WO2022188829A1 - Sars-cov-2 antibody and application thereof - Google Patents

Sars-cov-2 antibody and application thereof Download PDF

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WO2022188829A1
WO2022188829A1 PCT/CN2022/080100 CN2022080100W WO2022188829A1 WO 2022188829 A1 WO2022188829 A1 WO 2022188829A1 CN 2022080100 W CN2022080100 W CN 2022080100W WO 2022188829 A1 WO2022188829 A1 WO 2022188829A1
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seq
amino acid
acid sequence
chain variable
variable region
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PCT/CN2022/080100
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French (fr)
Chinese (zh)
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李理
刘洪川
张静
周岳华
林志妙
施春花
冯辉
姚盛
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Publication of WO2022188829A1 publication Critical patent/WO2022188829A1/en

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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • A61K47/6841—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses the antibody targeting a RNA virus
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • 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]
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    • C12P21/00—Preparation of peptides or proteins
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/56983—Viruses
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • C07K2317/622—Single chain antibody (scFv)
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    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention belongs to the field of medical technology, and particularly relates to an antibody or an antigen-binding fragment thereof of a novel coronavirus SARS-CoV-2 or a variant thereof with high neutralizing activity and application thereof. Also provided are nucleic acid molecules encoding the antibodies of the invention or antigen-binding fragments thereof, vectors and host cells for expressing the antibodies of the invention or antigen-binding fragments thereof, and therapeutic and diagnostic methods and uses of the antibodies of the invention or antigen-binding fragments thereof.
  • Therapeutic antibody drugs play an important role not only in tumors and autoimmune diseases, but also in the treatment of infectious diseases.
  • Drugs currently on the market for the treatment and prevention of viral infections include palivizumab (Synagis) for the prevention of pediatric respiratory syncytial virus (RSV) infection, ibalizumab (Trogarzo) for HIV infection, and rabies Rabishield for post-exposure prophylaxis.
  • RSV respiratory syncytial virus
  • ibalizumab Trogarzo
  • rabies Rabishield for post-exposure prophylaxis.
  • monoclonal antibodies against numerous viruses in various stages of clinical research There are also monoclonal antibodies against numerous viruses in various stages of clinical research.
  • 2019-nCoV is a coronavirus. Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), both of which are coronaviruses, also caused outbreaks in 2002-2003 and 2012, respectively. According to the World Health Organization (WHO), SARS-CoV caused a total of 8,000 infections and 794 deaths (https://www.who.int/). Since 2012, cases of MERS-CoV infection have continued to increase. As of the end of 2019, 2,499 infections and 861 deaths have been confirmed worldwide.
  • SARS-CoV Severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the World Health Organization officially named the new coronavirus "2019 novel coronavirus (2019-nCoV)", and later on February 11-12, 2020, the International Committee on Taxonomy of Viruses (International Committee on Taxonomy) of Viruses, ICTV) announced that the official classification of the new coronavirus (2019-nCoV) is called severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), the World Health Organization (WHO) on the same day
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • WHO World Health Organization
  • Antibodies especially neutralizing antibodies, block viral infection by binding to envelope proteins and blocking the binding of viruses to cellular receptors.
  • the antibody binds to the envelope protein to label free virus or infected cells, and recruits immune cells and immune molecules such as macrophages or complement through the Fc region of the antibody, thereby removing free virus and infected cells. infected cells. Therefore, antibodies targeting the receptor binding domain (RBD) not only have the activity of neutralizing virus infection, but can also act through the Fc region to promote the clearance of viruses and infected cells.
  • RBD receptor binding domain
  • S spike protein
  • S1 and S2 The role of S2 is to mediate membrane fusion.
  • NTD N-terminal
  • CTD C-terminal
  • an antibody targeting RBD which is an antibody that blocks the binding of S to ACE2, may become a neutralizing antibody that inhibits viral infection.
  • the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to SARS-CoV-2 or its variant RBD, which has advantages such as high neutralization activity against SARS-CoV-2 or its variant.
  • Antibodies or antigen-binding fragments thereof that neutralize SARS-CoV-2 or variants thereof provided by the present invention can be used as stand-alone therapy or in combination with other therapies/or other drugs.
  • the invention provides an antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain (RBD) of SARS-CoV-2 or a variant thereof, wherein the antibody or antigen-binding fragment thereof comprises
  • the CDR sequences HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and/or the CDR sequences LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown below:
  • HCDR1 GFX 1 VX 2 X 3 NY (SEQ ID NO: 144), wherein X 1 is selected from L, T, E, R, Q, V, W, I or S, preferably, X 1 is selected from L, T, E, R, Q, V or I; X 2 is selected from Q, G, D, R, P, M, K, V, A, N or Y, preferably, X 2 is selected from Q, G, D , R, P, N or Y; X 3 is selected from R, W, Y, A, F, V or H, preferably, X 3 is selected from W, R, A, F or V;
  • HCDR2 IYPGGX 4 T (SEQ ID NO: 145), wherein X 4 is T or S;
  • HCDR3 ARVLPMYGDYLDY (SEQ ID NO: 3);
  • LCDR1 QX 5 IX 6 X 7 Y (SEQ ID NO: 146), wherein X 5 is selected from V, D, Q, A, W, R, N, S, D, M, K or P, preferably, X 5 is selected from Q, A, R, N, S, D or M; X 6 is selected from N, H, L, G, P, S, M, E, V, R, D, A or I, preferably , X 6 is selected from E, L, V, R, D, E or A; X 7 is selected from H, V, F, P, N, S, R, Q, G, Y or T, preferably, X 7 selected from Q, P, S, G, P, R or Y;
  • LCDR2 AAS (SEQ ID NO: 5);
  • LCDR3 QQSX 8 SX 9 X 10 PEYT (SEQ ID NO: 147), wherein X 8 is selected from G, Y, T, S, K, A, N, E or P, preferably, X 8 is selected from A, N, S or P; X 9 is selected from P, S, I, N, A, W or F, preferably, X 9 is selected from S, P or A; X 10 is selected from T, V, L, I, R , K, S, M or F, preferably X 10 is selected from S, R, T, K, V, L or F.
