGN-COV2首次进入临床研究阶段，并且基于I期临床良好的安全性数据，该研究目前得以直接进入III期临床。今年6月，君实生物与中国科学院微生物所共同开发的重组全人源抗SARS-CoV-2单克隆抗体注射液(JS016)获批进入I期临床试验。JS016是国内最早进入临床的新冠病毒中和抗体。在现在全球新冠疫情严峻的形势下，尽快研发出SARS-CoV-2冠状病毒S蛋白中和抗体，具有更高的特异性、更佳的临床药效和更低的治疗成本，这将给SARS-CoV-2感染患者提供更多的用药选择。At present, three neutralizing antibodies against the S protein of SARS-CoV-2 coronavirus have entered clinical research. On June 1, 2020, the neutralizing antibody LY-CoV555 developed by Eli Lilly and AbCellera completed the first patient administration and entered Phase I clinical research. LY-CoV555 is a potent neutralizing antibody against the SARS-CoV-2 spike protein S of the IgG1 subtype. On June 11, 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. In June this year, the recombinant fully human anti-SARS-CoV-2 monoclonal antibody injection (JS016) jointly developed by Junshi Biology and the Institute of Microbiology, Chinese Academy of Sciences was approved to enter the Phase I clinical trial. JS016 is the first new coronavirus neutralizing antibody to enter the clinic in China. Under the current severe situation of the global new crown epidemic, the 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.
发明内容SUMMARY OF THE INVENTION
本发明提供了一种能够特异性识别并高亲和力结合SARS-CoV-2冠状病毒S蛋白的抗体。本发明所述抗体能够阻断SARS-CoV-2感染宿主细胞。本发明公开的S蛋白抗体可以(单独或与其它制剂或治疗方法组合)用于治疗、预防和/或诊断由SARS-CoV-2引起的疾病，例如COVID-19。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.
本发明第一方面，提供一种能够特异性结合SARS-CoV-2冠状病毒S蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含的重链可变区(VH)包含至少一个、两个或三个选自下组的互补决定区(CDR)：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:
(i)HCDR1，其具有如SEQ ID NO：1、7、16、22、31、37、46、52、61、67、76、82、111、117、126或132所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(i) 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;
(ii)HCDR2，其具有如SEQ ID NO：2、8、17、23、32、38、47、53、62、68、77、83、101、104、112、118、127、133、165或167所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(ii) 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
(iii)HCDR3，其具有如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或168所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) 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;
和/或，其包含的轻链可变区(VL)包含至少一个、两个或三个选自下组的互补决定区(CDR)：And/or, 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:
(iv)LCDR1，其具有如SEQ ID NO：4、10、19、25、34、40、49、55、64、70、79、85、91、92、103、106、114、120、129、135、152或159所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iv) 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;
(v)LCDR2，其具有如SEQ ID NO：5、11、20、26、35、41、50、56、65、71、80、86、115、121、130、136或141所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(v) 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
(vi)LCDR3，其具有如SEQ ID NO：6、21、36、51、66、81、116、131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列。(vi) 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.
在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.
在某些优选的实施方案中，所述重链可变区中含有的HCDR1、HCDR2及HCDR3，和/或所述轻链可变区中含有的LCDR1、LCDR2及LCDR3由Kabat或IMGT编号系统定义。实施例6中的表5示例性地给出了鼠源抗体按Kabat或IMGT编号系统定义出的CDR氨基酸序列。In certain preferred embodiments, 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.
在某些优选的实施方案中，所述抗体或其抗原结合片段包含3个VH可变区CDR和3个VL可变区CDR，其选自下述26组：In certain preferred embodiments, 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:
(1)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：1、2、3、4、5或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(1) Its 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);
(2)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：7、8、9、10、11或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(2) Its 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);
(3)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：16、17、18、19、20或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(3) Its 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);
(4)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：22、23、24、25、26或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(4) Its 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);
(5)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：31、32、33、34、35或36所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(5) Its 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);
(6)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：37、38、39、40、41或36所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(6) Its 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);
(7)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：46、47、48、49、50或51所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(7) Its 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);
(8)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：52、53、54、55、56或51所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(8) Its 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);
(9)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：61、62、63、64、65或66所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(9) Its 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);
(10)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：67、68、69、70、71或66所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(10) Its 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);
(11)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：76、77、78、79、80或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(11) Its 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);
(12)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：82、83、84、85、86或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(12) Its 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);
(13)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：1、2、3、91、5或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(13) Its 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);
(14)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：7、8、9、92、11或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(14) Its 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);
(15)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：16、101、 102、103、20或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(15) Its 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);
(16)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：22、104、105、106、26或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(16) Its 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);
(17)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：111、112、113、114、115或116所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(17) Its 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);
(18)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：117、118、119、120、121或116所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(18) Its 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);
(19)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：126、127、128、129、130或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(19) Its 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);
(20)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：132、133、134、135、136或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(20) Its 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);
(21)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：1、2、3、4、141或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(21) Its 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);
(22)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：22、23、146、25、26或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(22) Its 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);
(23)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：76、77、151、152、80或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(23) Its 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);
(24)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：82、83、153、85、86或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(24) Its 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);
(25)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：76、77、158、159、80或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(25) Its 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);
(26)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：82、83、160、85、86或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1 个，2个或3个置换、缺失或添加)的序列；(26) Its 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);
(27)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：126、165、166、129、130或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(27) Its 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);
(28)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：132、167、168、135、136或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列。(28) Its 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).
在某些实施方案中，所述抗体或其抗原结合片段为鼠源的或嵌合的，其重链可变区包含鼠源IgG1、IgG2、IgG3或其变体的重链FR区；和其轻链可变区包含鼠源κ、λ链或其变体的轻链FR区。实施例6中的表6中给出了一些优选的鼠源抗体的可变区氨基酸序列编号。In certain embodiments, 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.
在某些优选的实施方案中，所述鼠源抗体或其抗原结合片段包含选自下述11组的VH和VL结构域：In certain preferred embodiments, the murine antibody or antigen-binding fragment thereof comprises VH and VL domains selected from the following 11 groups:
(1)VH结构域包含如SEQ ID NO：12所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：13所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(1) 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)) ;
(2)VH结构域包含如SEQ ID NO：27所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：28所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(2) 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)) ;
(3)VH结构域包含如SEQ ID NO：42所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：43所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(3) 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)) ;
(4)VH结构域包含如SEQ ID NO：57所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：58所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(4) 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)) ;
(5)VH结构域包含如SEQ ID NO：72所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、 85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：73所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(5) 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)) ;
(6)VH结构域包含如SEQ ID NO：87所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：88所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(6) 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)) ;
(7)VH结构域包含如SEQ ID NO：93所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：94所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(7) 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)) ;
(8)VH结构域包含如SEQ ID NO：97所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：98所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(8) 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)) ;
(9)VH结构域包含如SEQ ID NO：107所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：108所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(9) 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)) ;
(10)VH结构域包含如SEQ ID NO：122所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：123所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(10) 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)) ;
(11)VH结构域包含如SEQ ID NO：137所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：138所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列。(11) 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)) .
在某些实施方案中，所述抗体或其抗原结合片段为人源化的。实施例6给出了人源化策略的基本流程，表6中给出了一些优选的人源化抗体的可变区氨基酸序列编号。In certain embodiments, 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.
在某些优选的实施方案中，所述人源化抗体或其抗原结合片段包含选自下述9组的VH和VL结构域：In certain preferred embodiments, the humanized antibody or antigen-binding fragment thereof comprises VH and VL domains selected from the following 9 groups:
(1)VH结构域包含如SEQ ID NO：14所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：15所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(1) 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)) ;
(2)VH结构域包含如SEQ ID NO：44所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：45所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(2) 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)) ;
(3)VH结构域包含如SEQ ID NO：74所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：75所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列(3) 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))
(4)VH结构域包含如SEQ ID NO：142所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：143所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(4) 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)) ;
(5)VH结构域包含如SEQ ID NO：147所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：148所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(5) 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)) ;
(6)VH结构域包含如SEQ ID NO：154所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：155所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(6) 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)) ;
(7)VH结构域包含如SEQ ID NO：161所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：162所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(7) 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)) ;
(8)VH结构域包含如SEQ ID NO：124所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：125所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(8) 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)) ;
(9)VH结构域包含如SEQ ID NO：169所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：170所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列。(9) 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)) .
在某些实施方案中，所述抗体包含来源于人免疫球蛋白的重链恒定区和轻链恒定区。In certain embodiments, the antibody comprises a heavy chain constant region and a light chain constant region derived from human immunoglobulin.
较优选地，所述抗体包含人κappa链恒定区氨基酸序列(氨基酸序列如SEQ ID NO：95所示)。More preferably, the antibody comprises the human kappa chain constant region amino acid sequence (amino acid sequence shown in SEQ ID NO: 95).
较优选地，所述抗体包含选自人IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；更优选地，包含选自人IgG1、IgG2和IgG4的重链恒定区；并且，所述重链恒定区具有天然序列或与其所源自的天然序列相比具有一个或多个氨基酸的置换、缺失或添加的序列。例如，在一种实施方案中，人源化抗体分子包含野生型人IgG1的重链恒定区(氨基酸序列如SEQ ID NO：96所示)。在一种实施方案中，人源化抗体分子包括在根据EU编号的含有M252Y、S254T、T256E和M428L突变的人IgG1的重链恒定区(氨基酸序列如SEQ ID NO：190所示)。在另一种实施方案中，人源化抗体分子包含野生型人IgG2的重链恒定区(氨基酸序列如SEQ ID NO：99所示)。在一种实施方案中，人源化抗体分子包括在根据EU编号的铰链区修饰的的人IgG2(例如缺失ERKCC，氨基酸序列如SEQ ID NO：100所示)，参见中国专利号CN104177496B。在另一种实施方案中，人源化抗体分子包含野生型人IgG4的重链恒定区(氨基酸序列如SEQ ID NO：109所示)。或采用修饰的人IgG4恒定区序列；在一种实施方案中，人源化抗体分子包括在根据EU编号的第228位突变(例如S变为P)的人IgG4(氨基酸序列如SEQ ID NO：110所示)。More preferably, 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. For example, in one embodiment, 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). In one embodiment, 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. In another embodiment, 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).
在某些优选的实施方案中，所述抗体的重链具有如SEQ ID NO：29、59、89、139、144、149、156、163或171所示的氨基酸序列；或与上述序列中的任何相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或与上述序列中的任何相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至 少99％、或更高同一性的序列；和/或，所述抗体的轻链具有如SEQ ID NO：30、60、90、140、145、150、157、164或172所示的氨基酸序列；或与上述序列中的任何相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或与上述序列中的任何相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或更高同一性的序列。In certain preferred embodiments, 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 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 more identical the sequence of.
在某些较优选的实施方案中，上述的置换是保守置换。In certain more preferred embodiments, the aforementioned substitutions are conservative substitutions.
在上述任何实施方案中，本发明所述抗体或其抗原结合片段，其中，SARS-CoV-2冠状病毒S蛋白具有：In any of the above embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the SARS-CoV-2 coronavirus S protein has:
(a)如SEQ ID NO：189所示的氨基酸序列；(a) the amino acid sequence shown in SEQ ID NO: 189;
(b)是对SEQ ID NO：189所示的氨基酸序列置换、缺失或添加一个或几个氨基酸残基而得的氨基酸序列。(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.
在某些优选的实施方案中，所述置换包括K417和/或L452和/或E484和/或N501。In certain preferred embodiments, the substitutions include K417 and/or L452 and/or E484 and/or N501.
在某些优选的实施方案中，所述置换的K417是K417N，和/或L452是L452R，和/或E484是E484K，和/或N501是N501Y。In certain preferred embodiments, the substituted K417 is K417N, and/or L452 is L452R, and/or E484 is E484K, and/or N501 is N501Y.
在上述任何实施方案中，本发明所述抗体或其抗原结合片段能够以10nM或更低的K D结合SARS-CoV-2冠状病毒S蛋白，较优选地，以1nM或更低的K D结合S蛋白；较优选地，以100pM或更低的K D结合S蛋白；更优选地，以10pM或更低的K D结合S蛋白；最优选地，以1pM或更低的K D结合S蛋白。 In any of the above embodiments, 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 . .
本发明的第二方面，提供了编码上述抗体或其抗原结合片段的DNA分子。The second aspect of the present invention provides a DNA molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.
本发明的优选实施例中，编码所述抗体重链的DNA分子具有如SEQ ID NO：173、175、177、179、181、183、185或187所示的核苷酸序列，和编码所述抗体轻链的DNA分子具有如SEQ ID NO：174、176、178、180、182、184、186或188所示的核苷酸序列。In a preferred embodiment of the present invention, 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.
本发明的第三方面，提供了包含上述DNA分子的载体。A third aspect of the present invention provides a vector comprising the above DNA molecule.
本发明的第四方面，提供了包含上述载体的宿主细胞；所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，优选为CHO细胞；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;
本发明的第五方面，提供了一种药物组合物，所述组合物包含上述抗体或其抗原结合片段以及可药用赋形剂、载体或稀释剂。The fifth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient, carrier or diluent.
本发明的第六方面，还提供了制备本发明所述抗体或其抗原结合片段的方法，其包括：(a)获得抗体或其抗原结合片段的基因，构建抗体或其抗原结合片段的表达载体；(b)通过基因工程方法将上述表达载体转染到宿主细胞中；(c)在允许产生所述抗体或其抗原结合片段的条件下培养上述宿主细胞；(d)分离、纯化产生的所述抗体或其抗原结合片段。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.
其中，步骤(a)中所述表达载体选自质粒、细菌和病毒中的一种或多种，优选地，所述表达载体为pcDNA3.1；Wherein, the expression vector in step (a) is selected from one or more of plasmids, bacteria and viruses, preferably, the expression vector is pcDNA3.1;
其中，步骤(b)通过基因工程方法将所构建的载体转染入宿主细胞中，所述宿主细胞包括原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，优选为CHO细胞。Wherein, in step (b), 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.
其中，步骤(d)通过常规的免疫球蛋白纯化方法，包含蛋白质A亲和层析和离子交换、疏水层析或分子筛方法分离、纯化所述抗体或其抗原结合片段。Wherein, 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.
本发明的第七方面，提供了所述抗体或其抗原结合片段在制备治疗和预防由SARS-CoV-2冠状病毒引起的疾病的药物中的用途。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.
优选地，所述疾病为新型冠状病毒肺炎(COVID-19)；例如，所述疾病为由B.1.351突变株和/或B.1.1.7病毒株引起的新型冠状病毒肺炎(COVID-19)。Preferably, 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 .
本发明的第八方面，提供了一种利用上述抗体检测或测定生物样品中SARS-CoV-2病毒或其抗原的存在情况或进行定量的免疫测定方法；该方法包括将待测生物样品与本发明的抗SARS-CoV-2病毒S蛋白单克隆抗体或其抗原结合片段孵育，形成抗原-抗体复合物，对形成的结合复合物进行定性检测和定量测定，该复合物的存在或数量指示了SARS-CoV-2病毒的存在情况或者含量；具体地，所述方法包括如下步骤：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:
将待测生物样品与本发明的至少一种单克隆抗体或其抗原结合片段在合适的条件下孵育；incubating the biological sample to be tested with at least one monoclonal antibody or antigen-binding fragment thereof of the present invention under suitable conditions;
(2)检测上述步骤中结合复合物的存在。(2) Detecting the presence of the binding complex in the above step.
根据本发明的单克隆抗体或其抗原结合片段可不依赖于使用的标记物(例如，酶、荧光等)，并且不依赖于检测模式(例如，荧光免疫测定法、酶联免疫吸附测定法或化学发光测定法等)或测定法原理(例如，夹心法、竞争法等)而被用于上述免疫测定方法中；其中，所述抗原结合片段的实例包括但不限于F(ab')2、Fab'、Fab和Fv。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.
上述免疫测定法，包括酶免疫测定、放射免疫测定、荧光免疫测定、化学发光免疫测定、蛋白质印迹、免疫层析、乳胶凝集实验等等；并且，上述免疫测定法都可用于以竞争法或夹心法，用标记物标记的抗原或抗体测定生物样品中的靶抗原。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.
上述竞争性方法是基于检测标本中SARS-CoV-2病毒和已知量的标记的SARS-CoV-2病毒S蛋白与本发明单克隆抗体或其抗原结合片段进行定量竞争性结合反应；具体地，上述竞争法包括：将预定量的针对SARS-CoV-2病毒S蛋白的本发明单克隆抗体或其抗原结合片段包埋在固相载体上，然后加入待检测的含有SARS-CoV-2病毒的生物样品和预定量的用标记物标记的SARS-CoV-2病毒S蛋白，并于适当的条件下孵育足够长的时间；反应后充分洗涤所述的固相并检测保留在载体上的或测定未保留在载体上的标记物的信号值；然后将所测得的信号值与平行测得的预定量的对照样品的信号值相比较以确定样品中SARS-CoV-2病毒的存在及其相对量；优选地，几乎同时加入标记抗原和待检测生物样品。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. and a predetermined amount of the SARS-CoV-2 virus S protein labeled with a marker, and incubated for a long enough time under appropriate conditions; after the reaction, 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.
上述夹心法是基于作为俘获抗体(或固相抗体)的本发明的单克隆抗体或其抗原结合片段以及能够配合使用的标记抗体都能特异性结合生物样品中的SARS-CoV-2病毒，通过对标记抗体进行定量来测定样品中SARS-CoV-2病毒的含量；具体地，上述夹心法包括：将本发明的针对SARS-CoV-2病毒S蛋白的特异性单克隆抗体或其抗原结合片段结合到固相载体上形成固相抗体(也称俘获抗体或第一抗体)，然后向被包被的固相载体上分别加入待检生物样品和对照样品并于适当的条件下孵育足够长的时间；反应后充分洗涤 所述的固相并加入适量标记物标记的、可与SARS-CoV-2病毒S蛋白结合的第二抗体并再次孵育；反应后充分洗涤所述的固相并以适合的方法检测与第二抗体结合的标记物的信号值；将所测得的信号值与平行测得的预定量的对照样品的信号值相比较以确定样品中SARS-CoV-2病毒的存在及其相对量。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. Quantify the labeled antibody to determine the content of the SARS-CoV-2 virus in the sample; specifically, 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. time; fully wash the solid phase after the reaction and add a secondary antibody labeled with an appropriate amount of marker that can bind to the S protein of SARS-CoV-2 virus and incubate again; fully wash the solid phase after the reaction and use a suitable The method of detecting the signal value of the label bound to the second antibody; 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 amount.
第二抗体也可以是其它多克隆抗体；优选地，第二抗体为单克隆抗体。The second antibody can also be other polyclonal antibodies; preferably, the second antibody is a monoclonal antibody.
更优选地，所述第二抗体选自本发明的任意一个能够与第一抗体配合使用的单克隆抗体或其抗原结合片段。More preferably, 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.
其中，标记物可以是放射性同位素(例如125I)、酶、酶底物、磷光物质、荧光物质、生物素和着色物质。Among them, 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.
优选地，本发明所用的的标记物包括如碱性磷酸酶、辣根过氧化物酶、β-半乳糖苷酶、脲酶和葡萄糖氧化酶；标记物还可以是荧光物质，如荧光素衍生物和罗丹明衍生物；另外，标记物还可以是允许时间分辨荧光测定的稀土元素或稀土元素复合物，如铕或铕复合物；另外，标记物可以是磷光物质，如丫啶酯和异鲁米诺；或者是放射性同位素如125I、3H、14C和32P；另外，标记物可以是着色物质，如乳胶颗粒和胶体金。即本发明包括利用测定颜色、荧光、时间分辨荧光、化学发光、电化学荧光或放射性而定性或定量测定生物成分中的SARS-CoV-2病毒的存在情况或含量。Preferably, the labels used in the present invention include alkaline phosphatase, horseradish peroxidase, β-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, electrochemical fluorescence or radioactivity.
根据上述竞争法和夹心法进行SARS-CoV-2免疫测定时，都需要充分洗涤固相，测量与标记物结合的活性。当标记物是放射性同位素时，用孔计数器或液相闪烁计数器进行测量。当标记物是酶时，加入底物，在显色后用比色或荧光测定酶活性。当标记物是荧光物质、磷光物质或着色物质时，可分别通过本领域已知的方法进行测量。For SARS-CoV-2 immunoassays according to the competition method and the sandwich method described above, the solid phase needs to be washed sufficiently to measure the activity of binding to the label. When the label is a radioisotope, the measurement is performed with a pore counter or a liquid scintillation counter. When the label is an enzyme, the substrate is added and the enzyme activity is measured colorimetrically or fluorometrically after color development. When the label is a fluorescent substance, a phosphorescent substance, or a coloring substance, it can be measured by methods known in the art, respectively.
上述提到的生物样品选自血浆、全血、嗽口液、咽拭子、尿液、粪便和支气管灌流液。The biological samples mentioned above are selected from plasma, whole blood, mouthwash, throat swabs, urine, feces and bronchial perfusate.
上述提到的固相载体包括但不限于硝酸纤维素膜、胶乳颗粒、磁性颗粒、胶体金、珠子或诸如玻璃、纤维玻璃或聚合物(如聚苯乙烯或聚氯乙烯)或纤维光学传感器。The 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.
本发明的第九方面，提供了上述单克隆抗体在制备SARS-CoV-2病毒检测试剂盒中的用途。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.
本发明的第十方面，提供了一种SARS-CoV-2病毒的检测试剂盒，其包含本发明的至少一种单克隆抗体或其抗原结合片段；用于制备该检测试剂的单克隆抗体不受特别限制，可以是以上述本发明任一单克隆抗体或其抗原结合片段(如F(ab')2、Fab'、Fab和scFv)作为固相抗体或标记抗体中的其中一种被单独使用；也可以是上述本发明中针对不同抗原表位的两个单克隆抗体或其抗原结合片段分别作为固相抗体或标记抗体被组合使用。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.