  • the LCDR1 shown in SEQ ID NO:146 does not include the LCDR1 shown in SEQ ID NO:4.
  • the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 144, 145, and 3 and LCDRl, LCDR2, and LCDR3 set forth in SEQ ID NOs: 146, 5, and 147 .
  • the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 144, 145, and 3 and LCDRl, LCDR2, and LCDR3 set forth in SEQ ID NOs: 4, 5, and 6 .
  • the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 1, 2, and 3 and LCDRl, LCDR2, and LCDR3 set forth in SEQ ID NOs: 146, 5, and 147 .
  • the HCDR1 shown in SEQ ID NO: 144 described in any of the embodiments herein is selected from SEQ ID NO: 26, 28, 29, 31, 32, 33, 34, 35, 104, 105, 106, 107,
  • the HCDR1 shown in any one of 108, 109, 110 and 111 is preferably selected from the HCDR1 shown in any one of SEQ ID NOs: 26, 28, 29, 31, 32, 33, 34 and 35.
  • the HCDR2 set forth in SEQ ID NO: 145 is selected from the HCDR2 set forth in any one of SEQ ID NO: 27 and 30.
  • the LCDR1 shown in SEQ ID NO: 146 is selected from SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 112, LCDR1 shown in any one of 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140 and 142, preferably selected from SEQ ID NOs: 36, 38, 40, LCDR1 shown in any of 42, 44, 46, 48, 50, 52, 54 and 56.
  • the LCDR3 shown in SEQ ID NO: 147 is selected from SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 113, LCDR3 shown in any one of 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 and 143, preferably selected from SEQ ID NOs: 37, 39, 41, LCDR3 shown in any of 43, 45, 47, 49, 51, 53, 55 and 57.
  • the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention contains HCDR1, HCDR2, and HCDR3 selected from the group consisting of: SEQ ID NOs: 104, 27, and 3; SEQ ID NO: 26 , 27 and 3; SEQ ID NO: 28, 27 and 3; SEQ ID NO: 29, 30 and 3; SEQ ID NO: 105, 27 and 3; SEQ ID NO: 31, 30 and 3; SEQ ID NO: 32 , 30 and 3; SEQ ID NO: 106, 30 and 3; SEQ ID NO: 107, 30 and 3; SEQ ID NO: 108, 27 and 3; SEQ ID NO: 33, 30 and 3; SEQ ID NO: 109 , 27 and 3; SEQ ID NO: 110, 30 and 3; SEQ ID NO: 111, 30 and 3; SEQ ID NO: 34, 30 and 3; and SEQ ID NO: 35, 30 and 3.
  • SEQ ID NOs: 104, 27, and 3 SEQ ID NO: 26 , 27 and 3; SEQ ID
  • amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention are shown in SEQ ID NOs: 31, 30 and 3, respectively.
  • amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 4, 5 and 6, respectively.
  • the light chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises LCDR1, LCDR2, and LCDR3 selected from the group consisting of: SEQ ID NO: 112, 5, 113; SEQ ID NO: 114 , 5, 115; SEQ ID NO: 116, 5, 117; SEQ ID NO: 118, 5, 119; SEQ ID NO: 120, 5, 121; SEQ ID NO: 122, 5, 123; SEQ ID NO: 124 , 5, 125; SEQ ID NO: 126, 5, 127; SEQ ID NO: 128, 5, 129; SEQ ID NO: 130, 5, 131; SEQ ID NO: 132, 5, 133; SEQ ID NO: 134 , 5, 135; SEQ ID NO: 136, 5, 137; SEQ ID NO: 138, 5, 139; SEQ ID NO: 140, 5, 141; SEQ ID NO: 142, 5, 143; SEQ ID NO: 36 , 5, 37; SEQ ID NO: 38, 5, 39; SEQ ID NO:
  • the light chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises LCDR1, LCDR2, and LCDR3 selected from the group consisting of: SEQ ID NO: 48, 5, 49; and SEQ ID NO: 54, 5, 55.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 1, 2 and 3, respectively.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and/or a light chain variable region, wherein:
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 3 respectively; or with SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 28, SEQ ID NO: 27 and SEQ ID NO: 3 respectively; or with SEQ ID NO: 28, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3 respectively; : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • (V) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO32, SEQ ID NO:30 and SEQ ID NO:3, respectively; or with SEQ ID NO:32, SEQ ID NO:30 and SEQ ID NO:3 HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in the indicated amino acid sequences, respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 34, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 34, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:35, SEQ ID NO:30 and SEQ ID NO:3, respectively; or with SEQ ID NO:35, SEQ ID NO:30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 104, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 104, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • (X) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 105, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 105, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 106, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 106, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 107, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 107, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 108, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 108, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 109, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 109, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 whose amino acid sequences shown in 3 have 1, 2 or 3 amino acid differences, respectively
  • HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO: 110, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 110, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 111, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 111, SEQ ID NO: 30 and SEQ ID NO HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 3;
  • the light chain variable region comprises:
  • (V) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:44, SEQ ID NO:5 and SEQ ID NO:45, respectively; or with SEQ ID NO:44, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 45; or
  • VI LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 46, SEQ ID NO: 5 and SEQ ID NO: 47, respectively; or with SEQ ID NO: 46, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 47; or
  • (X) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or with SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 55; or
  • the antibody or antigen-binding fragment thereof of the present invention does not include the amino acid sequences of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 35, 30 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 as shown in The antibodies or antigen-binding fragments thereof set forth in SEQ ID NOs: 54, 5 and 55.