本发明的一优选实施例中，所述检测试剂盒包括：In a preferred embodiment of the present invention, the detection kit includes:
(1)选自以下任一：(1) Choose from any of the following:
a.固相载体和第一抗体；a. Solid phase carrier and primary antibody;
b.包被有第一抗体的固相载体；b. A solid phase carrier coated with a primary antibody;
所述第一抗体为选自本发明的任意一个单克隆抗体或其抗原结合片段；The first antibody is any monoclonal antibody or antigen-binding fragment thereof selected from the present invention;
(2)第二抗体；(2) secondary antibody;
所述第二抗体任选地进行了适当标记，并且第二抗体选自本发明所述的能够与(1)中所述第一抗体配合 使用的单克隆抗体或其抗原结合片段。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).
上述检测试剂所包含的本发明的单克隆抗体或其抗原结合片段可预先固定在固相载体上，形成固相抗体，所述固相载体包括但不限于硝酸纤维素膜、胶乳颗粒、磁性颗粒、胶体金、珠子或诸如玻璃、纤维玻璃或聚合物(如聚苯乙烯或聚氯乙烯)或纤维光学传感器；本发明的一优选实施例中，所述固相载体为微量滴定板。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, and 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.
上述免疫测定试剂所包含的本发明的单克隆抗体或其抗原结合片段可预先用标记物进行标记，形成标记抗体，所述标记物包括但不限于放射性同位素(例如125I)、酶、酶底物、磷光物质、荧光物质、生物素和着色物质；优选地，所述酶包括如碱性磷酸酶、辣根过氧化物酶、β-半乳糖苷酶、脲酶和葡萄糖氧化酶；所述荧光物质包括如荧光素衍生物和罗丹明衍生物以及稀土元素或稀土元素复合物，如铕或铕复合物；所述磷光物质包括如丫啶酯和异鲁米诺；所述放射性同位素包括如125I、3H、14C和32P；所述着色物质包括如乳胶颗粒和胶体金；本发明的一优选实施例中，所述标记物为生物素。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, β-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, latex particles and colloidal gold; in a preferred embodiment of the present invention, the marker is biotin.
本发明的第十一方面，提供了上述免疫测定试剂在诊断SARS-CoV-2病毒感染所致疾病中的用途。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.
优选地，所述疾病为新型冠状病毒肺炎(COVID-19)；例如，所述疾病为由B.1.351突变株和/或B.1.1.7病毒株引起的新型冠状病毒肺炎(COVID-19)。Preferably, 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 .
本发明公开的技术方案，取得了有益的技术效果，概括如下：The technical solutions disclosed in the present invention have achieved beneficial technical effects, which are summarized as follows:
通过小鼠杂交瘤平台，用S蛋白(氨基酸326-685)和S三聚体(氨基酸16-1213)作为免疫抗原免疫小鼠，获得了一系列SARS-CoV-2冠状病毒S蛋白的鼠源抗体，这些抗体能够特异性识别并高亲和力结合S蛋白，KD值达到pM级。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.
ACE2体外竞争实验结果表明，这些鼠源抗体与人ACE2受体竞争S蛋白的结合位点，IC50值低至nM级。The results of ACE2 in vitro competition experiments showed that these murine antibodies competed with the human ACE2 receptor for the binding site of S protein with IC50 values as low as nM.
3、体外假病毒抑制实验结果表明，这些鼠源抗体能够有效抑制SARS-CoV-2冠状病毒感染宿主细胞，IC50值低至nM级。3. The results of in vitro pseudovirus inhibition experiments show that these murine antibodies can effectively inhibit the infection of host cells by SARS-CoV-2 coronavirus with IC50 values as low as nM.
4、进一步，对鼠源抗体进行了人源化改造，降低了免疫原性。人源化抗体保留了鼠源抗体的亲和力和假病毒抑制活性，结合亲和力KD值达到pM级，假病毒抑制活性相当于nM级。以上这些特性为抗体在临床上的应用奠定了基础。4. Further, 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.
5、本发明提供的抗体还可以用于检测样品中SARS-CoV-2病毒或者其相应抗原存在情况，所述抗体的检出灵敏度低于100pg/ml；更优地，低于10pg/ml。5. 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.
发明详述Detailed description of the invention
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同 的含义。Before the present invention is described in detail below, it is to be understood that this invention is not limited to the specific methodology, protocols and reagents described herein. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which shall be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
缩写和定义Abbreviations and Definitions
CDR   Complementarity-Determining Region，免疫球蛋白可变区中的互补决定区，用Kabat、IMGT、Chothia或AbM编号系统界定(参见术语“超变区”或“CDR区”或“互补决定区”)。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").
EC 50  产生50％功效或结合的浓度 EC 50 Concentration that produces 50% efficacy or binding
ELISA 酶联免疫吸附测定ELISA enzyme-linked immunosorbent assay
FR    抗体框架区(Framework)，将CDR区排除在外的免疫球蛋白可变区FR Antibody framework region (Framework), immunoglobulin variable region excluding CDR regions
HRP   辣根过氧化物酶HRP horseradish peroxidase
IC 50  产生50％抑制的浓度 IC 50 Concentration that produces 50% inhibition
IgG   免疫球蛋白GIgG Immunoglobulin G
Kabat 由Elvin A Kabat倡导的免疫球蛋白氨基酸序列比对及编号系统。Kabat An immunoglobulin amino acid sequence alignment and numbering system advocated by Elvin A Kabat.
mAb   单克隆抗体mAb monoclonal antibody
PCR   聚合酶链式反应PCR polymerase chain reaction
V区   在不同抗体之间序列可变的IgG链区段。其延伸到轻链的109位Kabat残基和重链的第113位残基。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.
VH    免疫球蛋白重链可变区VH immunoglobulin heavy chain variable region
VL    免疫球蛋白轻链可变区VL immunoglobulin light chain variable region
K D    平衡解离常数 K D equilibrium dissociation constant
k a    结合速率常数 ka association rate constant
k d    解离速率常数 k d dissociation rate constant
术语“EC 50”是指在使用抗体或其抗原结合片段进行的体外或体内测定中，诱导50％应答的抗体或其抗原结合片段的浓度，即在最大响应和基线之间一半时的浓度。 The term " 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.
术语“EU编号系统”(EU Numbering System or Scheme)：EU是指上个世纪60年代末(1968-1969)，Gerald M Edelman等人分离纯化得到第一个人IgG1免疫球蛋白，命名为EU，测定了其氨基酸序列并为其编号(Edelman GM et al,1969,Proc Natl Acad USA,63:78-85)。其它的免疫球蛋白的重链恒定区与EU进行氨基酸序列比对，对应氨基酸位置就是EU编号。EU编号系统主要针对的是免疫球蛋白重链恒定区，包括CH1，CH2，CH3和铰链区。The term "EU Numbering System or Scheme": 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.
术语“Kabat编号系统”(Kabat Numbering System or Scheme)：1979年，Kabat等人首先提出了标准化的人免疫球蛋白可变区的编号方案(Kabat EA,Wu TT,Bilofsky H,Sequences of Immunoglobulin Chains:Tabulation and Analysis of Amino Acid Sequences of Precursors,V-regions,C-regions,J-Chain andβ 2-Microglobulins.1979.Department of Health,Education,and Welfare,Public Health Service,National Institutes of Health)。在“免疫学相关蛋白质序列”一书中(Kabat EA,Wu TT,Perry HM,Gottesman KS,Foeller C.1991.Sequences of Proteins of Immunological Interest,5th edition.Bethesda,MD:US Department of Health and Human  Services,National Institutes for Health),Kabat等人对抗体轻链和重链的氨基酸序列进行了比对并编号。他们发现这些被分析序列表现出可变的长度，缺省和插入的氨基酸或氨基酸片段只能出现在特定的位置。有趣的是，插入点多位于CDR内部，但也可能出现在在框架区的某些位置。在Kabat编号方案中，轻链可变区编号到109位置，重链可变区编号到113位置，轻重链的插入氨基酸通过字母识别并注释(例如，27a，27b...)。所有Lambda轻链不包含位置10残基，而Lambda和Kappa轻链由两个不同的基因编码，位于不同的染色体上。Lambda和Kappa轻链可以通过它们的恒定区氨基酸序列的不同来区分。与EU编号系统只针对重链恒定区不同，Kabat编号系统的编号范围覆盖全长免疫球蛋白序列，包括免疫球蛋白轻链和重链的可变区和恒定区。 Term "Kabat Numbering System or Scheme": In 1979, Kabat et al first proposed a standardized numbering scheme for the variable regions of human immunoglobulins (Kabat EA, Wu TT, Bilofsky H, Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain and β 2 -Microglobulins. 1979. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health). In "Sequences of Immunologically Associated Proteins" (Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C. 1991. Sequences of Proteins of Immunological Interest, 5th edition. Bethesda, MD: US Department of Health and Human Services , National Institutes for Health), Kabat et al. aligned and numbered the amino acid sequences of antibody light and heavy chains. They found that the analyzed sequences exhibited variable lengths, with default and inserted amino acids or amino acid fragments only occurring at specific positions. Interestingly, insertion points are mostly located inside CDRs, but may also occur at certain positions in the framework region. In the Kabat numbering scheme, the light chain variable region is numbered to position 109, the heavy chain variable region is numbered to position 113, and the intervening amino acids of the light and heavy chains are identified by letter and annotated (eg, 27a, 27b...). All 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.
术语“结合”定义抗原上的特定表位与其对应抗体之间的亲和性相互作用，一般也理解为“特异性识别”。“特异性识别”的意思是本发明的抗体不与或基本上不与目标抗原以外的任意多肽交叉反应。其特异性的程度可以通过免疫学技术来判断，包括但不限于免疫印迹，免疫亲和层析，流式细胞分析等。在本发明中，特异性识别优选通过流式细胞技术来确定，而具体情况下特异性识别的标准可由本领域一般技术人员根据其掌握的本领域常识来判断。The term "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. In the present invention, 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.
术语“抗原”是外来能引发生物自身或人产生抗体的物质，是任何可诱发免疫反应的物质，如细菌、病毒等。外来抗原分子经过B细胞或抗原呈递细胞(如巨噬细胞、树突状细胞、内皮细胞和B细胞等)的辨识和加工处理，并与主要组织相容性复合体(如MHC II分子)结合成复合物再活化T细胞，引发连续的免疫反应。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.
术语“抗原表位”或“抗原决定簇”指具有抗原性(即可诱发特异性免疫应答)的分子上的特定化学基团或肽序列，是免疫球蛋白或抗体特异性结合的抗原(例如SARS-CoV-2的S蛋白)上的位点。表位决定区通常由分子的化学活性表面基团(如氨基酸或糖基侧链)组成且通常有特定的三维结构性质以及特定的电荷性质。抗原有两种表位或抗原决定簇(epitope)，B细胞表位和T细胞表位，分别为B细胞和T细胞所识别。我们通常所说抗原表位，一般是指B细胞抗原表位。B细胞表位位于抗原分子的表面，是与B细胞受体(BCR，一种位于B细胞膜上的抗体)结合的抗原部位，B细胞表位不需要加工处理即可直接被B细胞识别。然后B细胞吞噬抗原分子，加工处理成小肽(15个氨基酸左右大小，抗原T细胞表位)，呈递给Th细胞(辅助性T细胞)。同时，抗原分子也可以通过另一途径，如通过巨噬细胞吞噬，加工处理成小肽，呈递给Th细胞。Th受到B细胞和巨噬细胞的共刺激，三种细胞共同相互作用，Th细胞发出反馈信号给B细胞，指示B细胞增殖，分化成浆细胞和记忆细胞。浆细胞具有分泌抗体的功能，介导体液适应性免疫。抗体通过其可变区Fv部分结合抗原分子，通过其恒定区Fc部分与各种免疫细胞上的受体FcR结合，从而指引各种免疫细胞对抗原分子进行杀伤，行使ADCC(通过NK细胞)，CDC(通过补体)和ADCP(通过巨噬细胞)功能。每种B细胞具有特异性，只能分泌一种抗体。B细胞表位依据其在蛋白氨基酸序列中的连续性可分为连续表位和构象表位(或称不连续表位)。B细胞抗原表位大小不定，有5-20个氨基酸大小。T细胞抗原表位为T细胞所识别，与B细胞表位不同，T细胞表位可以位于抗原分子(例如病毒蛋白)中的任何位置，因此T细胞抗原表位贯穿于整个蛋白的序列之中。T细胞表位是连续性决定簇，一般为10-20个氨基酸大小。T细胞表位与I类(MHC I)或II类(MHC II)MHC分子结合并呈递在细胞表面，分别被T细胞的两个不 同子集CD8 +T细胞(杀伤性T细胞)和CD4 +T细胞(辅助性Th细胞)识别。因此，T细胞表位有CD8 +和CD4 +T细胞表位两种。MHC I分子几乎为所有细胞所表达，可以提供细胞内的一些状况，比如该细胞遭受病毒感染，则将病毒碎片之小肽分子通过MHC I提示在细胞表面，可以供杀手CD8 +T细胞等辨识，以进行扑杀。MHCⅡ分子大多位于抗原呈递细胞上，如巨噬细胞等。这类MHC II分子提供细胞外部(如体液中)的情况，像是组织中有细菌侵入，则巨噬细胞进行吞食后，把细菌碎片利用MHC II提示给辅助性Th细胞，启动免疫反应。B细胞和T细胞只能对外来抗原分子的抗原表位进行辨识并结合，对源于生物自身的抗原片段，如蛋白分子及其片段，不具有结合能力，这是因为在B细胞和T细胞的分化、发育和成熟过程中，对自身蛋白分子或片段有高亲和力的B细胞和T细胞，被抑制发育成熟或发生凋亡。 The term "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. We usually refer to antigenic epitopes, generally referring to B cell antigenic epitopes. B cell 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. 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. Each type of B cell is specific and can only secrete one type of antibody. 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. During the process of differentiation, development and maturation, B cells and T cells with high affinity for self-protein molecules or fragments are inhibited from maturation or undergo apoptosis.
术语“抗体”通常指具有免疫球蛋白一类功能的蛋白质结合分子。抗体的典型实例是免疫球蛋白，以及其衍生物或功能片段，只要其显示所需的结合特异性即可。用于制备抗体的技术是本领域熟知的。“抗体”包括不同类的天然免疫球蛋白(例如IgA、IgG、IgM、IgD和IgE)和亚类(如IgG1、lgG2、IgA1、IgA2等)。“抗体”还包括非天然免疫球蛋白，包括例如单链抗体，嵌合抗体(例如，人源化鼠抗体)和异源偶联抗体(例如，双特异性抗体)，以及其抗原结合片段(例如，Fab'，F(ab') 2，Fab，Fv和rIgG)。还可参见，例如，Pierce Catalog and Handbook,1994-1995(Pierce Chemical Co,Rockford,Ill)；Kuby J,Immunology,3rd Ed,WH Freeman&Co,New York,1997。抗体可以结合至一种抗原，称为“单特异性”；或结合至两种不同的抗原，称为“双特异性”；或结合至多于一种的不同的抗原，称为“多特异性”。抗体可以是单价、二价或多价的，即抗体可以一次结合至一个、两个或多个抗原分子。抗体“单价地”结合至某特定蛋白质，即一分子的抗体仅结合至一分子的蛋白质，但是该抗体也可以结合到不同的蛋白质。当抗体仅结合至两种不同蛋白质的每一种分子时，该抗体为“单价地”结合至每一种蛋白质，并且该抗体是“双特异性的”且“单价地”结合至两种不同蛋白质的每一种。抗体可以是“单体的”，即其包含单个多肽链。抗体可包含多个多肽链(“多聚体的”)或可包含两个(“二聚体的”)、三个(“三聚体的”)或四个(“四聚体的”)多肽链。若抗体为多聚体的，则该抗体可以是同源多聚体(homomulitmer)，即抗体包含多于一分子的仅一种多肽链，包括同源二聚体、同源三聚体或同源四聚体。可选的，多聚体抗体可以是异源多聚体，即抗体包含多于一种不同的多肽链，包括异源二聚体、异源三聚体或异源四聚体。 The term "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.). "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.
术语“单克隆抗体(mAb)”指获自基本均一抗体群体的抗体，例如除了可能少量存在的突变如天然产生的突变外，群体包含的单独抗体是相同的。因此，定语“单克隆”表示所述抗体特征为不是离散抗体的混合物。单克隆抗体由本领域技术人员所知晓的方法产生，例如通过将骨髓瘤细胞和免疫脾细胞融合制备杂合的抗体产生细胞。通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。单克隆抗体也可以用如重组技术、噬菌体展示技术、合成技术，或其它现有技术得到。The term "monoclonal antibody (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. Thus, 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.
术语“单链Fv抗体”(或“scFv抗体”)是指包含抗体的VH和VL结构域的抗体片段，是通过接头(linker)连接的重链可变区(VH)和轻链可变区(VL)的重组蛋白，接头使得这两个结构域相交联以形成抗原结合位点，接头序列一般由柔性肽组成，例如但不限于G2(GGGGS) 3。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一个核苷酸链编码的一条氨基酸链序列。对于scFv综述，可参见Pluckthun A,1994.Antibodies from Escherichia coli,in The Pharmacology of Monoclonal Antibodies,Vol 113,Rosenberg M and  Moore GP(EDs.),Springer-Verlag,New York,pp 269-315。还可参见国际专利申请公开号WO 88/01649和美国专利第4946778号和第5260203号。 The term "single-chain Fv antibody" (or "scFv 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. For a review of scFv, see Pluckthun A, 1994. Antibodies from Escherichia coli, in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenberg M and Moore GP (EDs.), Springer-Verlag, New York, pp 269-315. See also International Patent Application Publication No. WO 88/01649 and US Patent Nos. 4,946,778 and 5,260,203.
术语“完整抗体”是指由两条抗体重链和两条抗体轻链组成的抗体。“完整抗体重链”是在N-端到C-端方向上由抗体重链可变结构域(VH)、抗体恒定重链结构域1(CH1)、抗体铰链区(HR)、抗体重链恒定结构域2(CH2)和抗体重链恒定结构域3(CH3)组成，缩写为VH-CH1-HR-CH2-CH3；并且在IgE亚类的抗体的情形中，任选地还包括抗体重链恒定结构域4(CH4)。优选地“完整抗体重链”是在N-端到C-端方向上由VH、CH1、HR、CH2和CH3组成的多肽。“完整抗体轻链”是在N-端到C-端方向上由抗体轻链可变结构域(VL)和抗体轻链恒定结构域(CL)组成的多肽，缩写为VL-CL。所述抗体轻链恒定结构域(CL)可以是κ(kappa)或λ(lambda)。完整抗体链通过在CL结构域和CH1结构域之间(即轻链和重链之间)的多肽间二硫键和完整抗体重链的铰链区之间的多肽间二硫键连接在一起。典型的完整抗体的实例是天然抗体如IgG(例如，IgG1和IgG2)、IgM、IgA、IgD和IgE。The term "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). Preferably 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.
术语“抗体片段”或“抗原结合片段”是指保留与抗原(如，SARS-CoV-2冠状病毒的S蛋白)特异性结合能力的抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(Parental Antibody)的抗原结合区或可变区。抗体片段保留母体抗体的至少某些结合特异性。通常，当用摩尔单位(K D)来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％的母体抗体对靶标的结合亲和力。抗体片段包括但不限于：Fab片段、Fab'片段、F(ab') 2片段、Fv片段、Fd片段、互补决定区(CDR)片段、二硫键稳定性蛋白(dsFv)等；线性抗体(Linear Antibody)、单链抗体(例如scFv单抗体)、单抗体(Unibody，技术来自Genmab)、二价单链抗体、单链噬菌体抗体、单域抗体(Single Domain Antibody)(例如VH结构域抗体)、结构域抗体(Domantis，技术来自Domantis)、纳米抗体(nanobodies，技术来自Ablynx)；由抗体片段形成的多特异性抗体(例如三链抗体、四链抗体等)；和工程改造抗体如嵌合抗体(Chimeric Antibody)(例如人源化鼠抗体)、异缀合抗体(Heteroconjugate Antibody)等。这些抗体片段用本领域技术人员已知的常规技术获得，并用与完整抗体相同的方法对这些片段的实用性进行筛选。 The term "antibody fragment" or "antigen-binding 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. Typically, antibody fragments retain at least 10% of the parent binding activity when the activity is expressed in molar units ( KD ). Preferably, 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 art, and these fragments are screened for utility in the same manner as intact antibodies.
术语“VL结构域”是指免疫球蛋白轻链的氨基末端可变区结构域。The term "VL domain" refers to the amino-terminal variable region domain of an immunoglobulin light chain.
术语“VH结构域”是指免疫球蛋白重链的氨基末端可变区结构域。The term "VH domain" refers to the amino-terminal variable region domain of an immunoglobulin heavy chain.
术语“铰链区”包括重链分子的将CH1结构域连接至CH2结构域的那一部分。该铰链区包含约25个残基并且是柔性的，从而使两个N-末端抗原结合区独立地移动。铰链区可分为三个不同的结构域：上部、中部、和下部铰链结构域(Roux KH et al,1998,J Immunol,161:4083-4090)。The term "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).
术语“功能域”指的是能够特异性识别和/或结合到表位上的三维结构，例如抗体或抗体片段，包括天然完整抗体、单链抗体(scFv)、Fd片段、Fab片段、F(ab') 2片段、单结构域抗体片段、分离的CDR片段及其衍生物。此处“单链”意思是第一和第二功能域共价连接，可以以一个核酸分子编码的共线性氨基酸序列表示。 The term "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. Herein, "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.
术语“Fab片段”由一条重链的可变区及CH1区和一条轻链组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fab抗体”的大小是完整抗体的1/3，其只包含一个抗原结合位点。The term "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.
术语“Fab'片段”含有一条轻链、一条重链的VH结构域和CH1结构域、以及CH1和CH2结构域之间的 恒定区部分。The term "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.
术语“F(ab') 2片段”含有两条轻链和两条重链的VH结构域和CH1结构域以及CH1和CH2结构域之间的恒定区部分，由此在两条重链间形成链间二硫键。因此，F(ab′) 2片段由通过两条重链间的二硫键保持在一起的两个Fab'片段组成。 The term "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. Thus, an F(ab') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
术语“Fd片段”由一条重链的可变区和CH1组成，是Fab片段除去轻链后剩下的重链部分。The term "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.