  • the antibody or antigen-binding fragment thereof of the present invention also does not include the amino acid sequences of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
  • VI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:32, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
  • VII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
  • VIII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or
  • (IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:34, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or
  • (X) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
  • XI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:31, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:48, SEQ ID NO:5 and SEQ ID NO:49, respectively.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is selected from the heavy chain variable region shown in any of SEQ ID NOs: 58-73 or can be combined with any of the heavy chain variable regions.
  • Variable region heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity, preferably selected from SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 and 73 Any of the indicated heavy chain variable regions or heavy chain variable regions having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said heavy chain variable regions, more preferably Has at least 95%, 96%, 97%, 98% or 99% from or with any of the heavy chain variable regions set forth in SEQ ID NOs: 59, 61, 64, 72 and 73 % sequence identity of heavy chain variable regions.
  • the light chain variable region of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO:8.
  • the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention is selected from the light chain variable region shown in any one of SEQ ID NOs: 74-100 or the light chain variable region of any of the light chain A light chain variable region having a variable region of at least 95%, 96%, 97%, 98% or 99% sequence identity, preferably selected from SEQ ID NOs: 83, 84, 85, 86, 88, 89, 90, A light chain variable region shown in any one of 93, 94, 95 and 98 or a light chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said light chain variable regions A chain variable region, more preferably selected from the light chain variable region shown in any of SEQ ID NOs: 83, 90 and 95 or having at least 95%, 96%, 97% with any of said light chain variable regions , 98% or 99% sequence identity of the light chain variable region.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is selected from the heavy chain variable region shown in any of SEQ ID NOs: 58-73 or can be combined with any of the heavy chain variable regions.
  • Variable region heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity, preferably selected from SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 and 73 Any of the indicated heavy chain variable regions or heavy chain variable regions having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said heavy chain variable regions, more preferably Has at least 95%, 96%, 97%, 98% or 99% from or with any of the heavy chain variable regions set forth in SEQ ID NOs: 59, 61, 64, 72 and 73 % heavy chain variable regions of sequence identity; light chain variable regions are selected from or have at least 95% of the light chain variable regions shown in any of SEQ ID NOs: 74-100 A light chain variable regions
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region:
  • the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 or 73, or the amino acid sequence shown in any one of SEQ ID NOs: 59, 60 , 61, 63, 64, 68, 72 or 73 amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity; and the light chain can be
  • the variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, 85, 86, 88, 89, 90, 93, 94, 95, or 98, or the same as SEQ ID NOs: 83, 84, 85 , 86, 88, 89, 90, 93, 94, 95, or 98 amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or has at least 95%, 96%, 97%, 98% or 99% with the amino acid sequence shown in SEQ ID NO: 61 an amino acid sequence of % sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 95, or has at least 95%, 96%, 97%, or at least 95%, 96%, 97 amino acid sequences of %, 98% or 99% sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:64, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:64 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:59, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:59 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:73 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 90 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:61 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:64, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:64 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 68 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 68 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 8 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:72, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:72 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 8 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:63 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:63 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 90 , 97%, 98% or 99% amino acid sequence identity; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, 64 or 59, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 95; or the The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:90; or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:90;
  • the amino acid sequence set forth in SEQ ID NO: 61, 64 or 68, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 or 72, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof of the present invention does not include the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:73, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:95 antibodies or antigen-binding fragments thereof.
  • the antibody or antigen-binding fragment thereof of the present invention also does not include the amino acid sequence of the variable region of the heavy chain as shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the light chain as shown in SEQ ID NO:84 The indicated antibody or antigen-binding fragment thereof.
  • the antibody of the invention comprises a heavy chain and a light chain:
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 101, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 101 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 90%, 92 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 103, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 103 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 90%, 92 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 101 or 103, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 148, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 148 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 149, or has at least 90%, 92% with the amino acid sequence shown in SEQ ID NO: 149 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 148, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 148 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 150, or has at least 90%, 92% with the amino acid sequence shown in SEQ ID NO: 150 Amino acid sequences of %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antibodies of the invention are fully human antibodies or humanized antibodies.
  • the antigen-binding fragments of the present invention are selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, sdAb, or diabodies.
  • the antibodies or antigen-binding fragments thereof of the invention are of any IgG subtype, such as IgGl, IgG2, IgG3, or IgG4. In some embodiments, the antibody or antigen-binding fragment thereof of the invention is of the IgGl type.
  • the antibodies of the invention are monoclonal antibodies.
  • the present invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
  • the present invention also provides a single chain antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
  • the invention also provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof as described herein conjugated to a therapeutic or diagnostic agent.
  • the present invention provides a polynucleotide molecule encoding an antibody or antigen-binding fragment thereof as claimed herein.
  • the present invention provides an expression vector comprising the polynucleotide molecule described herein, preferably, the vector is a eukaryotic expression vector.
  • the present invention provides host cells comprising a polynucleotide or expression vector described herein, or expressing an antibody or antigen-binding fragment thereof described herein.
  • the host cells are eukaryotic cells, more preferably mammalian cells.
  • the present invention provides a method of making an antibody or antigen-binding fragment thereof described herein, the method comprising culturing a host described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof cells to express the antibody or antigen-binding fragment thereof, and recover the expressed antibody or antigen-binding fragment thereof.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule, the expression vector and/or the host cell, and a pharmaceutically acceptable carrier or excipients.
  • the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment or pharmaceutical composition thereof as described herein, and one or more additional therapeutic agents.
  • the present invention provides an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, a pharmaceutical combination as described herein Use of the drug and/or the pharmaceutical combination described herein in the manufacture of a medicament for the treatment and/or prevention of infection by SARS-CoV-2 or a variant thereof.
  • the present invention provides a method of treating and/or preventing infection by SARS-CoV-2 or a variant thereof, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, The polynucleotide molecule, the expression vector, the host cell, the pharmaceutical composition and/or the pharmaceutical combination.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein Or a pharmaceutical combination as described herein for the treatment and/or prevention of a disease infected by SARS-CoV-2 or a variant thereof; preferably, the SARS-CoV-2 variant comprises an alpha mutant (Alpha mutant) , Beta mutants (Beta mutants), Gamma mutants (Gamma mutants), Delta mutants (Delta mutants), Epsilon mutants, Zeta mutants, Eta mutants, Theta mutants, Iota mutants, At least one of a Kappa mutant (Kappa mutant), a Mu mutant (Mu mutant), and an Omicron mutant (omicron mutant); preferably an Omicron mutant.
  • the SARS-CoV-2 variant comprises an alpha mutant (Alpha mutant) , Beta mutants (Beta mutant
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule, the expression vector, the host cell and/or the pharmaceutical combination thing.
  • the present invention provides the use of the kit in the manufacture of a medicament for diagnosing infection by SARS-CoV-2 or a variant thereof.
  • the present invention provides a method of detecting the presence of SARS-CoV-2 or a variant thereof in a sample using the antibody or antigen-binding fragment thereof or the polypeptide described herein, the method comprising causing the The antibody or antigen-binding fragment thereof is contacted with the sample, and the presence of a conjugate or a binding signal produced by the binding of the antibody or antigen-binding fragment thereof to SARS-CoV-2 or its variant CBD is detected.
  • Figure 1 JS016 light and heavy chain CDR sequences.
  • FIG. 1 Mutation library construction workflow.
  • Figures 3A-3D Binding ELISA to detect binding of JS016 affinity matured antibody.
  • Figures 4A-4D Blocking ELISA to detect blocking effect of JS016 affinity matured antibody.
  • Figure 5 In vitro neutralizing activity of JS016-38 and JS016-40 antibodies against live SARS-CoV-2 virus.
  • Figure 6A ELISA profile of JS016 binding to RBD and RBD muteins.
  • Figure 6B ELISA profile of JS016-38 binding to RBD and RBD muteins.
  • Figure 6C ELISA profile of JS016-40 binding to RBD and RBD muteins.
  • FIG. 7A JS016 inhibits binding of RBD and RBD muteins to ACE2.
  • Figure 7B JS016-38 inhibits binding of RBD and RBD muteins to ACE2.
  • Figure 7C JS016-40 inhibits binding of RBD and RBD muteins to ACE2.
  • Figure 8A Kinetic parameters of CB6 binding to RBD-his, RBD-Omicron-his.
  • Figure 8B Kinetic parameters of JS016-40 binding to RBD-his, RBD-Omicron-his.
  • Figure 8C Kinetic parameters of binding of JS016-41-YTE to RBD-his, RBD-Omicron-his.
  • Figure 8D Kinetic parameters of JS016-77-YTE binding to RBD-his, RBD-Omicron-his.
  • Figure 9 In vitro binding activity of the antibody to the S protein of the omicron mutant strain of SARS-CoV-2.
  • Figure 10A Pseudovirus neutralizing activity of CB6 and JS016-40.
  • Figure 10B Pseudovirus neutralizing activity of JS016-41-YTE.