术语“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区，是包含完整抗原识别和结合位点的最小片段。The term "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.
术语“二硫键稳定性蛋白(dsFv)”在VH和VL区分别引入一个半胱氨酸突变点，从而在VH和VL之间形成二硫键而实现结构稳定性。术语“二硫键”包括两个硫原子之间形成的共价键。氨基酸半胱氨酸含有巯基，该巯基可以与第二个巯基形成二硫键或桥连。在大多数天然存在的IgG分子中，CH1和CK区由二硫键连接并且两个重链由两个二硫键连接，在对应于使用Kabat编号系统的239和242处(位置226或229，EU编号系统)连接。The term "disulfide stabilizing protein (dsFv)" 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. The term "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. In most naturally occurring IgG molecules, 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.
术语“重链恒定区”包括来自免疫球蛋白重链的氨基酸序列。包含重链恒定区的多肽至少包含以下一种：CH1结构域，铰链(例如，上部铰链区、中间铰链区，和/或下部铰链区)结构域，CH2结构域，CH3结构域，或其变体或片段。例如，本申请中使用的抗原结合多肽可包含具有CH1结构域的多肽链；具有CH1结构域、至少一部分的铰链结构域和CH2结构域的多肽；具有CH1结构域和CH3结构域的多肽链；具有CH1结构域、至少一部分铰链结构域和CH3结构域的多肽链，或者具有CH1结构域，至少一部分铰链结构，CH2结构域，和CH3结构域的多肽链。在另一个实施例中，本申请的多肽包括具有CH3结构域的多肽链。另外，在本申请中使用的抗体可能缺少至少一部分CH2结构域(例如，所有的或一部分的CH2结构域)。如上文所述，但本技术领域的普通技术人员应理解，重链恒定区可能会被修改，使得它们在氨基酸序列上与天然存在的免疫球蛋白分子不同。The term "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. For example, 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. In another embodiment, the polypeptide of the present application includes a polypeptide chain having a CH3 domain. Additionally, 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). As described above, but as will be understood by those of ordinary skill in the art, heavy chain constant regions may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.
术语“轻链恒定区”包括来自抗体轻链的氨基酸序列。优选地，所述轻链恒定区包括恒定kappa结构域和恒定lambda结构域中的至少一个。The term "light chain constant region" includes amino acid sequences from antibody light chains. Preferably, the light chain constant region comprises at least one of a constant kappa domain and a constant lambda domain.
术语“Fc区”或“Fc片段”是指免疫球蛋白重链的C端区，其含有铰链区的至少一部分、CH2结构域和CH3结构域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体结合或与经典补体系统的第一组分(C1q)结合。Fc区包括天然序列Fc区和变异Fc区。The term "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.
通常，人IgG重链Fc区为自其Cys 226或Pro 230位置的氨基酸残基至羧基末端的区段，但其边界可能有变化。Fc区的C末端赖氨酸(残基447，依照EU编号系统)可以存在或可以不存在。Fc还可以指独立存在的，或在包含Fc的蛋白多肽的情况下的这一区域，例如“包含Fc区的结合蛋白”，还称为“Fc融合蛋白”(例如，抗体或免疫粘合素)。本发明的抗体中天然序列Fc区来自包括哺乳动物(例如人)的IgG1、IgG2(IgG2A，IgG2B)、IgG3和IgG4。在某些实施方案中，相对于哺乳动物Fc多肽氨基酸序列，两条Fc多肽链的氨基酸序列中每100个氨基酸中具有10个左右氨基酸的单一氨基酸的置换、插入和/或缺失。在一些实施方案中，上述Fc区氨基酸差异可以是延长半衰期的Fc改变、增加FcRn结合的改变、增强Fcγ受体(FcγR)结合的改变和/或增强ADCC、ADCP和/或CDC的改变。Typically, 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). In certain embodiments, there are single amino acid substitutions, insertions and/or deletions of about 10 amino acids out of every 100 amino acids in the amino acid sequence of the two Fc polypeptide chains relative to the mammalian Fc polypeptide amino acid sequence. In some embodiments, the aforementioned Fc region amino acid differences may be Fc alterations that prolong half-life, alterations that increase FcRn binding, alterations that enhance Fcγ receptor (FcyR) binding, and/or alterations that enhance ADCC, ADCP, and/or CDC.
在IgG、IgA和IgD抗体同种型中，Fc区包含抗体两条重链中的每一条的CH2和CH3恒定结构域；IgM和IgE Fc区包含在每条多肽链中的三个重链恒定结构域(CH2-4结构域)。In IgG, IgA, and IgD antibody isotypes, 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).
术语“Fc受体”或“FcR”指结合免疫球蛋白Fc区的受体。FcR可以是天然序列人FcR，例如，可以是结合IgG抗体的FcR(γ受体)，以及这些受体的等位基因变体和可变剪接形式。FcγR家族由三种活化受体(小鼠中的FcγRI，FcγRIII和FcγRIV；人类中的FcγRIA，FcγRIIA和FcγRIIIA)和一种抑制性受体(小鼠中的FcγRIIb或等同的人类中的FcγRIIB)组成。FcγRII受体包括FcγRIIA(“活化受体”)和FcγRIIB(“抑制受体”)，它们具有相似的氨基酸序列。FcγRIIA的胞质结构域中包含免疫受体基于酪氨酸的活化基序(ITAM)。FcγRIIB的胞质结构域中包含免疫受体基于酪氨酸的抑制基序(ITIM)(参见Daeron M,1997,Annu Rev Immunol,15:203-234)。大多数天然效应细胞类型共表达一种或多种活化FcγR以及抑制性FcγRIIb，而NK细胞选择性只表达一种活化性Fc受体(小鼠中的FcγRIII和人类中的FcγRIIIA)，但不表达小鼠和人类中的抑制性FcγRIIb或FcγRIIB。人类IgG1与大多数人类Fc受体结合，在其结合的活化性Fc受体的类型方面被认为等同于鼠类IgG2a。术语“FcR”在本文中涵盖其它FcR，包括那些未来将会被鉴定的。术语“Fc受体”或“FcR”还包括新生儿受体FcRn，它负责将母体IgG转移给胎儿(Guyer RL et al,1976,J Immunol,117:587-593)。测量对FcRn结合的方法是已知的(参见例如Ghetie V and Ward ES,1998,Immunol Today,18:592-598；Ghetie V et al,1997,Nat Biotechnol,15:637-640)。可测定人FcRn高亲和力结合多肽与FcRn的体内结合和血清半衰期，例如在表达人FcRn的转基因小鼠或经转染的人细胞系中。The term "Fc receptor" or "FcR" 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 FcγRIIA 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). Most natural effector cell types co-express one or more activating FcγRs as well as inhibitory FcγRIIb, whereas NK cells selectively express only one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but not Inhibitory FcyRIIb or 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. The term "FcR" herein encompasses other FcRs, including those to be identified in the future. The term "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.
术语“嵌合抗体”是指重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性(美国专利US4816567；Morrison SL et al,1984,Proc Natl Acad Sci USA,81:6851-6855)。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。The term "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). For example, the term "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).
术语“人抗体”是指具有可变区的抗体，其中框架区和CDR区均源自人种系免疫球蛋白序列。此外，如果抗体含有恒定区，恒定区也来源于人种系免疫球蛋白序列。本发明的人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，如本文所用，术语“人抗体”不打算包括其中来源于另一种哺乳动物物种如小鼠的种系的CDR序列已经嫁接到人框架序列上的抗体。The term "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.
术语“人源化抗体”是指经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。非人抗体的CDR域外的大部分或全部氨基酸，例如小鼠抗体被来自人免疫球蛋白的相应氨基酸置换，而一个或多个CDR区内的大部分或全部氨基酸未改变。氨基酸的添加，删除，插入，替换或修饰是允许的，只要它们不会消除抗体结合特定抗原的能力。“人源化”抗体保留与原始抗体类似的抗原特异性。CDR的来源没有特别限制，可来源于任何动物。例如，可以利用源于小鼠抗体、大鼠抗体、兔抗体或非人灵长类动物(例如，食蟹猴)抗体的CDR区。框架区可以通过搜索IMGT antibody germline database(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)获得人类抗体胚系序列，一般选取与被改造 的非人源抗体同源度高的人类胚系抗体序列做人源化抗体的框架区。The term "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. For example, 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.
术语“超变区”或“CDR区”或“互补决定区”是指负责抗原结合的抗体氨基酸残基，是非连续的氨基酸序列。CDR区序列可以由Kabat、Chothia、IMGT(Lefranc et al,2003,Dev Comparat Immunol,27:55-77)和AbM(Martin ACR et al,1989,Proc Natl Acad Sci USA，86:9268–9272)方法来定义或本领域熟知的任何CDR区序列确定方法而鉴定的可变区内的氨基酸残基。例如，超变区包含以下氨基酸残基：来自序列比对所界定的“互补决定区”或“CDR”的氨基酸残基(Kabat编号系统)，例如，轻链可变结构域的24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)位残基和重链可变结构域的31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)位残基，参见Kabat et al,1991,Sequences of Proteins of Immunological Interest,5th Edition，Public Health Service，National Institutes of Health，Bethesda,Md.；和/或来自根据结构来界定的“超变环”(HVL)的残基(Chothia编号系统)，例如，轻链可变结构域的26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)位残基和重链可变结构域的26-32(HCDR1)、53-55(HCDR2)和96-101(HCDR3)位残基，参见Chothia C and Lesk AM,1987,J Mol Biol,196:901-917；Chothia C et al,1989,Nature,342:878-883。“框架”残基或“FR”残基为除本文定义的超变区残基之外的可变结构域残基。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、IMGT或Chothia编号系统确定。本领域技术人员可以明确地将每种编号系统赋予任何可变结构域序列，而不依赖于超出序列本身之外的任何实验数据。例如，给定抗体的Kabat残基编号方式可通过将抗体序列与每种“标准”编号序列对比同源区来确定。基于本文提供的序列编号方案，确定序列表中任何可变区序列的编号完全在本领域技术人员的常规技术范围内。The term "hypervariable 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. For example, 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), 53-55 (HCDR2) and 96-101 (HCDR3), see Chothia C and Lesk AM, 1987, J Mol Biol, 196:901-917; Chothia C et al, 1989, Nature, 342:878-883. "Framework" residues or "FR" residues are variable domain residues other than hypervariable region residues as defined herein. In certain embodiments, 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. One of skill in the art can unambiguously assign each numbering system to any variable domain sequence without reliance on any experimental data beyond the sequence itself. For example, 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.
术语“重组”，在涉及多肽或多核苷酸时指自然状态下不存在的多肽或多核苷酸的形式，其中一个非限制性的例子，可以通过将通常不会一起出现的多核苷酸或多肽组合在一起来实现。The term "recombinant", when referring to a 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.
术语“分离的抗体分子”指的是已经从其自然环境的组分中识别和分离和/或回收的抗体分子。其自然环境的污染组分是会干扰抗体的诊断或治疗用途的物质，并可能包括酶、激素和其它蛋白质的或非蛋白质的溶质。The term "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.
本文使用的关于核酸(如DNA或RNA)的术语“分离的”，是指分别从其它的以天然来源的大分子存在的DNA或RNA分离的分子。本文使用的术语“分离的”也指通过重组DNA技术生产时基本不含细胞材料，病毒材料或培养基的核酸或多肽，或经化学合成制备时基本不含化学前体或其它化学品。此外，“分离的核酸”是指包括不是天然存在的片段并且不会以天然状态发现的核酸片段。本文中术语“分离的”也用于指从其它细胞蛋白或组织分离的细胞或多肽。分离的多肽是指包括纯化的和重组的多肽。As used herein, the term "isolated" with respect to nucleic acid (eg, DNA or RNA) refers to a molecule that is separated from other DNA or RNA, respectively, which occurs as a macromolecule of natural origin. The term "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. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring fragments and are not found in their natural state. The term "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.
术语“交叉反应”是指本文所述的抗体结合来自不同物种的抗原的能力。例如，本文所述的结合SARS-CoV-2冠状病毒S蛋白的抗体还可结合来自其它物种的S蛋白(例如，SARS-CoV的S蛋白)。交叉反应性可通过检测在结合测定法(例如，SPR、ELISA)中与纯化抗原的特定反应性，或与生理表达抗原的细胞的结合或以其它方式与生理表达抗原的细胞的功能相互作用来测量。本领域中已知测定结合亲和力的分析的实例包括表面等离子共振(例如，Biacore)或类似技术(例如，Kinexa或Octet)。The term "cross-reactivity" refers to the ability of the antibodies described herein to bind antigens from different species. For example, 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).
术语“抗体依赖性细胞介导的细胞毒性(ADCC)”是指一种细胞毒性形式，抗体作为一种桥联形式，通过与免疫细胞或细胞毒性细胞(例如NK细胞、中性粒细胞或巨噬细胞)上存在的FcR结合，使这些细胞 毒性效应细胞特异性结合到抗体附着的靶细胞上，然后通过分泌细胞毒素杀死靶细胞。检测抗体的ADCC活性的方法是本领域已知的，例如可通过测定待测抗体与FcR(例如CD16a)之间的结合活性来评价。The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which antibodies act as a bridging form by interacting with immune cells or 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).
术语“抗体依赖细胞介导的吞噬作用(ADCP)”指一种细胞介导的反应，在该反应中，表达FcγR的非特异性细胞毒活性细胞识别靶细胞上结合的抗体并随后引起该靶细胞的吞噬。The term "antibody-dependent cell-mediated phagocytosis (ADCP)" refers to a cell-mediated reaction in which non-specific cytotoxicly active cells expressing FcγRs recognize bound antibodies on a target cell and subsequently elicit that target cell devoured.
术语“补体系统”指在血液中发现的大量的小蛋白质，称为补体因子，其通常以不活跃的前体(前蛋白)循环。这一术语指的是这一系统的下述能力：“补充”抗体和吞噬细胞从生物体中清除病原体如细菌以及抗原-抗体复合物的能力。补体因子的一个实例是复合物C1，其包含C1q和两个丝氨酸蛋白酶C1r和C1s。复合物C1是CDC途径的组分。C1q是分子量约为460,000的六价分子，且其结构类似郁金香的花束，其中六个胶原“茎”连接至六个球状的头部区域。为了激活补体级联，C1q必须结合至IgG1、IgG2或IgG3的至少两个分子。The term "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. In order to activate the complement cascade, C1q must bind to at least two molecules of IgG1, IgG2 or IgG3.
术语“补体依赖的细胞毒性(CDC)”是指通过使补体成分C1q与抗体Fc结合来激活补体级联的细胞毒性形式。检测抗体的CDC活性的方法是本领域已知的，例如可通过测定待测抗体与Fc受体(例如C1q)之间的结合活性来评价。The term "complement-dependent cytotoxicity (CDC)" 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).
术语“免疫结合”和“免疫结合性质”是指一种非共价相互作用，其发生在免疫球蛋白分子和抗原(对于该抗原而言免疫球蛋白为特异性的)之间。免疫结合相互作用的强度或亲和力可以用相互作用的平衡解离常数(K D)表示，其中K D值越小，表示亲和力越高。所选多肽的免疫结合性质可使用本领域中公知的方法测定。一种测定方法涉及测量抗原/抗体复合物形成和解离的速度。“结合速率常数”(K a或K on)和“解离速率常数”(K d或K off)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,1993,Nature,361:186-187)。k d/k a的比率等于平衡解离常数K D(参见Davies DR et al,1990,Annual Rev Biochem,59:439-473)。可用任何有效的方法测量K D、k a和k d值。 The terms "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. 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. Both "association rate constants" (K a or K on ) and "dissociation rate constants" (K d or K off ) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, 1993 , Nature, 361:186-187). The ratio of kd / ka is equal to the equilibrium dissociation constant KD (see Davies DR et al, 1990, Annual Rev Biochem, 59:439-473). KD , ka , and kd values can be measured by any effective method.
术语“免疫细胞”包括具有造血起源并在免疫应答中起作用的细胞，包括淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞，巨噬细胞，嗜酸性粒细胞，肥大细胞，嗜碱性粒细胞和粒细胞。The term "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.
术语“免疫应答”是指免疫系统的细胞(例如T淋巴细胞，B淋巴细胞，自然杀伤(NK)细胞，巨噬细胞，嗜酸性粒细胞，肥大细胞，树突状细胞和嗜中性粒细胞)和由这些细胞或肝脏中的任何一种产生的可溶性大分子(包括抗体，细胞因子和补体)的作用，该作用导致选择性地靶向，结合，损伤，破坏和/或从脊椎动物体内清除入侵的病原体，感染病原体的细胞或组织，癌细胞或其他异常细胞，或者在自身免疫或病理性炎症的情形下，是正常的人类细胞或组织。免疫反应包括例如T细胞(例如效应T细胞或Th细胞，如CD4 +或CD8 +T细胞)的活化或抑制，或Treg细胞的抑制。 The term "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.
术语“免疫原性”指特定物质引发免疫应答的能力。The term "immunogenicity" refers to the ability of a particular substance to elicit an immune response.
术语“宿主细胞”指在其中载体可以增殖并且其DNA可以表达的细胞，所述细胞可以是原核细胞或者真核细胞。该术语还包括受试宿主细胞的任何后代。应理解，并不是所有的后代都与亲本细胞相同，因为在复制过程中可能会发生突变，这类后代被包括在内。宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞。The term "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.
术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行,通过使用Needleman和Wunsch的方法(Needleman SB and Wunsch CD,1970,J Mol Biol,48:443-453)来实现。The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When 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), then 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. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6). Typically, comparisons are made when two sequences are aligned for maximum identity. Such alignment can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.), by using the method of Needleman and Wunsch (Needleman SB and Wunsch CD, 1970, J Mol Biol, 48:443-453).
术语“突变的”、“突变体”和“突变”分别指与天然核酸或多肽相比(即可以用来定义野生型的参照序列)，置换、缺失或插入一个或多个核苷酸或氨基酸。The terms "mutant", "mutant" and "mutation" respectively refer 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) .
术语“效应子功能”是指，那些可归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性，且其随抗体不同种型而变化。抗体效应子功能的例子包括但不限于：Fc受体结合亲和性、ADCC、ADCP、CDC、细胞表面受体(例如B细胞受体)的下调、B细胞活化、细胞因子分泌、抗体和抗原/抗体复合物的半衰期/清除率等。改变抗体的效应子功能的方法是本领域已知的，例如通过在Fc区引入突变来完成。The term "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. Examples of 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.
术语“药学上可接受的载体和/或赋形剂和/或稳定剂”，是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂和/或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。The term "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. For example, 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.
术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤或感染)在受试者体内的发生或如果它发生作用减到最小而实施的方法。The term "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.
术语“载体”是指能够运输与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指其中可以连接另外的DNA区段的环状双链DNA环。另一种类型的载体是病毒载体，其中额外的DNA区段可以连接到病毒基因组中。某些载体能够在它们被导入的宿主细胞中自主复制(例如，具有细菌复制起点和游离型哺乳动物载体的细菌载体)。其它载体(例如非附加型哺乳动物载体)可以在导入宿主细胞后整合到宿主细胞的基因组中，并由此与宿主基因组一起复制。此外，某些载体能够指导它们有效连接的基因的表达。 这种载体在本文中被称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常以质粒的形式存在。然而，也包括其它形式的表达载体，如病毒载体(例如，复制缺陷型逆转录病毒，腺病毒和腺伴随病毒)，其起到等同的功能。The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a 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. In addition, certain 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"). In general, expression vectors useful in recombinant DNA technology typically exist in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (eg, replication-defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。有益或所需的临床结果包括但不限于，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后，无论是可检测或是不可检测的。有效缓解任何特定疾病症状的治疗剂的量可以根据诸如患者的疾病状态，年龄和体重以及药物在受试者中引起期望的反应的能力等因素而变化。疾病症状是否得到缓解可以通过任何临床测量来评估，这些测量通常由医生或其它熟练的医疗保健提供者用于评估该症状的严重程度或进展状态。The term "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.
针对SARS-CoV-2冠状病毒S蛋白抗体的治疗用途Therapeutic use of antibodies against SARS-CoV-2 coronavirus S protein
可将本发明的抗体(其包括双特异性、多克隆、单克隆、人源化抗体)用作治疗剂。这些药剂可通常用于在受试者中治疗或预防新型冠状肺炎COVID-19，增加疫苗功效或提高天然免疫应答。将抗体制剂(优选地对其靶抗原S蛋白具有高特异性和高亲和力的抗体制剂)给予受试者并且通常因其与靶标的结合而具有效果。抗体的给予可消除或抑制或干扰SARS-CoV-2冠状病毒S蛋白的活性。在使用抗体片段的情况下，特异性结合至靶蛋白的结合结构域的最小的抑制性片段是优选的。例如，基于抗体的可变区序列，其保持结合靶蛋白序列的能力。这类肽可化学合成和/或通过重组DNA技术制备(参见，例如，Marasco WA et al,1993,Proc Natl Acad Sci USA,90:7889-7893)。The antibodies of the invention, which include bispecific, polyclonal, monoclonal, humanized antibodies, 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. For example, based on 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).
本发明的特异性结合SARS-CoV-2冠状病毒S蛋白的抗体或其片段可以药物组合物的形式给予。制剂可根据治疗的特定适应症的需要而含有超过一种活性化合物，优选相互间无不利影响的具有互补活性的那些。备选地或另外，组合物可包含增强它的功能的作用剂。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. Alternatively or additionally, the composition may contain an agent that enhances its function.
针对SARS-CoV-2冠状病毒S蛋白抗体的诊断用途Diagnostic use of antibodies against SARS-CoV-2 coronavirus S protein
本发明的单克隆抗体或其抗原结合性片段可用于SARS-CoV-2病毒的检测或定量用免疫测定。免疫测定方法本身是公知的，可以采用任何公知的免疫测定方法。即，如果以测定形式进行分类，有夹心法、竞争法、凝聚法、蛋白质印迹法等，如果以所使用的标记进行分类，则有荧光法、酶法、放射法、生物素法等，这些都可使用。还可通过免疫组织染色进行诊断。免疫测定方法中使用标记抗体时，抗体的标记方法本身是公知的，可以采用公知的任何方法。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. When a labeled antibody is used in the immunoassay method, the method for labeling the antibody itself is known, and any known method can be used.
这些免疫测定法本身是公知的，本说明书中无须赘述，简单来讲，例如夹心法是将本发明的抗体或抗原结合性片段作为第一抗体固定为固相，与待测生物样品反应，漂洗后，再使其与第二抗体反应，漂洗后测定与固相结合的第二抗体。将第二抗体用酶、荧光物质、放射性物质、生物素等标记，可以测定与固相结合的第二抗体。通过上述方法对已知浓度的多个标准品进行测定，根据测定的标记量与标准品含量的关系制作标准曲线，将对未知浓度的受检样品的测定结果对照该标准曲线，可以对受检样品中SARS-CoV-2病毒抗原进行定量。还可以将第一抗体和第二抗体在上述说明中替换。凝聚法中，将本发明的抗体或其抗原结合性片段固定在胶乳等颗粒上，与样品反应，测定吸光度。通过上述方法对已知浓度的多个标准品进 行测定，根据测定的标记量与标准品含量的关系制作标准曲线，将对未知浓度的受检样品的测定结果对照该标准曲线，可以对受检样品中SARS-CoV-2病毒抗原进行定量。These immunoassays themselves are well-known and need not be described in this specification. In simple terms, for example, 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. Through the above method, 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 first antibody and the second antibody can also be substituted in the above description. In the aggregation method, 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. Through the above method, 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.
供给上述免疫测定法的生物样品只要是含有SARS-CoV-2病毒的S蛋白的样品即可，没有特别限定，例如可以来自人和动物的血清、血浆、全血，除此之外还有鼻腔拭液(鼻腔拭子)、鼻腔吸引液、咽拭液(咽拭子)等体液提取液、唾液、呼吸道分泌物、尿液、粪便、细胞或组织匀浆液等。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. For example, it can be derived from human and animal serum, plasma, and whole blood, as well as nasal cavity. Swabs (nasal swabs), nasal aspiration fluids, throat swabs (pharyngeal swabs) and other body fluid extracts, saliva, respiratory secretions, urine, feces, cell or tissue homogenate, etc.
通过使用上述本发明的单克隆抗体，可将该抗体用作固相抗体和标记抗体的至少一方，制备SARS-CoV-2病毒免疫测定试剂。与上述单克隆抗体结合的固相可以使用以往免疫测定中使用的各种固相，例如有：ELISA板、胶乳、明胶颗粒、磁性颗粒、聚苯乙烯、玻璃等各种固相，珠子，可传输液体的基质等不溶性载体等。另外，可用酶、胶体金属颗粒、着色胶乳颗粒、发光物质、荧光物质、放射性物质等标记抗体，来制备标记抗体。将这些固相抗体和/或标记抗体等试剂组合，可以制备在酶联免疫测定法、放射免疫测定法、荧光免疫测定法等中使用的试剂。这些测定试剂是通过夹心法或竞争性结合测定法测定试样中的目标抗原的试剂。By using the above-described monoclonal antibody of the present invention, 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. Various 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. In addition, 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. By combining reagents such as these solid-phase antibodies and/or labeled antibodies, reagents for use in enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, and the like can be prepared. These assay reagents are reagents for measuring the target antigen in a sample by a sandwich method or a competitive binding assay.
上述通过夹心法进行免疫测定的试剂可以使用以下的试剂：例如准备两种本发明的单克隆抗体，以其中一种为上述标记抗体，以另外一种为与上述固相结合的固相抗体。首先，使含有待测定的抗原的样品与该固相抗体反应，接着使标记抗体(第二抗体)与被捕捉到该固相抗体上的抗原反应，通过检测与不溶性载体结合的标记物的存在或活性，可以实施免疫鉴定。同样，使含有待测定的抗原的样品与固相抗体反应，接着使标记抗体(第二抗体)与被捕捉到该固相抗体上的抗原反应，通过测定与不溶性载体结合的标记物的存在或活性，即通过标记抗体的量对待测定的抗原的量进行定量，可以实施免疫测量。夹心法的免疫测定试剂中，可以使用一种单克隆抗体作为固相抗体和标记抗体(例如抗原为多聚物时)，但通常优选使用可分别识别待测定的抗原的两个不同的表位的2种或以上的抗体。并且，对于任何的固相抗体和标记抗体，可以从2种或以上的单克隆抗体中选择组合使用。As 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. First, 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. Similarly, 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. In the immunoassay reagent of the sandwich method, 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. Furthermore, any solid-phase antibody and labeled antibody can be selected and used in combination from two or more monoclonal antibodies.
作为采用竞争性结合测定法的免疫测定试剂，例如可以制备成用酶、胶体金属颗粒、着色胶乳颗粒、发光物质、荧光物质、放射性物质等标记的一定量的病毒抗原。使用该试剂，可以与例如含有一定量的本发明的单克隆抗体、上述标记病毒抗原和待测定的抗原的样品进行竞争性反应，由与抗体结合的或未结合的标记病毒抗原的量对待测定样品中抗原的量进行定量，从而实施免疫测定。As 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. Using this reagent, 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.
本发明中，使上述抗体或抗原与固相或标记物结合时，可以采用物理吸附法、化学结合法等方法(参照“蛋白质核酸酵素”，别册No.31，37-45(1987年))。In the present invention, when binding the above-mentioned antibody or antigen to a solid phase or a label, methods such as physical adsorption method, chemical binding method, etc. can be used (refer to "Protein Nucleic Acid Enzyme", Separate Volume No. 31, 37-45 (1987) ) ).
上述标记抗SARS-CoV-2病毒单克隆抗体可以通过使抗SARS-CoV-2病毒单克隆抗体与标记物结合来制备。标记物可以是酶、胶体金属颗粒、着色胶乳颗粒、荧光胶乳颗粒、发光物质、荧光物质等。酶可以是酶联免疫测定法(Enzyme immunoassays，EIA)中使用的各种酶，例如碱性磷酸酶、过氧化物酶、β-D-半乳糖苷酶等；胶体金属颗粒例如可以使用胶体金颗粒、胶体硒颗粒等。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, β-D-galactosidase, etc.; colloidal metal particles such as colloidal gold can be used Granules, colloidal selenium granules, etc.
标记物与抗SARS-CoV-2病毒单克隆抗体的结合方法可以利用公知的产生共价键或非共价键的方法。结合的方法例如有：戊二醛法、高碘酸法、马来酰亚胺法、二硫二吡啶法、使用各种交联剂的方法等(例如“蛋白质核酸酵素”，别册No.31，37-45(1985年))。使用交联剂的结合方法中，交联剂例如可使用N-琥珀 酰亚氨基-4-马来酰亚氨基丁酸(GMBS)、N-琥珀酰亚氨基-6-马来酰亚氨基己酸、N-琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸等。通过共价键结合的方法中，可以依赖使用存在于抗体中的官能团，除此之外可以按照常规方法例如将硫羟基、氨基、羧基、羟基等官能团导入抗体中，然后采用上述结合方法，使该官能团与标记物结合，由此制备标记的抗SARS-CoV-2病毒单克隆抗体。通过非共价键结合的方法还有物理吸附法等。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)). In the bonding method using 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. acid, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, and the like. In the method of covalent bonding, the functional groups present in the antibody can be used depending on the use of functional groups. In addition, 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. There are also physical adsorption methods through non-covalent bonding methods.
底物可以使用对应于标记物的酶并如下表示的各种显色底物、荧光底物、发光底物等。As 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.
(a)显色底物：与过氧化氢组合的2，2’-连氮基-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)、3，3’，5，5’-四甲基联苯胺(TMB)、二氨基联苯胺(DAB)用于过氧化物酶；5-溴-4-氯-3-吲哚基磷酸(BCIP)、对硝基苯磷酸酯(p-NPP)、5-溴-4-氯-3-吲哚基磷酸钠(BCIP·Na)用于碱性磷酸酶。(a) 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.
(b)荧光底物：4-甲基伞形苯基磷酸酯(4-MUP)用于碱性磷酸酶；4-甲基伞形基苯基-β-D-半乳糖苷(4MUG)用于β-D-半乳糖苷酶。(b) Fluorescent substrates: 4-methylumbelliferyl phenyl phosphate (4-MUP) for alkaline phosphatase; 4-methylumbelliferyl phenyl-β-D-galactoside (4MUG) for in β-D-galactosidase.
(c)发光底物：3-(2’-螺旋金刚烷)-4-甲氧基-4-(3”-磷酰氧基)苯基-1，2-二氧杂环丁烷·2钠盐(AMPPD)用于碱性磷酸酶；3-(2’-螺旋金刚烷)-4-甲氧基-4-(3”-β-D-吡喃半乳糖基)苯基-1，2-二氧杂环丁烷(AMGPD)用于β-D-半乳糖苷酶；与过氧化氢组合得到的鲁米诺、异鲁米诺用于过氧化物酶。(c) 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"-β-D-galactopyranosyl)phenyl-1, 2-Dioxetane (AMGPD) was used for β-D-galactosidase; luminol, isoluminol obtained in combination with hydrogen peroxide were used for peroxidase.
通过将使用本发明针对SARS-CoV-2病毒的S蛋白的单克隆抗体测定来自人或动物的各种生物样品，可以实施SARS-CoV-2病毒感染的诊断。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.
具有保守修饰的抗体Antibodies with conservative modifications
术语“保守修饰”意图指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。此类保守修饰包括氨基酸的取代、添加和缺失。修饰可以通过本领域已知的标准技术，例如定点突变和PCR介导的突变引入到本发明的抗体中。保守氨基酸取代指氨基酸残基用具有类似侧链的氨基酸残基替换。本领域中对具有类似侧链的氨基酸残基家族已有详细说明。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区中的一个或多个氨基酸残基。The term "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. tyrosine, phenylalanine) , tryptophan, histidine) amino acids. Thus, 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.
药物组合物pharmaceutical composition
本发明的抗体或编码本申请抗体的核酸或多核苷酸，可以应用于制备药物组合物或无菌组合物，例如，将抗体与药学上可接受的载体、赋形剂或稳定剂混合。药物组合物可包括一种或组合的(如两种或更多不同的)本发明的抗体。例如，本发明的药物组合物可包含与靶抗原上的不同表位结合的具有互补活性的抗体或抗体片段(或免疫缀合物)的组合。治疗和诊断剂的制剂可通过以例如冻干粉末、浆液、水性溶液或悬浮液的形式与药学可接受的载体、赋形剂或稳定剂混合来制备。术语“药学上可接受的”指当分子本体、分 子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括糖类(如乳糖)、淀粉、纤维素及其衍生物、植物油、明胶、多元醇(如丙二醇)、海藻酸等。本发明的抗体或编码本申请抗体的核酸或多核苷酸可单独使用，或可与一种或更多种其它治疗剂共同使用，所述治疗剂例如疫苗。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. For example, 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. The term "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.
附图说明Description of drawings
图1、SARS-CoV-2 S1抗原免疫小鼠血清滴度。Figure 1. Serum titers of mice immunized with SARS-CoV-2 S1 antigen.
图2、SARS-CoV-2 S1抗原免疫小鼠血清中和抗体滴度。Figure 2. Serum neutralizing antibody titers of mice immunized with SARS-CoV-2 S1 antigen.
图3-1、纯化的鼠源抗体S1B-73-3、S1B-34-4以及S1B-8-2与SARS-CoV-2 S1的结合能力测定。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.
图3-2、纯化的鼠源抗体S1B-48-2和S1B-64-2与SARS-CoV-2 S1的结合能力测定。Figure 3-2. Determination of the binding ability of purified murine antibodies S1B-48-2 and S1B-64-2 to SARS-CoV-2 S1.
图3-3、纯化的鼠源抗体S1B-91-3与SARS-CoV-2 S1的结合能力测定。Figure 3-3. Determination of the binding ability of purified murine antibody S1B-91-3 to SARS-CoV-2 S1.
图3-4、纯化的鼠源抗体S1B-82-5与SARS-CoV-2 S1的结合能力测定。Figure 3-4. Determination of the binding ability of purified murine antibody S1B-82-5 to SARS-CoV-2 S1.
图3-5、纯化的鼠源抗体S1B-30-3与SARS-CoV-2 S1的结合能力测定。Figure 3-5. Determination of the binding ability of purified murine antibody S1B-30-3 to SARS-CoV-2 S1.
图3-6、纯化的鼠源抗体S1B-14-3与SARS-CoV-2 S1的结合能力测定。Figure 3-6. Determination of the binding ability of purified murine antibody S1B-14-3 to SARS-CoV-2 S1.
图3-7、纯化的鼠源抗体ST-10-4和ST-35-4与S-CoV-2 ST的结合能力测定。Figure 3-7. Determination of the binding ability of purified murine antibodies ST-10-4 and ST-35-4 to S-CoV-2 ST.
图4-1、纯化的鼠源抗体S1B-73-3、S1B-34-4以及S1B-8-2与SARS-CoV S的交叉反应性测定。Figure 4-1. Cross-reactivity determination of purified murine antibodies S1B-73-3, S1B-34-4 and S1B-8-2 with SARS-CoV S.
图4-2、纯化的鼠源抗体S1B-48-2和S1B-64-2与SARS-CoV S的交叉反应性测定。Figure 4-2. Cross-reactivity determination of purified murine antibodies S1B-48-2 and S1B-64-2 with SARS-CoV S.
图4-3、纯化的鼠源抗体S1B-91-3与SARS-CoV S的交叉反应性测定。Figure 4-3. Cross-reactivity determination of purified murine antibody S1B-91-3 with SARS-CoV S.
图4-4、纯化的鼠源抗体S1B-82-5与SARS-CoV S的交叉反应性测定。Figure 4-4. Cross-reactivity determination of purified murine antibody S1B-82-5 with SARS-CoV S.
图4-5、纯化的鼠源抗体S1B-30-3与SARS-CoV S的交叉反应性测定。Figure 4-5. Cross-reactivity determination of purified murine antibody S1B-30-3 with SARS-CoV S.
图5-1、鼠源抗体S1B-73-3、S1B-34-4以及S1B-8-2与ACE2-HRP竞争结合SARS-CoV-2 S1的能力。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.
图5-2、鼠源抗体S1B-48-2和S1B-64-2与ACE2-HRP竞争结合SARS-CoV-2 S1的能力。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.
图5-3、鼠源单抗S1B-91-3与ACE2-HRP竞争结合SARS-CoV-2 S1的能力。Figure 5-3. The ability of mouse monoclonal antibody S1B-91-3 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
图5-4、鼠源单抗S1B-82-5与ACE2-HRP竞争结合SARS-CoV-2 S1的能力。Figure 5-4. The ability of mouse monoclonal antibody S1B-82-5 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
图5-5、鼠源单抗S1B-30-3与ACE2-HRP竞争结合SARS-CoV-2 S1的能力。Figure 5-5. The ability of mouse monoclonal antibody S1B-30-3 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
图5-6、鼠源单抗S1B-14-3与ACE2-HRP竞争结合SARS-CoV-2 S1的能力。Figure 5-6. The ability of mouse monoclonal antibody S1B-14-3 to compete with ACE2-HRP for binding to SARS-CoV-2 S1.
图6-1、鼠源抗体S1B-73-3、S1B-48-2、S1B-91-3、S1B-82-5、S1B-30-3和阻断刺突S蛋白与293T-ACE2细胞的结合。Figure 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.
图6-2、鼠源抗体S1B-14-3阻断刺突S蛋白与293T-ACE2细胞的结合。Figure 6-2. Murine antibody S1B-14-3 blocks the binding of spike S protein to 293T-ACE2 cells.
图6-3、鼠源抗体ST-10-4和ST-35-4阻断刺突S蛋白与293T-ACE2细胞的结合。Figure 6-3. Murine antibodies ST-10-4 and ST-35-4 block the binding of spike S protein to 293T-ACE2 cells.
图7、鼠源抗体S1B-73-3、S1B-48-2、S1B-82-5、S1B-91-3/RBD分子对接模型、ACE2/RBD结构(PDB 6M0J)以及抗体CR3022/RBD结构(PDB 6W41)三个结构叠合图。Figure 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.
图8、鼠源抗体S1B-30-3、S1B-14-3、ST-10-4、ST-35-4/RBD分子对接模型、ACE2/RBD结构(PDB 6M0J)以及抗体CR3022/RBD结构(PDB 6W41)三个结构叠合图。Figure 8. 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.
图9-1、人源化抗体hS1B-73-3与其亲本鼠源抗体的重链可变区氨基酸序列比对结果。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.
图9-2、人源化抗体hS1B-73-3与其亲本鼠源抗体的轻链可变区氨基酸序列比对结果。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.
图10-1、人源化抗体hS1B-48-2与其亲本鼠源抗体的重链可变区氨基酸序列比对结果。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.
图10-2、人源化抗体hS1B-48-2与其亲本鼠源抗体的轻链可变区氨基酸序列比对结果。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.
图11-1、人源化抗体hS1B-91-3与其亲本鼠源抗体的重链可变区氨基酸序列比对结果。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.
图11-2、人源化抗体hS1B-91-3与其亲本鼠源抗体的轻链可变区氨基酸序列比对结果。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.
图12-1、人源化抗体hS1B-82-5与其亲本鼠源抗体的重链可变区氨基酸序列比对结果。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.
图12-2、人源化抗体hS1B-82-5与其亲本鼠源抗体的轻链可变区氨基酸序列比对结果。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.
图13-1、人源化抗体hS1B-30-3与其亲本鼠源抗体的重链可变区氨基酸序列比对结果。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.
图13-2、人源化抗体hS1B-30-3与其亲本鼠源抗体的轻链可变区氨基酸序列比对结果。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.
图14-1、人源化抗体hS1B-14-3-1和hS1B-14-3-2与其亲本鼠源抗体的重链可变区氨基酸序列比对结果。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.
图14-2、人源化抗体hS1B-14-3-1和hS1B-14-3-2与其亲本鼠源抗体的轻链可变区氨基酸序列比对结果。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.
图15-1、间接ELISA法测定人源化抗体hS1B-73-3与SARS-CoV-2 S三聚体抗原的结合能力。Figure 15-1. Indirect ELISA method to determine the binding ability of humanized antibody hS1B-73-3 to SARS-CoV-2 S trimer antigen.
图15-2、间接ELISA法测定人源化抗体hS1B-48-2与SARS-CoV-2 S三聚体抗原的结合能力。Figure 15-2. Indirect ELISA method to determine the binding ability of humanized antibody hS1B-48-2 to SARS-CoV-2 S trimer antigen.
图15-3、间接ELISA法测定人源化抗体hS1B-91-3与SARS-CoV-2 S三聚体抗原的结合能力。Figure 15-3. Indirect ELISA method to determine the binding ability of humanized antibody hS1B-91-3 to SARS-CoV-2 S trimer antigen.
图15-4、间接ELISA法测定人源化抗体hS1B-82-5与SARS-CoV-2 S三聚体抗原的结合能力。Figure 15-4. Indirect ELISA method to determine the binding ability of humanized antibody hS1B-82-5 to SARS-CoV-2 S trimer antigen.
图15-5、间接ELISA法测定人源化抗体hS1B-30-3与SARS-CoV-2 S三聚体抗原的结合能力。Figure 15-5. Indirect ELISA method to determine the binding ability of humanized antibody hS1B-30-3 to SARS-CoV-2 S trimer antigen.
图16-1、竞争ELISA法测定人源化抗体hS1B-73-3阻断SARS-CoV-2 S三聚体结合人ACE2的能力。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.
图16-2、竞争ELISA法测定人源化抗体hS1B-48-2阻断SARS-CoV-2 S三聚体结合人ACE2的能力。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.
图16-3、竞争ELISA法测定人源化抗体hS1B-91-3阻断SARS-CoV-2 S三聚体结合人ACE2的能力。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.
图16-4、竞争ELISA法测定人源化抗体hS1B-82-5阻断SARS-CoV-2 S三聚体结合人ACE2的能力。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.
图16-5、竞争ELISA法测定人源化抗体hS1B-30-3阻断SARS-CoV-2 S三聚体结合人ACE2的能力。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.
图17-1、人源化抗体hS1B-73-3、hS1B-48-2、hS1B-91-3、hS1B-82-5、hS1B-30-3阻断刺突S蛋白与293T-ACE2细胞的结合。Figure 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.
图17-2、人源化抗体hS1B-14-3阻断刺突S蛋白与293T-ACE2细胞的结合。Figure 17-2. Humanized antibody hS1B-14-3 blocks the binding of spike S protein to 293T-ACE2 cells.
图18-1、人源化抗体hS1B-91-3体外抑制假病毒的活性测定。Figure 18-1. Measurement of the activity of humanized antibody hS1B-91-3 for inhibiting pseudovirus in vitro.
图18-2、人源化抗体hS1B-30-3体外抑制假病毒的活性测定。Figure 18-2. Measurement of the activity of humanized antibody hS1B-30-3 for inhibiting pseudovirus in vitro.
图18-3、人源化抗体hS1B-48-2、hS1B-82-5、hS1B-14-3体外抑制假病毒突变株的活性测定。Figure 18-3. In vitro activity assay of humanized antibodies hS1B-48-2, hS1B-82-5 and hS1B-14-3 to inhibit pseudovirus mutants.
图19-1、已公开抗体与RBD结构结合图，以及RBD残基K417、E484和N501的空间位置。1A：LY-CoV555/RBD结构取自PDB 7KMG；1B：LY-CoV016/RBD结构取自PDB 7C01；1C：REGN-10933/RBD结构取自PDB 6XDG；1D：COV2-2196/RBD结构来源自COV2-2196结构建模，以及通过ZDOCK软件与RBD结构(PDB 6M0J)进行的分子对接计算模拟。Figure 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.