  • Figure 10C Pseudovirus neutralizing activity of JS016-77-YTE.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” should be construed as inclusive, that is, including at least one of the number or list of elements, but also including more than one, and optionally , additional unlisted items. Only under terms expressly stated to the contrary, such as “only one” or “indeed one” or when “consisting of” is used in a claim, will refer to only one of the listed numbers or one element of the list.
  • percent (%) amino acid sequence identity or simply “identity” is defined as the maximum percent sequence identity obtained when amino acid sequences are aligned (and where necessary gaps are introduced), and no conservative substitutions are considered to be Following the portion of sequence identity, the percentage of amino acid residues in the candidate amino acid sequence that are identical to those in the reference amino acid sequence.
  • Sequence alignments to determine percent amino acid sequence identity can be performed using various methods in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • immune response refers to the action by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines and complement) by these cells or by the liver, which results in selective Damage, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of signals from one part of a cell to another of the cell. part.
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions leading to signal transduction.
  • the penultimate process usually involves nuclear events that lead to changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein, including but not limited to epitope/antigen affinity and specificity, neutralization or antagonism of SARS in vivo or in vitro - Capacity for CoV-2 activity, IC50, in vivo stability of antibodies and immunogenic properties of antibodies.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (ie, generally with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of The ability of proteins to be expressed at high levels in mammalian cells.
  • antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media .
  • isolated does not mean the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.
  • antigen-binding fragment of an antibody includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody that retains the parental At least some binding specificity of an antibody.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • a binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • a “Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • a “Fab'fragment” contains a light chain and a portion of a heavy chain comprising a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F(ab') 2 molecules.
  • an “F(ab') 2 fragment” contains two light chains and two parts of a heavy chain comprising a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby the two heavy chains are Interchain disulfide bonds are formed between chains.
  • an F(ab') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • Fv regions comprise variable regions from both heavy and light chains, but lack constant regions.
  • Single-chain Fv antibody refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, these domains being contained in a single polypeptide chain.
  • scFv polypeptides contain a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present (numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. .Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • diabody refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Variable domain
  • humanized antibody refers to a form of antibody that contains sequences from human and non-human (eg, mouse, rat) antibodies.
  • humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • FR hypervariable loops Framework
  • a humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • binding refers to determining the presence or absence of a protein, eg, an antibody of the invention, in a heterogeneous population of proteins and/or other biological agents that binds to Binding response of 2019-nCoV RBD protein.
  • a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in significant amounts.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4). Isotypes also include modified forms of one of these species, wherein modifications have been made to alter Fc function, eg, to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4
  • epitope refers to a protein determinant capable of specific binding by an antibody.
  • Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is the loss of binding to the former but not to the latter in the presence of a denaturing solvent.
  • cross-reactivity refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Thus, “cross-reactivity” should be understood as an inter-species reaction with the same molecule X expressed in different species.
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art.
  • One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • does not bind to a protein or cell means that it does not bind to a protein or cell, or does not bind to it with high affinity, i.e. binds the protein or cell with a KD of 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 - 5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, more preferably 1.0 ⁇ 10 -2 M or higher.
  • high affinity for IgG antibodies means a KD for antigen of 1.0x10-6 M or less, preferably 5.0x10-8 M or less, more preferably 1.0x10-8 M or lower, 5.0 ⁇ 10 ⁇ 9 M or lower, more preferably 1.0 ⁇ 10 ⁇ 9 M or lower.
  • high affinity binding may vary.
  • “high affinity” binding of an IgM subtype refers to a KD of 10-6 M or lower, preferably 10-7 M or lower, more preferably 10-8 M or lower.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form.
  • RNA ribonucleic acid
  • nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Human US Patent No.
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements thereof, as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from Any source, capable of integrating with the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked).
  • the recombinant construct will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be directed using both heterologous and non-heterologous (ie, endogenous) promoters.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • 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).
  • vectors After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.
  • Activation can have the same meaning, eg, activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
  • a “response/response”, eg, the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (eg, concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), wherein changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
  • the terms “treating” or “treating” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treating” or “treating” refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernible by a patient. In another embodiment, “treating” or “treating” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to a cynomolgus monkey.
  • Administration "in combination with” one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” mean that the novel coronavirus antibody or antigen-binding fragment thereof of the present invention, when administered to cells, tissues or subjects alone or in combination with other therapeutic drugs, is effective in preventing Or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition.
  • a therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • the therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of the therapeutic agent will result in an improvement in the diagnostic criterion or parameter by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.
  • “Pharmaceutically acceptable carrier” refers to ingredients other than the active ingredient in a pharmaceutical formulation or composition that are not toxic to a subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • cancer is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers can be benign (also called benign tumors), premalignant, or malignant. The cancer cells can be solid cancer cells or leukemia cancer cells.
  • tumor refers to one or more cells comprising cancer.
  • tumor growth is used herein to refer to the proliferation or growth of one or more cells comprising the cancer, which results in a corresponding increase in the size or extent of the cancer.
  • the invention provides antibodies or antigen-binding fragments thereof that specifically bind to the RBD of SARS-CoV-2 or a variant thereof.
  • variants of SARS-CoV-2 include, but are not limited to, alpha mutants (Alpha mutants), beta mutants (Beta mutants), gamma mutants (Gamma mutants), delta mutants (Delta mutants) strains), Epsilon mutants, Zeta mutants, Eta mutants, Theta mutants, Iota mutants, Kappa mutants (Kappa mutants), Mu mutants (Mu mutants) and Omicron mutants (omicron mutants) at least one of the mutants).