图19-2、ACE2、抗体hS1B-48-2或ST-35-4与RBD结构结合图。2A：ACE2/RBD结构取自PDB 6M0J；2B：抗体hS1B-48-2/RBD结构来源自hS1B-48-2结构建模，以及通过ZDOCK软件与RBD结构(PDB 6M0J) 进行的分子对接计算模拟；2C：抗体ST-35-4/RBD结构来源自ST-35-4结构建模，以及通过ZDOCK软件与RBD结构(PDB 6M0J)进行的分子对接计算模拟。Figure 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.
具体实施方式detailed description
实施例1：抗SARS-CoV-2 S蛋白鼠源单克隆抗体制备Example 1: Preparation of anti-SARS-CoV-2 S protein mouse monoclonal antibody
抗原制备：SARS-CoV-2抗原制备过程：根据Uniprot中公布的新型冠状病毒S蛋白全长氨基酸序列(Uniprot Entry  P0DTC2)，选取其中326-685aa区段(S1蛋白，标记为S1B)，及其中16-1213aa区段(S三聚体，标记为ST)，用作本实施例中筛选抗体的抗原。为了获得高效表达的目的蛋白，对S1B和ST的编码基因进行人工改造和优化，并按照常规分子生物学方法构建该目的基因的真核表达载体pcDNA3.1-S1B和pcDNA3.1-ST，将测序正确的重组表达质粒转染CHO细胞，并按常规方法进行表达和纯化，获得纯化后的抗原用于免疫。 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. In order to obtain the target protein with high expression, 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.
动物免疫：将上述SARS-CoV-2 S1蛋白抗原和S三聚体蛋白抗原分别以完全弗氏佐剂充分乳化后，采用多点免疫方式免疫雄性Balb/C小鼠(上海斯莱克实验动物有限责任公司)，50μg/只，免疫周期为三周一次。在第3次免疫后第10天，眼眶取血，以实施例2.1中描述的间接ELISA法测试血清抗SARS-CoV-2抗体滴度以监测小鼠免疫应答程度，结果显示于图1，竞争ELISA法测试血清SARS-CoV-2中和抗体水平,结果显示于图2。然后在融合前3天，对产生抗SARS-CoV-2抗体滴度最高以及中和抗体水平最高的小鼠加强免疫一次。3天后，处死小鼠并取出该小鼠脾脏与小鼠骨髓瘤Sp2/0细胞株融合。Animal immunization: After fully emulsifying the above SARS-CoV-2 S1 protein antigen and S trimeric protein antigen with complete Freund's adjuvant, male Balb/C mice (Shanghai Slack Laboratory Animal Co., Ltd.) were immunized by multi-point immunization. responsible company), 50 μg/only, and the immunization cycle is once every three weeks. On the 10th day after the third immunization, the orbital blood was collected, and the serum anti-SARS-CoV-2 antibody titer was tested by the indirect ELISA method described in Example 2.1 to monitor the degree of immune response in mice. The results are shown in Figure 1. Competition Serum SARS-CoV-2 neutralizing antibody levels were tested by ELISA, and the results are shown in Figure 2. 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.
细胞融合与抗体筛选：混合2×10 8Sp2/0细胞与2×10 8脾细胞在50％聚乙二醇(分子量为1450)和5％二甲基亚砜(DMSO)溶液中融合。用Iscove培养基(含有10％胎牛血清、100单位/mL青霉素、100μg/mL链霉素、0.1mM次黄嘌呤、0.4μM氨基蝶呤和16μM胸苷)来调整脾脏细胞数至5×10 5/mL，以0.3mL加入96孔培养板孔内，并置于37℃，5％CO 2培养箱内。培养10天后，采用高通量ELISA法分别检测上清中抗体与HRP标记的人ACE2竞争结合SARS-CoV-2的能力，以此筛选出与人ACE2竞争的阳性孔(方法参见实施例2.3)。再将上述含有能够抑制HRP标记的ACE2与SARS-CoV-2结合的单克隆抗体的孔内融合细胞进行亚克隆，同样以竞争ELISA方法筛选得到表达高亲和力鼠单克隆抗体的杂交瘤细胞株。其中11个细胞株后来被证明是表达以下抗体的细胞株：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。在补充10％FCS的RPMI 1640培养基中培养产生特异性抗体的克隆。当细胞密度达到大约5×10 5个细胞/mL时，用无血清培养基替换该培养基。2至4天后，将培养过的培养基离心，收集培养上清液。用蛋白A柱纯化抗体，然后用150mM NaCl透析单克隆抗体洗脱液。通过0.2μm滤器将透析的溶液过滤除菌，获得待测试的纯化的鼠单克隆抗体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。 Cell fusion and antibody screening: 2×10 8 Sp2/0 cells were mixed with 2×10 8 splenocytes in 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) solution. Use Iscove's medium (containing 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) to adjust the spleen cell number to 5 x 10 5 /mL, add 0.3 mL to the wells of a 96-well culture plate, and place in a 37°C, 5% CO 2 incubator. After 10 days of culture, 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. Eleven of these cell lines were later shown to express the following antibodies: 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. Specific antibody-producing clones were grown in RPMI 1640 medium supplemented with 10% FCS. When the cell density reached approximately 5 x 105 cells/mL, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged and the culture supernatant was collected. 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 μm 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.
实施例2、抗SARS-CoV-2鼠源单克隆抗体的功能鉴定Example 2. Functional identification of anti-SARS-CoV-2 murine monoclonal antibodies
2.1间接ELISA法测定鼠源抗体与SARS-CoV-2抗原的结合能力2.1 Determination of the binding ability of mouse-derived antibodies to SARS-CoV-2 antigen by indirect ELISA
以SARS-CoV-2 S1和S三聚体(ACRO Biosystems)分别包被酶标板，室温过夜。弃去包被溶液，用 溶解在PBS缓冲液的脱脂奶粉封闭1h，用PBST(PBS含0.05％吐温20(Tween-20)，pH 7.4)洗板3～4次。然后每孔分别加入100μL纯化的待测抗SARS-CoV-2鼠源抗体及针对SARS-CoV和SARS-CoV-2 S1的人源抗体CR3022(其重链可变区和轻链可变区的公布于GenBank(Accession numbers:DQ168569和DQ168570)阳性对照，室温孵育1h，用含有0.05％吐温20的PBS洗孔，然后每孔加入100μL HRP标记的羊抗鼠或羊抗人IgG多克隆抗体(Jackson Laboratory)作为检测抗体，再以PBST洗板3～4次、加入底物TMB显色10分钟、然后加入0.2M H 2SO 4终止反应，后读取吸光度值(OD值)，其结果示于图3-1至图3-7。 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 μL 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. Published in GenBank (Accession numbers: DQ168569 and DQ168570) positive controls, incubated for 1 h at room temperature, washed with PBS containing 0.05% Tween 20, and then added 100 μL of HRP-labeled goat anti-mouse or goat anti-human IgG polyclonal antibody ( Jackson Laboratory) as the detection antibody, wash the plate 3-4 times with PBST, add the substrate TMB to develop color for 10 minutes, then add 0.2MH 2 SO 4 to stop the reaction, and then read the absorbance value (OD value), the results are shown in Figure 3-1 to Figure 3-7.
结果显示，鼠源抗体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都能以较高亲和力与抗原结合。The results showed that murine antibodies 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 can bind to antigen with higher affinity.
2.2抗SARS-CoV-2 S1鼠源抗体交叉反应测定2.2 Anti-SARS-CoV-2 S1 mouse antibody cross-reactivity assay
对获得的鼠源单克隆抗体与SARS-CoV S的结合能力进行测定。The binding ability of the obtained murine monoclonal antibodies to SARS-CoV S was determined.
用PBS缓冲液将SARS-CoV S蛋白(ACRO Biosystems)，稀释至0.1μg/mL，以100μL/孔的体积加于96孔板中，4℃放置16～20h。吸掉上清，用PBST缓冲液洗板1次后，每孔加入200μL含1％脱脂奶粉的PBST(PBST/1％脱脂奶粉)，室温孵育1h封闭。移去封闭液，用PBST缓冲液洗板3次后，加入上述抗SARS-CoV-2的鼠源抗体，100μL/孔，室温孵育1.5h。移去反应体系，PBST洗板3次后，50μL/孔加入1:4000稀释的HRP标记的羊抗鼠IgG二抗(The Jackson Laboratory)，室温孵育1h。再以PBST洗板3次后，每孔加入100μL TMB，室温孵育5-10min。最后，每孔加入50μL 0.2M H 2SO 4终止反应，并用酶标仪在双波长450/620nm处读取OD值。 SARS-CoV S protein (ACRO Biosystems) was diluted to 0.1 μg/mL with PBS buffer, added to a 96-well plate in a volume of 100 μL/well, and placed at 4°C for 16-20 h. Aspirate the supernatant, wash the plate once with PBST buffer, add 200 μL 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 μL/well, and incubate at room temperature for 1.5 h. The reaction system was removed, and after washing the plate three times with PBST, 50 μL/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 μL of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50 μL 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.
如图4-1至图4-5所示，鼠源抗体S1B-82-5与SARS-CoV有较强的交叉反应，而S1B-73-3、S1B-48-2、S1B-91-3、S1B-30-3、S1B-34-4、S1B-8-2、S1B-64-2与SARS-CoV S则不交叉。As shown in Figure 4-1 to Figure 4-5, 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.
2.3竞争ELISA法测定鼠源抗体阻断SARS-CoV-2 S1与ACE2的结合能力2.3 Competitive ELISA to determine the ability of mouse-derived antibodies to block the binding of SARS-CoV-2 S1 to ACE2
用PBS缓冲液将SARS-CoV-2 S1蛋白(ACRO Biosystems)稀释至0.1μg/mL，以100μL/孔加于96孔板中，室温过夜。弃去包被溶液，每孔加入200μL PBST/1％脱脂奶粉，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板3次后，然后每孔加入100μL以辣根过氧化物酶(HRP)标记的ACE2分别和待测鼠源抗体的混合物。以PBST作为空白对照。充分孵育后以PBS洗去未结合的HRP标记的ACE2，室温孵育1h。再以PBST洗板3次后，每孔加入100μL TMB，室温孵育5-10min。最后，每孔加入50μL 0.2M H 2SO 4终止反应，并用酶标仪在双波长450/620nm处读取OD值。 SARS-CoV-2 S1 protein (ACRO Biosystems) was diluted to 0.1 μg/mL with PBS buffer and added to 96-well plates at 100 μL/well, overnight at room temperature. The coating solution was discarded, 200 μL 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 μL 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. After sufficient incubation, unbound HRP-labeled ACE2 was washed away with PBS, and incubated at room temperature for 1 h. After washing the plate three times with PBST, 100 μL of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50 μL 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.
图5-1至图5-6中显示，鼠源单抗S1B-73-3、S1B-48-2、S1B-82-5、S1B-91-3、S1B-30-3、S1B-14-3、S1B-34-4、S1B-8-2、S1B-64-2，都能与ACE2竞争结合SARS-CoV-2 S1，即通过阻断SARS-CoV-2 S1和ACE2的结合发挥功能。As shown in Figure 5-1 to Figure 5-6, the murine monoclonal antibodies S1B-73-3, S1B-48-2, S1B-82-5, S1B-91-3, S1B-30-3, S1B-14- 3. 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.
2.4生物薄膜干涉技术测定抗SARS-CoV-2 S1鼠源抗体的动力学常数和亲和力2.4 Determination of kinetic constants and affinities of anti-SARS-CoV-2 S1 murine antibodies by biofilm interferometry
采用生物薄膜干涉技术(BLI)对纯化的鼠源抗体S1B-82-5、S1B-91-3、S1B-15-5、S1B-30-3、S1B-48-2、S1B-73-3、对照抗体CR3022以及人ACE2(ACRO Biosystems)与SARS-CoV-2 S1(ACRO Biosystems)的结合亲和力常数进行测定。测定时，将待测生物素化SARS-CoV-2 S1固定在SA(Streptavidin)传感器表面，用抗SARS-CoV-2 S1鼠源抗体作为分析物。处理数据，并用分析软件1:1结合的模型进行拟合，拟合数据 与实验数据基本重叠，得到结合和解离速率常数k a和k d，用k d除以k a得到平衡解离常数K D(见表1)。结果显示，鼠源抗体S1B-73-3、S1B-48-2、S1B-82-5、S1B-91-3、S1B-30-3、S1B-14-3、S1B-34-4、S1B-8-2、S1B-64-2的K D值均达到pM级。 The purified murine antibodies S1B-82-5, S1B-91-3, S1B-15-5, S1B-30-3, S1B-48-2, S1B-73-3, The binding affinity constants of the control antibody CR3022 and human ACE2 (ACRO Biosystems) to SARS-CoV-2 S1 (ACRO Biosystems) were determined. During the measurement, the biotinylated SARS-CoV-2 S1 to be tested was immobilized on the surface of the SA (Streptavidin) sensor, and the anti-SARS-CoV-2 S1 mouse antibody was used as the analyte. The data were processed and fitted with a 1:1 binding model of the analysis software. The fitted data substantially overlapped with the experimental data to obtain the association and dissociation rate constants ka and k d . Divide k d by ka to obtain the equilibrium dissociation constant K D (see Table 1). The results showed that murine antibodies S1B-73-3, S1B-48-2, S1B-82-5, S1B-91-3, S1B-30-3, S1B-14-3, S1B-34-4, S1B- The K D values of 8-2 and S1B-64-2 reached pM level.
表1、鼠源抗体的动力学常数和亲和力测定结果Table 1. Kinetic constants and affinity determination results of murine antibodies
鼠源单抗Mouse monoclonal antibody K D(M) K D (M) K a(M/s) K a (M/s) K d(1/s) K d (1/s)
S1B-73-3S1B-73-3 <1.0E-12<1.0E-12 3.87E+053.87E+05 <1.0E-07<1.0E-07
S1B-48-2S1B-48-2 1.56E-111.56E-11 2.73E+052.73E+05 4.27E-064.27E-06
S1B-91-3S1B-91-3 <1.0E-12<1.0E-12 2.80E+052.80E+05 <1.0E-07<1.0E-07
S1B-82-5S1B-82-5 <1.0E-12<1.0E-12 2.27E+052.27E+05 <1.0E-07<1.0E-07
S1B-30-3S1B-30-3 6.10E-116.10E-11 1.79E+051.79E+05 1.08E-051.08E-05
S1B-14-3S1B-14-3 2.22E-112.22E-11 6.09E+056.09E+05 1.35E-051.35E-05
S1B-34-4S1B-34-4 <1.0E-12<1.0E-12 4.42E+054.42E+05 <1.0E-07<1.0E-07
S1B-8-2S1B-8-2 3.54E-123.54E-12 1.95E+051.95E+05 6.90E-076.90E-07
S1B-64-2S1B-64-2 9.10E-119.10E-11 4.87E+054.87E+05 4.43E-054.43E-05
CR3022CR3022 8.66E-118.66E-11 2.20E+052.20E+05 1.90E-051.90E-05
hACE2-FchACE2-Fc 1.40E-091.40E-09 2.34E+042.34E+04 3.28E-053.28E-05
2.5抗SARS-CoV-2鼠源抗体体外抑制假病毒实验2.5 In vitro inhibition of pseudovirus by anti-SARS-CoV-2 mouse antibody
通过使用新冠Spike假病毒与待测抗体孵育后侵染293T-ACE2细胞，采用化学发光法检测Luciferase发光值RLU，根据RLU读值计算待检抗体的假病毒抑制率，评价待检抗体的中和效果。新冠Spike假病毒基因组编码萤火虫荧光素酶，当病毒基因组进入细胞整合后，萤火虫荧光素酶的表达和活性与转导的细胞数量成正比。假病毒与真病毒相比只能一次性感染细胞。By infecting 293T-ACE2 cells with the new coronavirus Spike pseudovirus after incubation with the antibody to be tested, 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.
鼠源抗体对野生型(WT)假病毒或突变株假病毒(B.1.1.7、B.1.351或E484K)感染的抑制实验，具体步骤如下。293T-ACE2细胞(上海翊圣生物)，用培养基DMEM+10％FBS培养至对数生长期，以3000个细胞/孔接种于384孔白板中，并放入37℃，5％CO 2培养箱中培养过夜。待测抗体样品S1B-14-3、ST-10-4或ST-35-4用含10％FBS的DMEM稀释，起始浓度为10μg/mL，5倍比稀释，9梯度；野生型(WT)假病毒或突变株假病毒(B.1.1.7、B.1.351或E484K)(吉满生物)从-80℃取出，4℃复融，将复融后的假病毒稀释125倍，作为工作液；稀释后的待测抗体和假病毒工作液分别以50μL/孔加入到96孔U底板中混匀，37℃预孵育30分钟；然后以20μL/孔加入到前一天细胞铺板的384孔白板中，阳性对照组：假病毒工作液和含10％FBS的DMEM培养基分别以10μL/孔加入到384孔白板中。阴性对照组：含10％FBS的DMEM培养基以20μL/孔加入到384孔白板中，设4复孔。24小时后，以30μL/孔加入含10％FBS的DMEM培养基，继续放入细胞培养箱中培养24小时；用移液器小心吸掉上清，以30μL/孔加入新配的荧光素酶显色液，室温孵育5分钟，将384孔板置于酶标仪，读取每孔的化学荧光信号。抑制率(％)＝[1-(样品RLU信号值-阴性对照RLU信号值)/(阳性对照RLU信号值-阴性对照RLU信号值)]×100。再以抑制率作为Y轴，抗体浓度作为X轴，通过软件GraphPad Prism 6进行分析，得出抗体的量效曲线，使用非线性回归曲 线计算半数抑制浓度(IC 50)。 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). 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 μg/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 μL/well and mixed well, and pre-incubated at 37°C for 30 minutes; then 20 μL/well was added to the 384-well white plate where the cells were plated on the previous day. In the middle, positive control group: pseudovirus working solution and DMEM medium containing 10% FBS were added to 384-well white plate at 10 μL/well, respectively. Negative control group: DMEM medium containing 10% FBS was added to 384-well white plate at 20 μL/well, and 4 wells were set up. After 24 hours, add DMEM medium containing 10% FBS at 30 μL/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 μL/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. Taking the inhibition rate as the Y axis and the antibody concentration as the X axis, 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 ).
表2-1至表2-4显示了抗SARS-CoV-2鼠源抗体体外抑制假病毒感染的实验结果。如结果所示，S1B-14-3能很好的抑制新冠假病毒，而ST-10-4或ST-35-4均能很好的抑制野生型、B.1.1.7、B.1.351或E484K的新冠假病毒感染，并且ST-10-4或ST-35-4在B.1.1.7或B.1.351突变株上的中和作用明显优于野生型的，ST-35-4在B.1.351上的中和作用更好。Table 2-1 to Table 2-4 show the experimental results of anti-SARS-CoV-2 murine antibodies inhibiting pseudovirus infection in vitro. As shown in the results, S1B-14-3 can well inhibit the new coronavirus pseudovirus, while 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, and 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.
表2-1、抗SARS-CoV-2鼠源抗体假病毒抑制结果Table 2-1. Anti-SARS-CoV-2 murine antibody pseudovirus inhibition results
Figure PCTCN2021086477-appb-000001
Figure PCTCN2021086477-appb-000001
表2-2、抗SARS-CoV-2鼠源抗体假病毒突变株(B.1.1.7)抑制结果Table 2-2. Inhibition results of anti-SARS-CoV-2 murine antibody pseudovirus mutant strain (B.1.1.7)
Figure PCTCN2021086477-appb-000002
Figure PCTCN2021086477-appb-000002
表2-3、抗SARS-CoV-2鼠源抗体假病毒突变株(B.1.351)抑制结果Table 2-3. Inhibition results of anti-SARS-CoV-2 murine antibody pseudovirus mutant (B.1.351)
Figure PCTCN2021086477-appb-000003
Figure PCTCN2021086477-appb-000003
表2-4、抗SARS-CoV-2 S鼠源抗体对假病毒单点突变(E484K)抑制结果Table 2-4. Inhibition results of anti-SARS-CoV-2 S murine antibody to pseudovirus single point mutation (E484K)
Figure PCTCN2021086477-appb-000004
Figure PCTCN2021086477-appb-000004
实施例3、抗SARS-CoV-2鼠源抗体阻断刺突S蛋白与293T-ACE2细胞的结合Example 3. Anti-SARS-CoV-2 murine antibody blocks the binding of spike S protein to 293T-ACE2 cells
细胞水平上，通过FACS法测定待测抗体竞争阻断刺突S-鼠Fc融合蛋白(S-mFc)与293T-ACE2细胞的结合，通过荧光标记的羊抗鼠第二抗体，检测细胞上结合的S-mFc平均荧光强度，计算待测抗体阻断S蛋白与细胞表面ACE2结合的IC 50，评价待检抗体的阻断效果。S-mFc抗原制备过程：根据Uniprot中公布 的新型冠状病毒S蛋白全长氨基酸序列(Uniprot Entry P0DTC2)，选取S蛋白全长区段，并在S蛋白C端连接鼠IgG2a Fc片段(Uniprot Entry P01863(107-330aa))，构建用作本实施例中评价中和抗体的抗原S-mFc融合蛋白。为了获得高效表达的目的蛋白，对S-mFc的编码基因进行人工改造和优化，并按照常规分子生物学方法构建该目的基因的真核表达载体pcDNA3.1-S-mFc，将测序正确的重组表达质粒转染CHO细胞，并按常规方法进行表达和纯化。 At the cellular level, 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 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. In order to obtain high-efficiency expression of the target protein, 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.