  • the variant of SARS-CoV-2 is an Omicron mutant.
  • the SARS-CoV-2 variant is a mutant having any one, any two, or all three selected from K417N, E484K, and N501Y.
  • the SARS-CoV-2 variant is SARS-CoV-2 South African mutant 501Y.V2, which contains three major characteristic mutation sites: K417N, E484K, and N501Y.
  • the South African mutant 501Y.V2 has the same N501Y mutation as the British mutant B.1.1.7 subtype, the difference is that it also contains two key loci of S protein E484K and K417N which have a potentially important influence on the virus infectivity. point mutation. These two sites may enhance the binding ability of S protein to human epidermal cell receptor.
  • the present invention provides antibodies or antigen-binding fragments thereof that bind to SARS-CoV-2 or a variant RBD thereof. In some embodiments, the present invention provides antibodies that block the binding of SARS-CoV-2 or its variant RBD to ACE2.
  • the present invention provides an antibody or antigen-binding fragment thereof that specifically binds the receptor binding domain (RBD) of SARS-CoV-2 or a variant thereof, wherein the antibody or antigen thereof binds
  • RBD receptor binding domain
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the fragment are:
  • HCDR1 GFX 1 VX 2 X 3 NY, wherein X 1 is selected from L, T, E, R, Q, V, W, I or S, preferably, X 1 is selected from L, T, E, R, Q , V or I; X 2 is selected from Q, G, D, R, P, M, K, V, A, N or Y, preferably, X 2 is selected from Q, G, D, R, P, N or Y; X is selected from R , W, Y, A, F, V or H, preferably, X is selected from W, R, A, F or V;
  • HCDR2 IYPGGX 4 T, where X 4 is T or S;
  • LCDR1 QX 5 IX 6 X 7 Y, wherein X 5 is selected from V, D, Q, A, W, R, N, S, D, M, K or P, preferably, X 5 is selected from Q, A , R, N, S, D or M; X 6 is selected from N, H, L, G, P, S, M, E, V, R, D, A or I, preferably, X 6 is selected from E, L, V, R, D, E or A ; X is selected from H, V, F, P, N, S, R, Q, G, Y or T, preferably, X is selected from Q, P, S , G, P, R or Y;
  • LCDR2 AAS
  • LCDR3 QQSX 8 SX 9 X 10 PEYT, wherein X 8 is selected from G, Y, T, S, K, A, N, E or P, preferably, X 8 is selected from A, N, S or P; X 9 is selected from P, S, I, N, A, W or F, preferably, X 9 is selected from S, P or A; X 10 is selected from T, V, L, I, R, K, S, M or F, preferably, X 10 is selected from S, R, T, K, V, L or F.
  • N, T and S are polar uncharged aliphatic amino acids
  • S, R, N and D are polar aliphatic amino acids
  • D, G, V and I are all aliphatic Amino acids
  • L and P are non-polar amino acids
  • Y and F are aromatic amino acids
  • Y and S are polar uncharged amino acids
  • N, Q and H are polar amino acids
  • H and R, etc. are polar charged amino acids.
  • the present invention obtains the following 43 clones through phage display technology:
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia based on the three-dimensional structure of the antibody and topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al, Sequences of Proteins of Immunological Interest, 4th Edition, U.S.
  • the CDRs of the antibodies of the invention can be bounded by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).
  • the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. different assignment systems or combinations), resulting in their claimed CDR boundaries being different from the specific CDR boundaries defined by the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • the boundaries of the CDRs of the antibodies or antigen-binding fragments of the invention are defined using the IMGT scheme.
  • amino acid changes include amino acid deletions, insertions or substitutions.
  • the anti-novel coronavirus antibodies or antigen-binding fragments thereof of the present invention include those that have been mutated by amino acid deletions, insertions or substitutions (particularly in the CDR regions depicted in the above sequences) but still have the same Those antibodies having amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions that have been mutated by amino acid deletions, insertions, or substitutions when compared to the CDR regions depicted in a particular sequence. In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the framework regions that have been mutated by amino acid deletions, insertions or substitutions when compared to the framework regions in a particular sequence.
  • the polynucleotide molecules encoding the antibodies of the invention include those that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60, Polynucleotide molecules of 70, 80, 90, 95 or 100% identity.
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants.
  • An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl - 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • the present invention provides a polynucleotide molecule encoding an antibody or antigen-binding fragment thereof as described herein.
  • the polynucleotide molecule may comprise a polynucleotide molecule encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or a polynucleotide comprising the amino acid sequence encoding the light chain and/or heavy chain of an antibody. Glycolic acid molecule.
  • the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the vector is a eukaryotic expression vector.
  • the polynucleotide molecules as described herein are contained in one or more expression vectors.
  • the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein or expressing an antibody or antigen-binding fragment thereof as described herein.
  • the host cells are eukaryotic cells, more preferably mammalian cells.