鼠源抗体体外阻断实验，具体步骤如下：293T-ACE2细胞胰酶消化，以细胞密度1×10 6个/mL，100μL/孔加入96孔U底板，4℃孵育30分钟；将S-mFc(安源医药科技(上海)有限公司)用1％PBSB稀释至一定浓度；待测鼠源抗体样品分别稀释为8μg/mL，4倍比稀释，9个梯度，然后将稀释后的S-mFc和不同浓度梯度的抗体1:1混合，室温预孵育30分钟，加入到上述96孔U底板中，4℃孵育1小时；离心去上清，用1％PBSB洗涤细胞3次；将AF647-羊抗鼠IgG Fc(Jackson Immuno)用1％PBSB以1：400稀释，100μL/孔，4℃孵育1小时；离心去上清，用1％PBSB洗涤细胞3次；用1％PBSB重悬细胞，150μL/孔；流式细胞仪检测信号强度。再以平均荧光强度作为Y轴，抗体浓度作为X轴，通过软件GraphPad Prism 6进行分析，计算抗SARS-CoV-2鼠源抗体阻断S-mFc蛋白与293T-ACE2细胞结合的IC 50值。 In vitro blocking experiments of mouse-derived antibodies, the specific steps are as follows: 293T-ACE2 cells were trypsinized, added to 96-well U-bottom plate at a cell density of 1×10 6 cells/mL, 100 μL/well, and incubated at 4°C for 30 minutes; (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd.) was diluted with 1% PBSB to a certain concentration; the mouse antibody samples to be tested were diluted to 8 μg/mL, 4-fold ratio dilution, 9 gradients, and then the diluted S-mFc Mixed with antibodies of different concentration gradients 1:1, pre-incubated at room temperature for 30 minutes, added to the above 96-well U-bottom plate, incubated at 4 °C for 1 hour; centrifuged to remove the supernatant, and washed the cells three times with 1% PBSB; Anti-mouse IgG Fc (Jackson Immuno) was diluted 1:400 with 1% PBSB, 100 μL/well, and incubated at 4°C for 1 hour; centrifuged to remove the supernatant, washed the cells three times with 1% PBSB; resuspended the cells with 1% PBSB, 150 μL/well; flow cytometry to detect signal intensity. Taking the mean fluorescence intensity as the Y-axis and the antibody concentration as the X-axis, 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.
如图6-1至图6-3和表3所示，细胞水平上，抗SARS-CoV-2鼠源抗体S1B-73-3、S1B-91-3、S1B-82-5、S1B-30-3、S1B-48-2、S1B-14-3、ST-10-4和ST-35-4均能很好的竞争阻断S蛋白与ACE2的结合，IC 50处于5-20ng/mL之间，其中鼠抗S1B-73-3阻断效果最好，其次鼠抗S1B-91-3、S1B-30-3、S1B-48-2、ST-35-4阻断效果相当，而鼠抗S1B-82-5、S1B-14-3、ST-10-4阻断作用较弱。 As shown in Figure 6-1 to Figure 6-3 and Table 3, at the cellular level, 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.
表3、抗SARS-CoV-2鼠源抗体阻断刺突S蛋白与293T-ACE2细胞的结合IC 50 Table 3. Anti-SARS-CoV-2 murine antibody blocking the binding IC 50 of Spike S protein to 293T-ACE2 cells
鼠抗名称Mouse Antibody Name IC 50(ng/mL) IC50 (ng/mL)
S1B-73-3S1B-73-3 6.136.13
S1B-48-2S1B-48-2 10.7710.77
S1B-91-3S1B-91-3 13.7013.70
S1B-82-5S1B-82-5 15.9615.96
S1B-30-3S1B-30-3 11.9311.93
S1B-14-3S1B-14-3 16.7916.79
ST-10-4ST-10-4 16.5516.55
ST-35-4ST-35-4 11.7911.79
实施例4、采用计算机分子对接技术评估抗SARS-CoV-2鼠源抗体对RBD/ACE2之间结合能力的影响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
采用计算机软件Discovery Studio对鼠源抗体S1B-73-3、S1B-15-5、S1B-30-3、S1B-48-2、S1B-82-5以及S1B-91-3进行结构建模，并对这些鼠源抗体与其抗原RBD结构域的分子对接空间构象进行模拟，预测S蛋白鼠源抗体在RBD结构域上的结合位点，评估抗体对RBD/ACE2之间结合能力的影响。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.
运用Discovery Studio软件，构建鼠源抗体三维结构模型。建模采取3个步骤进行：1、搜索与鼠源抗体轻链和重链可变区分别具有氨基酸序列高相似度的三维结构模板。搜索与整体鼠源抗体可变区(轻重链 合在一起)具有氨基酸序列高相似度的三维结构模板，以便确定鼠源抗体可变区中轻链和重链的相对取向；2、运用步骤1获得的3个结构模型模板以及鼠源抗体可变区轻重链氨基酸序列，构建鼠源抗体骨架区结构模型；3、在步骤2的基础上，构建六个CDR环状区的结构模型。分子对接模拟计算中的RBD结构模型采用蛋白质数据库中
Figure PCTCN2021086477-appb-000005
高分辨率的RBD结构(PDB ID 6M0J)。通过比较两个RBD结构(PDB ID 6M0J和6W41)，把6M0J中的F486残基的侧链构象调整为与6W41中的一致，即rotamer1构象。这个构象占有率最高，而且，6M0J中RBD由于结合ACE2的缘故，F486的侧链不以这个构象存在。分子对接的软件采用的是Discovery Studio软件包中的ZDOCK软件。分子对接模拟实验所用参数均采取默认值。鼠源抗体作为分子对接受体，RBD作为分子对接配体。受体封闭氨基酸(receptor blocked residues)选取远离CDR区、空间位置位于CDR区反方向的可变区氨基酸。受体结合位点氨基酸(receptor binding site residues)选取HCDR3loop暴露在蛋白表面最顶端的一个氨基酸。鼠源抗体S1B-73-3、S1B-48-2、S1B-82-5、S1B-91-3、S1B-30-3、S1B-14-3、ST-10-4和ST-35-4与RBD的分子对接结果参见图7和图8。通过RBD结构叠合引入ACE2(PDB 6M0J,Lan J et al,2020,Nature,581:215-220)和抗体CR3022结构(PDB 6W41,Yuan M et al,2020,Science,368:630-633)。分子对接结果表明，鼠源抗体S1B-73-3、S1B-48-2、S1B-82-5、S1B-91-3、S1B-30-3、S1B-14-3、ST-10-4和ST-35-4与ACE2竞争结合RBD，能够阻断RBD与ACE2之间的结合，为这些抗体能够抑制SARS-CoV-2病毒感染宿主细胞提供了分子水平上的合理解释。 Using the Discovery Studio software, 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
Figure PCTCN2021086477-appb-000005
High-resolution RBD structure (PDB ID 6MOJ). By comparing the two RBD structures (PDB ID 6M0J and 6W41), 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.
实施例5、SARS-CoV-2冠状病毒S1蛋白鼠源抗体的人源化Example 5. Humanization of SARS-CoV-2 coronavirus S1 protein murine antibody
我们采用CDR移植方法(CDR grafting)对鼠源抗体进行人源化改造。CDR grafting的基本原理是通过把鼠抗的CDR区移植到人源抗体模板上，同时把稳定CDR构象和对抗原-抗体结合重要的几个或一些关键鼠抗FR区残基，也一并引入到人源抗体模板上(backmutations)，从而达到既降低鼠抗的免疫原性又保持鼠抗的亲和力的目的。除了上述CDR grafting操作外，我们还进一步对CDR grafting后的人源化抗体的等电点(PI)、疏水聚集(aggregation)、翻译后修饰(PTM，如糖基化、断裂、异构化位点等)和免疫原性(immunogenicity)四方面进行计算，对于造成这四方面问题的氨基酸进行突变，以便使人源化抗体在临床使用时充分发挥出药效。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. On the human antibody template (backmutations), so as to achieve the purpose of reducing the immunogenicity of the mouse antibody and maintaining the affinity of the mouse antibody. In addition to the above CDR grafting operations, we also further investigated the isoelectric point (PI), hydrophobic aggregation (aggregation), and post-translational modification (PTM) of the humanized antibody after CDR grafting, such as glycosylation, fragmentation, and isomerization. Points, etc.) and immunogenicity (immunogenicity) are calculated, and the amino acids that cause these four problems are mutated, so that the humanized antibody can fully exert its efficacy in clinical use.
抗体人源化的具体流程如下。搜索IMGT网站的人抗体胚系数据库(IMGT human antibody germline database,http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)，获得与鼠抗具有高相似度的人源抗体模板(表4)。用Discovery Studio对鼠抗和人源抗体模板进行CDR区注释，按Kabat或IMGT方案定义出CDR区(表5)。用鼠抗的六个CDR区分别替换人源抗体模板的六个CDR区。移植的6个CDR区中的单独每个CDR区可以是按Kabat定义出的氨基酸区域，或按IMGT定义出的氨基酸区域。CDR移植后进行从鼠源抗体到人源化模板FR区的回复突变。稳定抗体CDR区构象和对抗原-抗体结合重要的关键鼠抗FR区氨基酸包括4类氨基酸残基：1)CDR区
Figure PCTCN2021086477-appb-000006
以内埋藏在抗体表面下的氨基酸；2)CDR区
Figure PCTCN2021086477-appb-000007
以内暴露在抗体表面的氨基酸；3)抗体轻链和重链结构域之间的界面氨基酸；和4)稳定抗体CDR区构象的vernier zone residues(Foote J and Winter G,1992,J Mol Biol,224:487-499)。以上4类关键鼠抗FR区残基是通过建立鼠抗三维结构模型确定的。对于与鼠抗序列不一致的人源模板的这4类氨基酸，通过三维结构分析，选择对保 持CDR构象和抗原-抗体结合重要的氨基酸，进行从鼠抗到人源模板的氨基酸移植或替换。然后，对4类氨基酸移植后产生的人源化抗体进一步计算等电点、疏水聚集、翻译后修饰和免疫原性，对问题氨基酸进行突变，从而得到最终的人源化抗体序列(表6)。图9-1至图14-2分别显示了上述人源化抗体与其亲本鼠源抗体的重链可变区和轻链可变区氨基酸序列的比对结果。 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. Backmutation of the FR regions from the murine antibody to the humanized template was performed after CDR grafting. 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
Figure PCTCN2021086477-appb-000006
Amino acids buried under the surface of the antibody; 2) CDR regions
Figure PCTCN2021086477-appb-000007
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. For these four types of amino acids in the human template that are inconsistent with the mouse antibody sequence, 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.
表4、示例性的鼠源抗体在人源化时所用人抗体胚系模板Table 4. Human Antibody Germline Templates for Humanization of Exemplary Murine Antibodies
   VHVH VLVL
S1B-73-3S1B-73-3 IGHV1-69*02IGHV1-69*02 IGKV1-16*01IGKV1-16*01
S1B-48-2S1B-48-2 IGHV1-46*01IGHV1-46*01 IGKV4-1*01IGKV4-1*01
S1B-91-3S1B-91-3 IGHV1-69*02IGHV1-69*02 IGKV2-28*01IGKV2-28*01
S1B-82-5S1B-82-5 IGHV3-11*01IGHV3-11*01 IGKV1-9*01IGKV1-9*01
S1B-30-3S1B-30-3 IGHV3-7*01IGHV3-7*01 IGKV1-39*01IGKV1-39*01
S1B-14-3S1B-14-3 IGHV1-69*02IGHV1-69*02 IGKV3-11*01IGKV3-11*01
表5、鼠源抗体可变区中的CDR区Table 5. CDR regions in the variable regions of murine antibodies
Figure PCTCN2021086477-appb-000008
Figure PCTCN2021086477-appb-000008
Figure PCTCN2021086477-appb-000009
Figure PCTCN2021086477-appb-000009
Figure PCTCN2021086477-appb-000010
Figure PCTCN2021086477-appb-000010
Figure PCTCN2021086477-appb-000011
Figure PCTCN2021086477-appb-000011
Figure PCTCN2021086477-appb-000012
Figure PCTCN2021086477-appb-000012
Figure PCTCN2021086477-appb-000013
Figure PCTCN2021086477-appb-000013
Figure PCTCN2021086477-appb-000014
Figure PCTCN2021086477-appb-000014
Figure PCTCN2021086477-appb-000015
Figure PCTCN2021086477-appb-000015
表6、鼠源抗体，对应的人源化抗体以及它们的可变区氨基酸序列Table 6. Murine antibodies, corresponding humanized antibodies and their variable region amino acid sequences
抗体编号Antibody number VH氨基酸序列VH amino acid sequence VL氨基酸序列VL amino acid sequence
S1B-73-3S1B-73-3 SEQ ID NO:12SEQ ID NO: 12 SEQ ID NO:13SEQ ID NO: 13
S1B-48-2S1B-48-2 SEQ ID NO:27SEQ ID NO: 27 SEQ ID NO:28SEQ ID NO: 28
S1B-91-3S1B-91-3 SEQ ID NO:42SEQ ID NO: 42 SEQ ID NO:43SEQ ID NO: 43
S1B-82-5S1B-82-5 SEQ ID NO:57SEQ ID NO: 57 SEQ ID NO:58SEQ ID NO: 58
S1B-30-3S1B-30-3 SEQ ID NO:72SEQ ID NO: 72 SEQ ID NO:73SEQ ID NO: 73
S1B-14-3S1B-14-3 SEQ ID NO:87SEQ ID NO: 87 SEQ ID NO:88SEQ ID NO: 88
S1B-34-4S1B-34-4 SEQ ID NO:93SEQ ID NO: 93 SEQ ID NO:94SEQ ID NO: 94
S1B-8-2S1B-8-2 SEQ ID NO:97SEQ ID NO: 97 SEQ ID NO:98SEQ ID NO: 98
S1B-64-2S1B-64-2 SEQ ID NO:107SEQ ID NO: 107 SEQ ID NO:108SEQ ID NO: 108
ST-10-4ST-10-4 SEQ ID NO:122SEQ ID NO: 122 SEQ ID NO:123SEQ ID NO: 123
ST-35-4ST-35-4 SEQ ID NO:137SEQ ID NO: 137 SEQ ID NO:138SEQ ID NO: 138
hS1B-73-3hS1B-73-3 SEQ ID NO:142SEQ ID NO: 142 SEQ ID NO:143SEQ ID NO: 143
hS1B-48-2hS1B-48-2 SEQ ID NO:147SEQ ID NO: 147 SEQ ID NO:148SEQ ID NO: 148
hS1B-91-3hS1B-91-3 SEQ ID NO:14SEQ ID NO: 14 SEQ ID NO:15SEQ ID NO: 15
hS1B-82-5hS1B-82-5 SEQ ID NO:44SEQ ID NO: 44 SEQ ID NO:45SEQ ID NO: 45
hS1B-30-3hS1B-30-3 SEQ ID NO:74SEQ ID NO: 74 SEQ ID NO:75SEQ ID NO: 75
hS1B-14-3-1hS1B-14-3-1 SEQ ID NO:154SEQ ID NO: 154 SEQ ID NO:155SEQ ID NO: 155
hS1B-14-3-2hS1B-14-3-2 SEQ ID NO:161SEQ ID NO: 161 SEQ ID NO:162SEQ ID NO: 162
hST-10-4hST-10-4 SEQ ID NO:124SEQ ID NO: 124 SEQ ID NO:125SEQ ID NO: 125
hST-35-4hST-35-4 SEQ ID NO:169SEQ ID NO: 169 SEQ ID NO:170SEQ ID NO: 170
为了获得由两条重链和两条轻链组成的全长抗体序列，将表6中所示VH和VL序列的与抗体重链恒定区(优选自人IgG1、IgG2或IgG4)和轻链恒定区(优选自人κ轻链，氨基酸序列如SEQ ID NO：95所示)序列采用常规技术进行拼接或组装。例如，在一种实施方案中，人源化抗体分子包含野生型人IgG1的重链恒定区(氨基酸序列如SEQ ID NO：96所示)。在一种实施方案中，人源化抗体分子包括在根据EU编号的含有M252Y、S254T、T256E和M428L突变的人IgG1的重链恒定区(氨基酸序列如SEQ ID NO：190所示)。在另一种实施方案中，人源化抗体分子包含野生型人IgG2的重链恒定区(氨基酸序列如SEQ ID NO：99所示)。或采用修饰的人IgG2恒定区序列；在一种实施方案中，人源化抗体分子包括在根据EU编号的铰链区修饰的的人IgG2(例如缺失ERKCC，氨基酸序列如SEQ ID NO：100所示)。在另一种实施方案中，人源化抗体分子包含野生型人IgG4的重链恒定区(氨基酸序列如SEQ ID NO：109所示)。或采用修饰的人IgG4恒定区序列；在一种实施方案中，人源化抗体分子包括在根据EU编号的第228位突变(例如S变为P)的人IgG4(氨基酸序列如SEQ ID NO：110所示)。To obtain a full-length antibody sequence consisting of two heavy chains and two light chains, 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) is spliced or assembled using conventional techniques. For example, in one embodiment, 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). In one embodiment, 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). Or 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) ). In another embodiment, 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).
表7、人κ轻链和突变的人IgG重链的恒定区氨基酸序列Table 7. Constant region amino acid sequences of human kappa light chain and mutated human IgG heavy chain
Figure PCTCN2021086477-appb-000016
Figure PCTCN2021086477-appb-000016
Figure PCTCN2021086477-appb-000017
Figure PCTCN2021086477-appb-000017
实施例6、人源化抗体表达载体的构建和蛋白表达Example 6. Construction and protein expression of humanized antibody expression vector
用上述方法中获得的人源化抗体的重链和轻链的编码cDNA插入到PcDNA3.1或其衍生质粒，或其它真核表达载体中，构建人源化抗体表达载体。优选地，使用的载体质粒应含有在哺乳动物细胞中高水平表达所需的巨细胞病毒早期基因启动因子-增强子。同时，载体质粒中含有可选择标记基因，从而在细菌中赋予氨苄青霉素抗性，而在哺乳动物细胞中赋予G418抗性。另外，载体质粒中含有DHFR基因，在合适的宿主细胞中，能以氨甲喋呤(Methotrexate,MTX,Sigma)共扩增人源化抗体基因和DHFR基因(例如，参见专利CN103333917B)。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. Preferably, the vector plasmid used should contain the cytomegalovirus early gene promoter-enhancer required for high-level expression in mammalian cells. At the same time, the vector plasmid contains a selectable marker gene that confers ampicillin resistance in bacteria and G418 resistance in mammalian cells. In addition, 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).
将上述已构建的重组表达载体质粒转染入哺乳动物宿主细胞系，以表达人源化抗体。为了稳定高水平的表达，优选的宿主细胞系是二氢叶酸还原酶(DHFR)缺陷型的中国仓鼠卵巢(CHO)细胞(参见，例如Chasin,L.等人的美国专利4818679号)。优选的转染方法是电穿孔，也可以使用其他方法，包括磷酸钙共沉降，脂转染和原生质融合等。在电穿孔中，用设为250V电场和960μFd电容的Gene Pulser(Bio-Rad Laboratories)，在比色杯内加入2×10 7个细胞悬浮在0.8ml的PBS中，并含有10μg用PvuI(Takara)线性化的表达载体质粒DNA。转染2天后，加入含有0.2mg/mL G418以及200nM氨甲喋呤(methotrexate或MTX)。为了实现较高水平的表达，用受MTX药物抑制的DHFR基因共扩增转染的人源化抗体基因。用极限稀释亚克隆转染子及ELISA的方法测定各细胞系的分泌率，选出高水平表达人源化抗体的细胞株。收集人源化抗体的条件培养基，用于测定其体外和体内生物学活性。 The recombinant expression vector plasmids constructed above are transfected into mammalian host cell lines to express humanized antibodies. For stable high-level expression, 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.). The preferred method of transfection is electroporation, but other methods can also be used, including calcium phosphate co-precipitation, lipofection, and protoplast fusion, among others. In electroporation, using a Gene Pulser (Bio-Rad Laboratories) set to an electric field of 250 V and a capacitance of 960 μFd, 2×10 7 cells were added to a cuvette suspended in 0.8 ml of PBS containing 10 μg of PvuI (Takara ) linearized expression vector plasmid DNA. Two days after transfection, G418 containing 0.2 mg/mL and 200 nM methotrexate (methotrexate or MTX) was added. To achieve higher levels of expression, the transfected humanized antibody gene was co-amplified with the DHFR gene inhibited by the MTX drug. 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.
例如，将表8所示编码人源化抗体的重链和轻链的核苷酸序列插入上述构建的表达载体中，经过加压筛选、亚克隆稳定、高表达目的抗体的细胞株，再经培养、纯化得到各目的抗体。For example, the 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.