  • the present invention provides a method for preparing an antibody or antigen-binding fragment thereof as described herein, the method comprising culturing the antibody or antigen-binding fragment thereof as described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof
  • the host cell is used to express the antibody or antigen-binding fragment thereof, and the expressed antibody or antigen-binding fragment thereof is recovered.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, Monkey Kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • ATCC American Type Culture Collection
  • the present invention provides a method of making an antibody as described herein, wherein the method comprises, when introducing an expression vector into a mammalian host cell, by culturing the host cell for a period of time sufficient to allow the antibody to grow in the host.
  • the antibody is produced by expression in the cell, or more preferably by secretion of the antibody into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • afucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic , because their carbohydrate structure is a normal component of native human serum IgG.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, or a host as described herein cells, and a pharmaceutically acceptable carrier or excipient.
  • the antibodies or pharmaceutical compositions thereof provided by the present invention may incorporate suitable carriers, excipients and other agents in formulations for co-administration to provide improved transfer, delivery, tolerance, and the like.
  • composition refers to a formulation that allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered.
  • Antibodies of the invention can be prepared by mixing an antibody of the invention of the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)) containing the compounds described herein.
  • Pharmaceutical formulations of antibodies preferably in the form of aqueous solutions or lyophilized formulations.
  • compositions or formulations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other .
  • the other active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a growth inhibitory agent, an antibiotic, or various anti-tumor or anti-cancer agents known, in a suitable amount effective for the intended use exist in combination.
  • the pharmaceutical compositions of the present invention further comprise compositions of polynucleotide molecules encoding the antibodies described herein.
  • the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or the pharmaceutical compositions described herein.
  • antibodies provided herein can be used in therapeutic methods. It should also be understood that when discussing an "antibody", compositions comprising the antibody are also included.
  • the antibodies of the present invention may be used in a therapeutically or prophylactically effective amount in any of the methods of treatment or prophylaxis described in any of the embodiments of the present invention.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical combination described herein
  • a compound in the preparation of a medicine for the treatment and/or prevention of infection by SARS-CoV-2 or its variants preferably, the SARS-CoV-2 variants include alpha mutants (Alpha mutants), beta mutants strain (Beta mutant), Gamma mutant (Gamma mutant), Delta mutant (Delta mutant), Epsilon mutant, Zeta mutant, Eta mutant, Theta mutant, Iota mutant, Kappa mutant At least one of a strain (Kappa mutant strain), a Mu mutant strain (Mu mutant strain), and an Omicron mutant strain (omicron mutant strain); preferably an Omicron mutant strain.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical combination described herein substances for the treatment and/or prevention of infection with SARS-CoV-2 or variants thereof.
  • the present invention provides a method of treating and/or preventing infection by SARS-CoV-2 or a variant thereof, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, A polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition or combination described herein.
  • Modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), by inhalation, aerosol delivery, or intralesional administration, and the like.
  • the invention also includes co-administration to a subject of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents).
  • the therapy includes surgery and/or radiation therapy.
  • the antibodies, antigen-binding fragments thereof, or pharmaceutical compositions of the invention can be used alone or in combination with other therapeutic agents in therapy.
  • the antibody, antigen-binding fragment or pharmaceutical composition thereof of the invention is co-administered with at least one additional therapeutic agent.
  • the present invention provides a method of detecting the presence of SARS-CoV-2 or a variant thereof in a sample using the antibody or antigen-binding fragment thereof described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the method comprises the steps of contacting the antibody or antigen-binding fragment thereof described herein, or a detection composition comprising the antibody or antigen-binding fragment thereof, with a sample, and detecting the presence or absence of the antibody or antigen-binding fragment thereof and SARS- Steps in which CoV-2 or its variant CBD binds to produce a conjugate or a binding signal.
  • the antibodies or antigen-binding fragments thereof described herein can be labeled to indicate whether the conjugate is formed.
  • the present invention provides a detection reagent comprising the antibody or antigen-binding fragment thereof of any of the embodiments herein.
  • the detection reagent may contain a suitable carrier such as a solvent such as water.
  • the detection reagents herein can be obtained by diluting the recombinant human ACE protein with murine Fc linked at its C-terminus with skim milk, and then diluting the antibody or antigen-binding fragment thereof described in any of the embodiments herein with the resulting dilution.
  • the antibody or antigen-binding fragment thereof in the detection reagent may be present at any suitable concentration, and may be diluted to the desired concentration for use.
  • the present invention provides a test strip or a test chip coated with the antibody or antigen-binding fragment thereof described in any of the embodiments herein.
  • the antibodies or antigen-binding fragments thereof described herein can be coated on commonly used test strips or chips using well-known methods.
  • the antibody or antigen-binding fragment thereof described in any of the embodiments herein is coated onto a Protein A chip via Protein A.
  • kits for detecting the presence of SARS-CoV-2 or a variant thereof in a sample may contain the antibody or antigen-binding fragment thereof described in any of the embodiments herein, detection reagents, and/or detection strips or chips.
  • kits for use in the therapeutic treatment and/or prevention of infection by SARS-CoV-2 or a variant thereof may contain the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector, host cell or pharmaceutical composition described in any of the embodiments herein.