表8、人源化抗体重链和轻链的氨基酸序列及其编码核苷酸序列Table 8. Amino acid sequences of humanized antibody heavy and light chains and their encoding nucleotide sequences
抗体编号Antibody number HC氨基酸序列HC amino acid sequence LC氨基酸序列LC amino acid sequence HC核苷酸序列HC nucleotide sequence LC核苷酸序列LC nucleotide sequence
hS1B-73-3hS1B-73-3 SEQ ID NO：144SEQ ID NO: 144 SEQ ID NO：145SEQ ID NO: 145 SEQ ID NO：173SEQ ID NO: 173 SEQ ID NO：174SEQ ID NO: 174
hS1B-48-2hS1B-48-2 SEQ ID NO：149SEQ ID NO: 149 SEQ ID NO：150SEQ ID NO: 150 SEQ ID NO：175SEQ ID NO: 175 SEQ ID NO：176SEQ ID NO: 176
hS1B-91-3hS1B-91-3 SEQ ID NO：29SEQ ID NO: 29 SEQ ID NO：30SEQ ID NO: 30 SEQ ID NO：177SEQ ID NO: 177 SEQ ID NO：178SEQ ID NO: 178
hS1B-82-5hS1B-82-5 SEQ ID NO：59SEQ ID NO: 59 SEQ ID NO：60SEQ ID NO: 60 SEQ ID NO：179SEQ ID NO: 179 SEQ ID NO：180SEQ ID NO: 180
hS1B-30-3hS1B-30-3 SEQ ID NO：89SEQ ID NO: 89 SEQ ID NO：90SEQ ID NO: 90 SEQ ID NO：181SEQ ID NO: 181 SEQ ID NO：182SEQ ID NO: 182
hS1B-14-3-1hS1B-14-3-1 SEQ ID NO：156SEQ ID NO: 156 SEQ ID NO：157SEQ ID NO: 157 SEQ ID NO：183SEQ ID NO: 183 SEQ ID NO：184SEQ ID NO: 184
hST-10-4hST-10-4 SEQ ID NO：139SEQ ID NO: 139 SEQ ID NO：140SEQ ID NO: 140 SEQ ID NO：185SEQ ID NO: 185 SEQ ID NO：186SEQ ID NO: 186
hST-35-4hST-35-4 SEQ ID NO：171SEQ ID NO: 171 SEQ ID NO：172SEQ ID NO: 172 SEQ ID NO：187SEQ ID NO: 187 SEQ ID NO：188SEQ ID NO: 188
实施例7、人源化抗体的功能鉴定Example 7. Functional identification of humanized antibodies
7.1、间接ELISA法测定人源化抗体与SARS-CoV-2 S三聚体抗原的结合能力7.1. Determination of the binding ability of humanized antibody to SARS-CoV-2 S trimer antigen by indirect ELISA
以SARS-CoV-2 S三聚体(ACRO BioSystem)包被酶标板，室温过夜。弃去包被溶液，用溶解在PBS缓冲液的脱脂奶粉封闭1h，用PBST(pH7.4，PBS含0.05％吐温-20)洗板3～4次。然后每孔分别加入100μl纯化的抗SARS-CoV-2 S1 RBD人源化抗体hS1B-73-3及针对SARS-CoV-2 S1 RBD的受体hACE2-Fc(ACRO BioSystem)，室温孵育1h，用含有0.05％吐温(Tween)20的PBS洗孔，然后每孔加入100μl HRP标记的羊抗人IgG多克隆抗体(Jackson Laboratory)作为检测抗体，再以PBST洗板3～4次、加入底物TMB显色10分钟、然后加入0.2M H 2SO 4终止反应，后读取吸光度值(OD值)，其结果示于图15-1至图15-5。 Plates were coated with SARS-CoV-2 S trimer (ACRO BioSystem) 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 (pH 7.4, PBS containing 0.05% Tween-20). Then, 100 μl of purified anti-SARS-CoV-2 S1 RBD humanized antibody hS1B-73-3 and the SARS-CoV-2 S1 RBD receptor hACE2-Fc (ACRO BioSystem) were added to each well, incubated at room temperature for 1 h, The wells were washed with PBS containing 0.05% Tween 20, and then 100 μl of HRP-labeled goat anti-human IgG polyclonal antibody (Jackson Laboratory) was added to each well as the detection antibody, and then the plate was washed 3 to 4 times with PBST, and the substrate was added. TMB developed color for 10 minutes, then 0.2MH 2 SO 4 was added to stop the reaction, and then the absorbance value (OD value) was read, and the results were shown in Figure 15-1 to Figure 15-5.
7.2、竞争ELISA法测定人源化抗体的阻断SARS-CoV-2 S三聚体结合ACE2的能力7.2. Competitive ELISA assay to determine the ability of humanized antibodies to block the binding of SARS-CoV-2 S trimer to ACE2
用PBS缓冲液将SARS-CoV-2 S三聚体蛋白(ACRO BioSystem)稀释至0.1μg/ml，以100μl/孔加于96孔板中，室温过夜。弃去包被溶液，每孔加入200μl PBST/1％脱脂奶粉，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板3次后，然后每孔加入100μl以辣根过氧化物酶(HRP)标记的hACE2-Fc分别和人源化抗体hS1B-73-3及针对SARS-CoV-2 S1 RBD的受体hACE2-Fc的混合物。以PBST作为空白对照。充分孵育后以PBS洗去未结合的HRP标记的hACE2-Fc，室温孵育1h。再以PBST洗板3次后，每孔加入100μl TMB，室温孵育5-10min。最后，每孔加入50μl 0.2M H 2SO 4终止反应，并用酶标仪在双波长450/620nm处读取OD值。其结果示于图16-1至图16-5。 SARS-CoV-2 S trimer protein (ACRO BioSystem) was diluted to 0.1 μg/ml with PBS buffer and added to 96-well plates at 100 μl/well, overnight at room temperature. The coating solution was discarded, 200 μl 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 μl 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. After sufficient incubation, the unbound HRP-labeled hACE2-Fc was washed away with PBS, and incubated at room temperature for 1 h. After washing the plate three times with PBST, 100 μl of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50 μl of 0.2M 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 results are shown in Figs. 16-1 to 16-5.
7.3、竞争ELISA法测定人源化抗体阻断SARS-CoV-2 S1与ACE2的结合能力7.3. Competitive ELISA to determine the ability of humanized antibodies to block the binding of SARS-CoV-2 S1 to ACE2
竞争ELISA法测定人源化抗体阻断SARS-CoV-2 S1与ACE2的结合能力实验方法参见实施例2.3。The competition ELISA method to determine the ability of humanized antibodies to block the binding of SARS-CoV-2 S1 to ACE2 is shown in Example 2.3.
结果如表9所示，上述人源化抗体能与hACE2竞争结合SARS-CoV-2 S1，即通过阻断SARS-CoV-2 S1和hACE2的结合发挥功能。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.
表9、人源化抗体竞争结合hACE2的能力Table 9. The ability of humanized antibodies to compete for binding to hACE2
   EC 50(μg/ml) EC50 (μg/ml)
hS1B-73-3hS1B-73-3 0.025390.02539
hS1B-48-2hS1B-48-2 0.019650.01965
hS1B-91-3hS1B-91-3 0.073770.07377
hS1B-82-5hS1B-82-5 0.165880.16588
hS1B-30-3hS1B-30-3 0.033070.03307
hACE2-FchACE2-Fc 1.314701.31470
7.4、生物薄膜干涉技术测定抗SARS-CoV-2 S1人源化抗体的动力学常数和亲和力7.4. Determination of kinetic constants and affinity of anti-SARS-CoV-2 S1 humanized antibody by biofilm interference technology
人源化抗体动力学常数和亲和力平衡解离常数测定的实验方法参见实施例2.4。实验结果见表10。与鼠抗相比，人源化抗体的亲和力平衡解离常数保持在同一pM级别。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.
表10、人源化抗体的动力学常数和亲和力测定结果Table 10. Kinetic constants and affinity determination results of humanized antibodies
人源化单抗Humanized mAb K D(M) K D (M) K a(M/s) K a (M/s) K d(1/s) K d (1/s)
hS1B-73-3hS1B-73-3 2.07E-112.07E-11 1.07E+061.07E+06 2.22E-052.22E-05
hS1B-48-2hS1B-48-2 1.13E-121.13E-12 8.67E+058.67E+05 9.81E-079.81E-07
hS1B-91-3hS1B-91-3 6.41E-126.41E-12 6.26E+056.26E+05 4.01E-064.01E-06
hS1B-82-5hS1B-82-5 <1.0E-12<1.0E-12 2.97E+052.97E+05 <1.0E-07<1.0E-07
hS1B-30-3hS1B-30-3 2.09E-122.09E-12 7.88E+057.88E+05 1.65E-061.65E-06
CR3022CR3022 8.66E-118.66E-11 2.20E+052.20E+05 1.90E-051.90E-05
hACE2-FchACE2-Fc 1.40E-091.40E-09 2.34E+042.34E+04 3.28E-053.28E-05
7.5、抗SARS-CoV-2 S1人源化抗体阻断刺突S蛋白与293T-ACE2细胞的结合7.5. Anti-SARS-CoV-2 S1 humanized antibody blocks the binding of spike S protein to 293T-ACE2 cells
刺突S蛋白与293T-ACE2细胞结合的阻断实验方法参见实施例3。实验结果见图17-1至图17-2和表11。细胞水平上，抗SARS-CoV-2 S1人源化抗体均能很好的竞争阻断S蛋白与ACE2的结合，IC 50处于4-20ng/mL之间，其中hS1B-73-3阻断效果最好，其次人源化抗体hS1B-91-3、hS1B-30-3、hS1B-48-2、hS1B-14-3阻断效果相当，而人源化抗体hS1B-82-5阻断作用较弱。与鼠源抗体相比较，人源化之后抗体的阻断效果没有显著变化。 See Example 3 for the experimental method of blocking the binding of spike S protein to 293T-ACE2 cells. The experimental results are shown in Figure 17-1 to Figure 17-2 and Table 11. At the cellular level, anti-SARS-CoV-2 S1 humanized antibodies can compete well to block the binding of S protein and ACE2, with IC 50 between 4-20ng/mL, among which hS1B-73-3 has a blocking effect. The best, secondly, humanized antibodies hS1B-91-3, hS1B-30-3, hS1B-48-2, hS1B-14-3 had comparable blocking effects, while humanized antibodies hS1B-82-5 had better blocking effects. weak. Compared with the murine antibody, the blocking effect of the antibody did not change significantly after humanization.
表11、抗SARS-CoV-2 S1人源化抗体阻断刺突S蛋白与293T-ACE2细胞的结合IC 50 Table 11. IC50 of anti-SARS-CoV-2 S1 humanized antibody blocking the binding of spike S protein to 293T-ACE2 cells
人源化抗体名称Humanized Antibody Name IC 50(ng/mL) IC50 (ng/mL)
hS1B-73-3hS1B-73-3 4.334.33
hS1B-91-3hS1B-91-3 8.138.13
hS1B-82-5hS1B-82-5 19.2919.29
hS1B-30-3hS1B-30-3 6.916.91
hS1B-48-2hS1B-48-2 5.825.82
hS1B-14-3hS1B-14-3 7.547.54
7.6、抗SARS-CoV-2 S1人源化单克隆抗体外抑制假病毒实验7.6. Anti-SARS-CoV-2 S1 Humanized Monoclonal Antibody Inhibition Pseudovirus Experiment
通过使用新冠S蛋白假病毒与待测抗体孵育后侵染293T-ACE2细胞，采用化学发光法检测Luciferase发光值RLU，根据RLU读值计算待检抗体的假病毒抑制率，评价待检抗体的中和效果。新冠S蛋白假病毒基因组编码萤火虫荧光素酶，当病毒基因组进入细胞整合后，萤火虫荧光素酶的表达和活性与转导的细胞数量成正比。假病毒与真病毒相比只能一次性感染细胞。By infecting 293T-ACE2 cells with the new coronavirus S protein pseudovirus after incubation with the antibody to be tested, 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.
人源化抗体对野生型(WT)假病毒或突变株假病毒(B.1.1.7、B.1.351或E484K)感染的抑制实验，具体步骤如下。293T-ACE2细胞(上海翊圣生物)，用培养基DMEM+10％FBS培养至对数生长期，以3000个细胞/孔接种于384孔白板中，并放入37℃，5％CO 2培养箱中培养过夜。待测抗体样品用含10％FBS的DMEM稀释，起始浓度为10μg/mL，5倍比稀释，9梯度；野生型(WT)假病毒或突变株假病毒(B.1.1.7、B.1.351或E484K)(吉满生物)从-80℃取出，4℃复融，将复融后的假病毒稀释125倍，作为工作液；稀释后的待测抗体和假病毒工作液分别以50μL/孔加入到96孔U底板中混匀，37℃预孵育30分钟；然后以 20μL/孔加入到前一天细胞铺板的384孔白板中，阳性对照组：假病毒工作液和含10％FBS的DMEM培养基分别以10μL/孔加入到384孔白板中。阴性对照组：含10％FBS的DMEM培养基以20μL/孔加入到384孔白板中，设4复孔。24小时后，以30μL/孔加入含10％FBS的DMEM培养基，继续放入细胞培养箱中培养24小时；用移液器小心吸掉上清，以30μL/孔加入新配的荧光素酶显色液，室温孵育5分钟，将384孔板置于酶标仪，读取每孔的化学荧光信号。抑制率(％)＝[1-(样品RLU信号值-阴性对照RLU信号值)/(阳性对照RLU信号值-阴性对照RLU信号值)]×100。再以抑制率作为Y轴，抗体浓度作为X轴，通过软件GraphPad Prism 6进行分析，得出抗体的量效曲线，使用非线性回归曲线计算半数抑制浓度(IC 50)。 Inhibition experiments of humanized antibodies on wild-type (WT) pseudovirus or mutant pseudovirus (B.1.1.7, B.1.351 or E484K) infection, the specific steps are as follows. 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 were diluted with DMEM containing 10% FBS, the initial concentration was 10 μg/mL, 5-fold 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, 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 μL/ The wells were added to a 96-well U-bottom plate and mixed, and pre-incubated at 37°C for 30 minutes; then 20 μL/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 μL/well. Negative control group: DMEM medium containing 10% FBS was added to 384-well white plate at 20 μL/well, and 4 wells were set up. After 24 hours, add DMEM medium containing 10% FBS at 30 μL/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 μL/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. Taking the inhibition rate as the Y axis and the antibody concentration as the X axis, 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 ).
表12-1至表12-5显示了抗SARS-CoV-2 S1人源化单克隆抗体外抑制假病毒的实验结果。如图18-1至图18-2和表12-1所示，人源化单克隆抗体hS1B-91-3和hS1B-30-3均能显著地，且呈剂量依赖性地的抑制野生型新冠假病毒侵染293T-ACE2细胞，能够阻断SARS-CoV-2的感染对宿主细胞的早期入侵，起到保护作用。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. As shown in Figure 18-1 to Figure 18-2 and Table 12-1, 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.
此外，如图18-3和表12-2至表12-5所示，hS1B-82-5、hS1B-48-2和hS1B-14-3均能很好的抑制野生型和B.1.1.7的新冠假病毒感染，同时hS1B-82-5、hS1B-48-2和hS1B-14-3也能很好的抑制B.1.351或E484K新冠假病毒突变株感染，并且hS1B-82-5或hS1B-48-2在B.1.351突变株上中和作用明显优于野生型的，hS1B-48-2的表现更好。In addition, as shown in Figure 18-3 and Table 12-2 to Table 12-5, 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.
表12-1、抗SARS-CoV-2 S1人源化抗体野生型假病毒抑制结果Table 12-1. Inhibition results of wild-type pseudovirus of anti-SARS-CoV-2 S1 humanized antibody
Figure PCTCN2021086477-appb-000018
Figure PCTCN2021086477-appb-000018
表12-2、抗SARS-CoV-2 S1人源化抗体野生型假病毒抑制结果Table 12-2. Inhibition results of wild-type pseudovirus of anti-SARS-CoV-2 S1 humanized antibody
Figure PCTCN2021086477-appb-000019
Figure PCTCN2021086477-appb-000019
表12-3、抗SARS-CoV-2 S1人源化抗体假病毒突变株(B.1.1.7)抑制结果Table 12-3. Inhibition results of anti-SARS-CoV-2 S1 humanized antibody pseudovirus mutant strain (B.1.1.7)
Figure PCTCN2021086477-appb-000020
Figure PCTCN2021086477-appb-000020
表12-4、抗SARS-CoV-2 S1人源化抗体假病毒突变株(B.1.351)抑制结果Table 12-4. Inhibition results of anti-SARS-CoV-2 S1 humanized antibody pseudovirus mutant strain (B.1.351)
Figure PCTCN2021086477-appb-000021
Figure PCTCN2021086477-appb-000021
表12-5、抗SARS-CoV-2 S1人源化抗体对假病毒单点突变(E484K)抑制结果Table 12-5. Inhibition results of anti-SARS-CoV-2 S1 humanized antibody to pseudovirus single point mutation (E484K)
Figure PCTCN2021086477-appb-000022
Figure PCTCN2021086477-appb-000022
实施例8、S蛋白RBD结构域上的突变对本发明的抗SARS-CoV-2抗体的影响Example 8. Effect of mutation on the RBD domain of S protein on the anti-SARS-CoV-2 antibody of the present invention
与野生型SARS-CoV-2病毒不同，B.1.351南非突变株S蛋白RBD结构域包含3个氨基酸突变，分别是K417N，E484K和N501Y。这三个氨基酸K417、E484和N501基本上位于S蛋白RBD结构域与这些抗体结合的界面上(图19-1)，这个界面也是S蛋白RBD结构域与人源ACE2分子结合的界面(图19-2A)。几个已公开的SARS-CoV-2冠状病毒抗体，针对SARS-CoV-2冠状病毒南非突变株B.1.351都失活或中和活性减弱了，例如，礼来公司的抗体LY-CoV55、LY-CoV016和再生元的REGN-10933抗体失活，以及阿斯利康的COV2-2196中和活性减弱14.6倍(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)。运用Discovery Studio软件，我们计算了这三个界面氨基酸突变导致的抗原/抗体结合自由能变化，可以很好地解释为什么以上这些已公开抗体对SARS-CoV-2病毒突变株B.1.351失活或活性减弱。例如，LY-CoV555抗体/RBD结合界面上主要的氨基酸突变E484K导致6.54Kcal/mol自由能变化，LY-CoV016抗体/RBD结合界面上主要的氨基酸突变K417N导致0.75Kcal/mol自由能变化，REGN-10933抗体/RBD结合界面上主要的氨基酸突变K417N和E484K分别导致了0.68Kcal/mol和2.0Kcal/mol自由能变化。自由能变化大于0.5Kcal/mol的突变，就对抗原/抗体结合力有明显影响；突变导致的自由能变化为正值，表明这种突变降低抗原/抗体结合力。由于以上这些突变导致这些抗体不再能够结合B.1.351病毒株S蛋白RBD结构域或结合减弱，这些抗体针对B.1.351突变株就失去中和活性或中和活性降低。Different from the wild-type SARS-CoV-2 virus, 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). Several published 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). Using Discovery Studio software, we calculated the changes in the free energy of antigen/antibody binding caused by amino acid mutations at these three interfaces, which can well explain why these published antibodies are inactive or inactive against SARS-CoV-2 virus mutant B.1.351. Reduced activity. For example, 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, and 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.
通过竞争ELISA法阻断ACE2/RBD结合的实验、抗体针对野生型及三种SARS-CoV-2假病毒突变株的中和能力比较、以及抗体/RBD的分子对接计算模拟，我们确定出本发明提供的抗体hS1B-48-2或ST-35-4在S蛋白RBD结构域上的结合表位。竞争ELISA法阻断ACE2/RBD结合的实验结果表明，抗体hS1B-48-2 或ST-35-4均能阻断ACE2与RBD之间的结合(参见实施例2.3和实施例3，分别为分子和细胞阻断)。这说明抗体hS1B-48-2或ST-35-4在RBD结构域上结合的抗原表位，与RBD和ACE2结合的界面相似或相同。这个RBD界面可以用空间上呈三角形分布的三个RBD残基E484、N501和K417来大致界定(图19-2A)。进一步地，如实施例2.5和实施例7.6所示，我们对抗体hS1B-48-2或ST-35-4针对野生型及三种SARS-CoV-2假病毒突变株的中和能力IC 50进行了测定(表13)。与野生型SARS-CoV-2病毒不同，B.1.1.7突变株S蛋白RBD结构域包含一个氨基酸突变N501Y，而B.1.351突变株包含3个氨基酸突变(K417N，E484K，N501Y)。从结果中可以看出，针对野生型以及包含单个氨基酸突变的E484K突变株和B.1.1.7病毒株，抗体hS1B-48-2的中和活性IC 50值并没有明显差别，表明N501Y和E484K突变对抗体hS1B-48-2的中和活性没有影响；但针对包含三个氨基酸突变的B.1.351病毒株，抗体hS1B-48-2的IC 50值降低为3.9ng/mL，表明K417N突变导致抗体hS1B-48-2的中和活性升高。一个合理的解释是，K417N突变导致hS1B-48-2抗体/RBD结合能力增强，从而使hS1B-48-2抗体能更有效地中和SARS-CoV-2假病毒突变株B.1.351。至于抗体ST-35-4，相较于野生型病毒，E484K突变对抗体中和活性无明显影响，而N501Y突变导致抗体中和活性增强，IC 50值降低，K417N突变导致抗体中和活性进一步增强，IC 50进一步降低。合理的解释是，N501Y和K417N突变导致抗体ST-35-4与RBD的亲和结合力增强，从而使抗体能更有效地中和SARS-CoV-2假病毒突变株B.1.351。综合以上结果，抗体hS1B-48-2与RBD结构域的K417或其同等位置的突变残基N417结合或有相互作用；抗体ST-35-4与RBD结构域的N501和K417或其同等位置的突变残基Y501和N417结合或有相互作用。进一步地，运用ZDOCK软件，我们对抗体hS1B-48-2或ST-35-4在RBD结构域上的结合表位进行了分子对接(docking)计算模拟。计算模拟结果表明，抗体hS1B-48-2或ST-35-4在RBD上的结合表位与ACE2在RBD上的结合界面大致重叠，抗体hS1B-48-2或ST-35-4与RBD残基E484、N501和K417有相互作用，此外，与RBD残基L452也有相互作用(图19-2A、19-2B以及19-2C)。 Through the experiment of blocking ACE2/RBD binding by competitive ELISA, the comparison of the neutralization ability of the antibody against wild-type and three SARS-CoV-2 pseudovirus mutants, and the molecular docking computational simulation of the antibody/RBD, we determined that the present invention Provided the binding epitope of the antibody hS1B-48-2 or ST-35-4 on the RBD domain of the S protein. The experimental results of blocking ACE2/RBD binding by competitive ELISA showed that either antibody hS1B-48-2 or ST-35-4 could block the binding between ACE2 and RBD (see Example 2.3 and Example 3, respectively Molecular and cell block). This indicates that the antigenic epitope that antibody hS1B-48-2 or ST-35-4 binds on the RBD domain is similar or identical to the interface between RBD and ACE2. This RBD interface can be roughly delimited by three RBD residues E484, N501 and K417 distributed in a spatially triangular distribution (Figure 19-2A). Further, as shown in Example 2.5 and Example 7.6, we tested the neutralizing ability IC50 of antibody hS1B-48-2 or ST-35-4 against wild-type and three SARS-CoV-2 pseudovirus mutants. determined (Table 13). Different from the wild-type SARS-CoV-2 virus, 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). It can be seen from the results that there is no significant difference in the neutralizing activity IC50 values of the antibody hS1B-48-2 against the wild type as well as the E484K mutant strain and the B.1.1.7 virus strain containing a single amino acid mutation, indicating that N501Y and E484K The mutation had no effect on the neutralizing activity of the antibody hS1B-48-2; however, against the B.1.351 strain containing three amino acid mutations, the IC50 value of the antibody hS1B-48-2 was reduced to 3.9 ng/mL, indicating that the K417N mutation caused the The neutralizing activity of antibody hS1B-48-2 was increased. A plausible explanation is that 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. As for the antibody ST-35-4, 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. A plausible explanation is that the N501Y and K417N mutations lead to enhanced affinity binding of the antibody ST-35-4 to RBD, allowing the antibody to more effectively neutralize the SARS-CoV-2 pseudovirus mutant strain B.1.351. Based on the above results, antibody hS1B-48-2 binds or interacts with K417 of the RBD domain or the mutated residue N417 at its equivalent position; antibody ST-35-4 binds to N501 and K417 of the RBD domain or its equivalent position. Mutated residues Y501 and N417 bind or interact. Further, using ZDOCK software, we performed molecular docking computational simulations on the binding epitope of antibody hS1B-48-2 or ST-35-4 on the RBD domain. Computational simulation results show that the binding epitope of antibody hS1B-48-2 or ST-35-4 on RBD roughly overlaps with the binding interface of ACE2 on RBD, and the binding interface of antibody hS1B-48-2 or ST-35-4 with RBD residue Groups E484, N501 and K417 interact, and in addition, interact with RBD residue L452 (Figures 19-2A, 19-2B and 19-2C).
表13、抗SARS-CoV-2抗体针对野生型及三种SARS-CoV-2假病毒突变株的中和活性Table 13. Neutralizing activity of anti-SARS-CoV-2 antibodies against wild-type and three SARS-CoV-2 pseudovirus mutants
Figure PCTCN2021086477-appb-000023
Figure PCTCN2021086477-appb-000023
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (27)

  1. 一种能够特异性结合SARS-CoV-2冠状病毒S蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含的重链可变区(VH)包含至少一个、两个或三个选自下组的互补决定区(CDR)：An antibody or an antigen-binding fragment thereof capable of specifically binding to the S protein of the 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 A complementarity determining region (CDR) selected from the group consisting of:
    (i)HCDR1，其具有如SEQ ID NO：1、7、16、22、31、37、46、52、61、67、76、82、111、117、126或132所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(i) 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;
    (ii)HCDR2，其具有如SEQ ID NO：2、8、17、23、32、38、47、53、62、68、77、83、101、104、112、118、127、133、165或167所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(ii) 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
    (iii)HCDR3，其具有如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或168所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) 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;
    和/或，其包含的轻链可变区(VL)包含至少一个、两个或三个选自下组的互补决定区(CDR)：And/or, 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:
    (iv)LCDR1，其具有如SEQ ID NO：4、10、19、25、34、40、49、55、64、70、79、85、91、92、103、106、114、120、129、135、152或159所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iv) 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;
    (v)LCDR2，其具有如SEQ ID NO：5、11、20、26、35、41、50、56、65、71、80、86、115、121、130、136或141所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(v) 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
    (vi)LCDR3，其具有如SEQ ID NO：6、21、36、51、66、81、116、131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) 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 (e.g. 1, 2 or 3 substitutions, deletions or additions) of sequences;
    优选的，(i)-(vi)任一项中所述的置换为保守置换。Preferably, the substitutions described in any of (i)-(vi) are conservative substitutions.
  2. 如权利要求1所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含3个VH可变区CDR和3个VL可变区CDR，其选自下组：The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 3 VH variable region CDRs and 3 VL variable region CDRs selected from the group consisting of:
    (1)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：1、2、3、4、5或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(1) Its 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);
    (2)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：7、8、9、10、11或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(2) Its 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);
    (3)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：16、17、18、19、20或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(3) Its 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);
    (4)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：22、23、24、25、26或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如 1个，2个或3个置换、缺失或添加)的序列；(4) Its 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);
    (5)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：31、32、33、34、35或36所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(5) Its 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);
    (6)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：37、38、39、40、41或36所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(6) Its 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);
    (7)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：46、47、48、49、50或51所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(7) Its 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);
    (8)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：52、53、54、55、56或51所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(8) Its 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);
    (9)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：61、62、63、64、65或66所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(9) Its 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);
    (10)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：67、68、69、70、71或66所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(10) Its 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);
    (11)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：76、77、78、79、80或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(11) Its 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);
    (12)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：82、83、84、85、86或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(12) Its 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);
    (13)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：1、2、3、91、5或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(13) Its 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);
    (14)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：7、8、9、92、11或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(14) Its 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);
    (15)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：16、101、102、103、20或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(15) Its 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);
    (16)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：22、104、 105、106、26或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(16) Its 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 compared with any of the above sequences Sequences of several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions);
    (17)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：111、112、113、114、115或116所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(17) Its 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);
    (18)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：117、118、119、120、121或116所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(18) Its 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);
    (19)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：126、127、128、129、130或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(19) Its 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);
    (20)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：132、133、134、135、136或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(20) Its 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);
    (21)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：1、2、3、4、141或6所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(21) Its 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);
    (22)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：22、23、146、25、26或21所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(22) Its 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);
    (23)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：76、77、151、152、80或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(23) Its 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);
    (24)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：82、83、153、85、86或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(24) Its 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);
    (25)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：76、77、158、159、80或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(25) Its 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);
    (26)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：82、83、160、85、86或81所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(26) Its 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);
    (27)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：126、165、166、129、130或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(27) Its 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);
    (28)其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有如SEQ ID NO：132、167、168、135、136或131所示的序列，或者与上述序列中的任何相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列。(28) Its 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).
  3. 如权利要求2所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段为鼠源的或嵌合的，其重链可变区包含鼠源IgG1、IgG2、IgG3或其变体的重链FR区；和其轻链可变区包含鼠源κ、λ链或其变体的轻链FR区。The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is murine or chimeric, and its heavy chain variable region comprises murine IgG1, IgG2, IgG3 or its The heavy chain FR region of the variant; and the light chain variable region thereof comprises the light chain FR region of a murine kappa, lambda chain or a variant thereof.
  4. 如权利要求3所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含选自下组的VH和VL序列：The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof comprises a VH and VL sequence selected from the group consisting of:
    (1)VH结构域包含如SEQ ID NO：12所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：13所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(1) 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)) ;
    (2)VH结构域包含如SEQ ID NO：27所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：28所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(2) 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)) ;
    (3)VH结构域包含如SEQ ID NO：42所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：43所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(3) 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)) ;
    (4)VH结构域包含如SEQ ID NO：57所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：58所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(4) 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)) ;
    (5)VH结构域包含如SEQ ID NO：72所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：73所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(5) 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)) ;
    (6)VH结构域包含如SEQ ID NO：87所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取 代))的序列；和其VL结构域包含如SEQ ID NO：88所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(6) 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)) ;
    (7)VH结构域包含如SEQ ID NO：93所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：94所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(7) 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)) ;
    (8)VH结构域包含如SEQ ID NO：97所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：98所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(8) 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)) ;
    (9)VH结构域包含如SEQ ID NO：107所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：108所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(9) 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)) ;
    (10)VH结构域包含如SEQ ID NO：122所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：123所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(10) 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)) ;
    (11)VH结构域包含如SEQ ID NO：137所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：138所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列。(11) 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)) .
  5. 如权利要求2所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段为人源化的。The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is humanized.
  6. 如权利要求5所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含选自下组的VH和VL序列：The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof comprises VH and VL sequences selected from the group consisting of:
    (1)VH结构域包含如SEQ ID NO：14所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：15所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代 (例如保守性取代))的序列；(1) 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)) ;
    (2)VH结构域包含如SEQ ID NO：44所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：45所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(2) 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)) ;
    (3)VH结构域包含如SEQ ID NO：74所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：75所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列(3) 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))
    (4)VH结构域包含如SEQ ID NO：142所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：143所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(4) 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)) ;
    (5)VH结构域包含如SEQ ID NO：147所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：148所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(5) 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)) ;
    (6)VH结构域包含如SEQ ID NO：154所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：155所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(6) 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)) ;
    (7)VH结构域包含如SEQ ID NO：161所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：162所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；(7) 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)) ;
    (8)VH结构域包含如SEQ ID NO：124所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：125所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代 (例如保守性取代))的序列；(8) 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)) ;
    (9)VH结构域包含如SEQ ID NO：169所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：170所示的氨基酸序列，或与上述序列基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性或具有一个或更多个氨基酸取代(例如保守性取代))的序列。(9) 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)) .
  7. 如权利要求6所述抗体，其特征在于，所述抗体包含来源于人免疫球蛋白的重链恒定区和轻链恒定区；较优选地，所述重链恒定区选自人IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；更优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4的重链恒定区；并且，所述重链恒定区具有天然序列或与其所源自的天然序列相比具有一个或多个氨基酸的置换、缺失或添加的序列；和所述轻链恒定区优选如SEQ ID NO：95所示的人κappa链的恒定区。The antibody of claim 6, wherein the antibody comprises a heavy chain constant region and a light chain constant region derived from human immunoglobulin; more preferably, the heavy chain constant region is selected from human IgG1, IgG2, Heavy chain constant regions of IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; more preferably, the heavy chain constant regions are selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 and IgG4; The 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; and the light chain constant region is preferably human kappa as shown in SEQ ID NO:95 chain constant region.
  8. 如权利要求7所述抗体，其特征在于，所述抗体包含的重链恒定区选自下组：The antibody of claim 7, wherein the antibody comprises a heavy chain constant region selected from the group consisting of:
    (i)如SEQ ID NO：96所示的野生型人IgG1的重链恒定区；(i) the heavy chain constant region of wild-type human IgG1 as shown in SEQ ID NO: 96;
    (ii)如SEQ ID NO：190所示的含有M252Y、S254T、T256E和M428L突变的人IgG1的重链恒定区；(ii) the heavy chain constant region of human IgG1 containing the M252Y, S254T, T256E and M428L mutations as set forth in SEQ ID NO: 190;
    (iii)如SEQ ID NO：99所示的野生型人IgG2的重链恒定区；(iii) the heavy chain constant region of wild-type human IgG2 as shown in SEQ ID NO: 99;
    (iv)如SEQ ID NO：100所示的铰链区修饰的人IgG2的重链恒定区；(iv) the heavy chain constant region of the hinge region modified human IgG2 as shown in SEQ ID NO: 100;
    (v)如SEQ ID NO：109所示的野生型人IgG4的重链恒定区；(v) the heavy chain constant region of wild-type human IgG4 as shown in SEQ ID NO: 109;
    (vi)如SEQ ID NO：110所示的含有S228P突变的人IgG4的重链恒定区。(vi) The heavy chain constant region of human IgG4 containing the S228P mutation as set forth in SEQ ID NO: 110.
  9. 如权利要求7或8所述的抗体，其特征在于，所述抗体的重链具有如SEQ ID NO：29、59、89、139、144、149、156、163或171所示的氨基酸序列；或与上述序列中的任何相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或与上述序列中的任何相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或更高同一性的序列；和/或，所述抗体的轻链具有如SEQ ID NO：30、60、90、140、145、150、157、164或172所示的氨基酸序列；或与上述序列中的任何相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或与上述序列中的任何相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或更高同一性的序列。The antibody of claim 7 or 8, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO: 29, 59, 89, 139, 144, 149, 156, 163 or 171; or a sequence with one or several substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the above sequences; or Any comparison has at least 80%, 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 a sequence of higher identity; 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; or A sequence having one or several substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the above sequences; or to any of the above sequences at least 80%, 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% compared to , or a sequence of higher identity.
  10. 如权利要求1所述的抗体或其抗原结合片段，其中，SARS-CoV-2冠状病毒S蛋白具有：The antibody or antigen-binding fragment thereof of claim 1, wherein the SARS-CoV-2 coronavirus S protein has:
    (a)如SEQ ID NO：189所示的氨基酸序列；(a) the amino acid sequence shown in SEQ ID NO: 189;
    (b)是对SEQ ID NO：189所示的氨基酸序列置换、缺失或添加一个或几个氨基酸残基而得的氨基酸序列。(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.
  11. 如权利要求10所述的抗体或其抗原结合片段，其中，置换包括K417和/或L452和/或E484和/或N501。The antibody or antigen-binding fragment thereof of claim 10, wherein the substitution comprises K417 and/or L452 and/or E484 and/or N501.
  12. 如权利要求10所述的抗体或其抗原结合片段，其中，K417是K417N，和/或L452是L452R，和/或E484是E484K，和/或N501是N501Y。The antibody or antigen-binding fragment thereof of claim 10, wherein K417 is K417N, and/or L452 is L452R, and/or E484 is E484K, and/or N501 is N501Y.
  13. 如权利要求1-12任一项权利要求所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段以10nM或更低的K D结合S蛋白，较优选地，以1nM或更低的K D结合S蛋白；较优选地，以100pM 或更低的K D结合S蛋白；更优选地，以10pM或更低的K D结合S蛋白；最优选地，以1pM或更低的K D结合S蛋白。 The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof binds to the S protein with a K D of 10 nM or lower, more preferably, with a K D of 1 nM or lower KD binds S protein; more preferably, with 100pM or lower KD binds S protein; more preferably, with 10pM or lower KD binds S protein; most preferably, with 1pM or more Low KD binds to the S protein.
  14. 编码如权利要求1-13任一项所述抗体或其抗原结合片段的DNA分子。A DNA molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-13.
  15. 如权利要求14所述的DNA分子，其特征在于，编码所述抗体重链的DNA分子具有如SEQ ID NO：173、175、177、179、181、183、185或187所示的核苷酸序列，和编码所述抗体轻链的DNA分子具有如SEQ ID NO：174、176、178、180、182、184、186或188所示的核苷酸序列。The DNA molecule of claim 14, wherein the DNA molecule encoding the antibody heavy chain has a nucleotide as shown in SEQ ID NO: 173, 175, 177, 179, 181, 183, 185 or 187 The sequence, and the DNA molecule encoding the antibody light chain has the nucleotide sequence set forth in SEQ ID NO: 174, 176, 178, 180, 182, 184, 186 or 188.
  16. 包含如权利要求14或15所述DNA分子的载体。A vector comprising the DNA molecule of claim 14 or 15.
  17. 包含如权利要求16所述载体的宿主细胞；所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，优选为CHO细胞。A host cell comprising the vector of claim 16; the host cell comprises a prokaryotic, yeast or mammalian cell, preferably a CHO cell.
  18. 一种药物组合物，所述组合物包含如权利要求1-13任一项所述的抗体或其抗原结合片段以及可药用赋形剂、载体或稀释剂。A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-13 and a pharmaceutically acceptable excipient, carrier or diluent.
  19. 制备如权利要求1-13任一项所述抗体或其抗原结合片段的方法，其包括：(a)获得抗体或其抗原结合片段的基因，构建抗体或其抗原结合片段的表达载体；(b)通过基因工程方法将上述表达载体转染到宿主细胞中；(c)在允许产生所述抗体或其抗原结合片段的条件下培养上述宿主细胞；(d)分离、纯化产生的所述抗体或其抗原结合片段；A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1-13, comprising: (a) obtaining a gene of the antibody or an antigen-binding fragment thereof, and constructing an expression vector for the antibody or an antigen-binding fragment thereof; (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 produced antibody or antigen-binding fragments thereof;
    其中，步骤(a)中所述表达载体选自质粒、细菌和病毒中的一种或多种，优选地，所述表达载体为PXY1A1M载体；Wherein, the expression vector in step (a) is selected from one or more of plasmids, bacteria and viruses, preferably, the expression vector is a PXY1A1M vector;
    其中，步骤(b)通过基因工程方法将所构建的载体转染入宿主细胞中，所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，优选为CHO细胞；Wherein, in step (b), the constructed vector is transfected into host cells by genetic engineering method, and the host cells comprise prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells;
    其中，步骤(d)通过常规的免疫球蛋白纯化方法，包含蛋白质A亲和层析和离子交换、疏水层析或分子筛方法分离、纯化所述抗体或其抗原结合片段。Wherein, 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.
  20. 如权利要求1-13任一项所述抗体或其抗原结合片段在制备治疗和预防由SARS-CoV-2冠状病毒引起的疾病的药物中的用途；优选地，所述疾病为新型冠状病毒肺炎(COVID-19)；例如，所述疾病为由B.1.351突变株和/或B.1.1.7病毒株引起的新型冠状病毒肺炎(COVID-19)。Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-13 in the preparation of a medicine for the treatment and prevention of a disease caused by SARS-CoV-2 coronavirus; preferably, 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 strain.
  21. 一种检测样品中SARS-CoV-2病毒或者其相应抗原存在情况的方法，包括如下步骤：A method for detecting the presence of SARS-CoV-2 virus or its corresponding antigen in a sample, comprising the following steps:
    (1)将待测生物样品与权利要求1-13任一项中所述的至少一种单克隆抗体或其抗原结合片段在合适的条件下孵育；(1) incubating the biological sample to be tested with at least one monoclonal antibody or its antigen-binding fragment described in any one of claims 1-13 under suitable conditions;
    (2)检测上述步骤中结合复合物的存在；(2) detecting the presence of the binding complex in the above steps;
    其中，所述生物样品选自血浆、全血、嗽口液、咽拭子、尿液、粪便和支气管灌流液。Wherein, the biological sample is selected from plasma, whole blood, mouthwash, throat swab, urine, feces and bronchial perfusate.
    其中，所述抗原结合片段选自F(ab') 2、Fab'、Fab和Fv。 Wherein, the antigen-binding fragment is selected from F(ab') 2 , Fab', Fab and Fv.
  22. 如权利要求1-13任一项所述的单克隆抗体在制备SARS-CoV-2病毒检测试剂盒中的用途。Use of the monoclonal antibody according to any one of claims 1-13 in the preparation of a SARS-CoV-2 virus detection kit.
  23. 一种检测试剂盒，其包含如权利要求1-13任一项所述的至少一种单克隆抗体或其抗原结合片段。A detection kit comprising at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-13.
  24. 如权利要求23所述的检测试剂盒，所述试剂盒包括：The detection kit of claim 23, comprising:
    (1)选自以下任一：(1) Select from any of the following:
    a.固相载体和第一抗体；a. Solid phase carrier and primary antibody;
    b.包被有第一抗体的固相载体；b. A solid phase carrier coated with a primary antibody;
    所述第一抗体为选自权利要求1-13任一项的任意一个单克隆抗体或其抗原结合片段；The first antibody is any one of the monoclonal antibodies or antigen-binding fragments thereof selected from any one of claims 1-13;
    (2)第二抗体；(2) secondary antibody;
    所述第二抗体任选地进行了适当标记，并且第二抗体选自权利要求1-13任一项所述能够与(1)中所述第一抗体配合使用的单克隆抗体或其抗原结合片段。The second antibody is optionally labeled appropriately, and the second antibody is selected from the monoclonal antibody or its antigen binding which can be used in conjunction with the first antibody in (1) according to any one of claims 1-13 Fragment.
  25. 如权利要求24所述的检测试剂盒，所述固相载体选自硝酸纤维素膜、胶乳颗粒、磁性颗粒、胶体金、珠子或诸如玻璃、纤维玻璃或聚合物(如聚苯乙烯或聚氯乙烯)或纤维光学传感器。The detection kit of claim 24, wherein the solid support is selected from nitrocellulose membranes, latex particles, magnetic particles, colloidal gold, beads, or materials such as glass, fiberglass or polymers such as polystyrene or polychlorinated vinyl) or fiber optic sensors.
  26. 如权利要求24所述的检测试剂盒，所述标记物可以是放射性同位素、酶、酶底物、磷光物质、荧光物质、生物素和着色物质；优选地，所述酶包括如碱性磷酸酶、辣根过氧化物酶、β-半乳糖苷酶、脲酶和葡萄糖氧化酶；所述荧光物质包括如荧光素衍生物和罗丹明衍生物以及稀土元素或稀土元素复合物，如铕或铕复合物；所述磷光物质包括如丫啶酯和异鲁米诺；所述放射性同位素包括如 125I、 3H、 14C和 32P；所述着色物质包括如乳胶颗粒和胶体金。 The detection kit according to claim 24, the label can be a radioisotope, an enzyme, an enzyme substrate, a phosphorescent substance, a fluorescent substance, a biotin and a coloring substance; preferably, the enzyme includes, for example, alkaline phosphatase , horseradish peroxidase, β-galactosidase, urease and glucose oxidase; the fluorescent substances include, for example, fluorescein derivatives and rhodamine derivatives and rare earth elements or rare earth element complexes, such as europium or europium complexes The phosphorescent substances include, for example, acridine esters and isoluminol; the radioisotopes include, for example, 125 I, 3 H, 14 C, and 32 P; and the coloring substances include, for example, latex particles and colloidal gold.
  27. 如权利要求23-26任一项所述检测试剂盒在诊断SARS-CoV-2病毒感染所致疾病中的用途；优选地，所述疾病为新型冠状病毒肺炎(COVID-19)；例如，所述疾病为由B.1.351突变株和/或B.1.1.7病毒株引起的新型冠状病毒肺炎(COVID-19)。Use of the detection kit according to any one of claims 23-26 in diagnosing a disease caused by SARS-CoV-2 virus infection; preferably, the disease is novel coronavirus pneumonia (COVID-19); for example, the The disease is novel coronavirus pneumonia (COVID-19) caused by B.1.351 mutant strain and/or B.1.1.7 strain.
PCT/CN2021/086477 2020-08-28 2021-04-12 Antibody against sars-cov-2 coronavirus s protein and application thereof WO2022041745A1 (en)

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CN202010889404.6A CN114106161A (en) 2020-08-28 2020-08-28 Antibodies against SARS-CoV-2 coronavirus S protein and uses thereof
CN202010887508.3A CN114106159A (en) 2020-08-28 2020-08-28 SARS-CoV-2 spike protein antibody and its application
CN202010889418.8A CN114106162A (en) 2020-08-28 2020-08-28 Novel coronavirus Spike protein antibody and application thereof
CN202010889404.6 2020-08-28
CN202010889425.8A CN114106163A (en) 2020-08-28 2020-08-28 SARS-CoV-2 virus neutralizing antibody and its use
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CN202010889400.8A CN114106160A (en) 2020-08-28 2020-08-28 Monoclonal antibody for resisting SARS-CoV-2 virus and its application
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