  • kits of any of the embodiments herein in the manufacture of a medicament for diagnosing infection by SARS-CoV-2 or a variant thereof; or of any of the embodiments herein
  • Antibodies or antigen-binding fragments thereof, polynucleotide molecules, expression vectors, host cells, detection reagents, and/or detection strips or detection chips of any of the embodiments are prepared for use in detecting the presence of SARS-CoV-2 or SARS-CoV-2 in a sample. Kit application of its variants.
  • the compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other methods, and equivalent substitutions known to those skilled in the art
  • preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the present invention adopts the following abbreviations:
  • OD optical density
  • HRP stands for horseradish peroxidase
  • IPTG stands for isopropyl- ⁇ -D-thiogalactoside
  • TMB stands for 3,3',5,5'-tetramethylbenzidine
  • PBS stands for Phosphate Buffered Saline
  • PBST stands for PBS + 0.05% Tween 20.
  • VH fragment of antibody variable region was obtained by PCR with the plasmid of CB6-HC (made by Junshi Biotechnology) as the template, and the VL fragment of antibody variable region was obtained by PCR with the plasmid of CB6-LC (made by Junshi Biotechnology), and then respectively passed through VH-CH1 (amino acid sequence shown in SEQ ID NO: 11) and VL-C ⁇ (amino acid sequence shown in SEQ ID NO: 10) were obtained by overlapping PCR with CH1 and C ⁇ . VH-CH1 and VL-CK were assembled into Fab fragments by overlapping PCR.
  • the Fab fragments were digested with pCOS vector (manufactured by Junshi Biotechnology) with SfiI (purchased from NEB Company) and recovered with a kit.
  • the above-mentioned restriction fragment and the vector were connected with T4 ligase (purchased from NEB Company) to obtain the pCOS-CB6 plasmid containing the Fab sequence, which was transformed into E. clone.
  • CDR key site shown in Figure 1 mutation library fragments.
  • the first round of PCR used pCOS-CB6 as a template to amplify fragment H1 with primers RSC-F and H1-R; amplify fragment H2 with primers H2-F and Dp-EX; use primers RSC-F and H3-R amplified fragment H3; amplified fragment H4 with primers H3-F and Dp-EX; amplified fragment H5 with primers H2-F and H3-R; amplified fragment L1 with primers RSC-F and L1-R; Fragment L2 was amplified with primers L1-F and L3-R; fragment L3 was amplified with primers L3-F and Dp-EX.
  • the second round of PCR took fragment H1 and fragment H2 as templates and used primers RSC-F and Dp-EX overlap PCR to obtain the combined mutation of HCDR1 and HCDR2.
  • Combination mutations of HCDR1, HCDR2 and HCDR3 were obtained by PCR with primers RSC-F and Dp-EX overlap using fragment H1 and fragments H4 and H5 as templates.
  • the mutation of HCDR3 was obtained by PCR with primers RSC-F and Dp-EX overlap using fragment H3 and fragment H4 as templates.
  • Combinatorial mutations of LCDR1, LCDR2 and LCDR3 were obtained by PCR with primers RSC-F and Dp-EX overlap using fragment L1 and fragments L2 and L3 as templates.
  • the PCR products were separated by 1% agarose gel electrophoresis, and the target fragments were recovered with a gel recovery kit.
  • the combined mutated fragment and pCOS vector were digested with SfiI and recovered by the kit.
  • the digested fragment and vector were then ligated with T4 ligase.
  • the ligation product was recovered by ethanol precipitation, and the ligation product was transferred into the TG1 strain by electroporation to obtain a mutant library.
  • the constructed heavy chain library and light chain library were packaged into phages.
  • CB6H-F GCT GCC CAA CCA GCC ATG GCC GAGGTGCAGCTGGTGGAG (SEQ ID NO: 12)
  • CB6L-F GGG CCC AGG CGG CCG AGC TC GACATCGTGATGACCCAG (SEQ ID NO: 14)
  • CB6H1-R CAGGCCCTTGCCGGGGGCCTGTCTCACCCAGCTCATGTAGTTSNNSNNCACSNNGAAGCCGCTGGC (SEQ ID NO: 16)
  • CB6H2-F GTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCTACNNSNNSGGCNNSACCTTCTACGCC (SEQ ID NO: 17)
  • CB6H3-R CACCAGGGTGCCCTGGCCCCAGTAGTCCAGGTASTCSNNSNNSNNSNNSNNCACTYTGGCGCAGTAGTA (SEQ ID NO: 18)
  • CB6H3-F GACTACTGGGGCCAGGGCACCCTGGTG (SEQ ID NO: 19)
  • CB6L1-R CAGCTTGGGGGCCTTGCCGGGCTTCTGCTGGTACCAGTTCAGGTASNNSNNGATSNNCTGGCTGGCTCT (SEQ ID NO: 20)
  • CB6L1-F CAGAAGCCCGGCAAGGCCCCCAAGCTG (SEQ ID NO: 21)
  • CB6L3-R CTTGATCTCCAGCTTGGTGCCCTGGCCGAAGGTGTACTCGGGSNNSNNGCTSNNGCTCTGCTGGCA (SEQ ID NO: 22)
  • CB6L3-F GGCCAGGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 23)
  • RSC-F GAG GAG GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TC (SEQ ID NO:24)
  • Dp-EX GAG GAG GAG GAG GAG GAG GAG AGA AGC GTA GTC CGG AAC GTC (SEQ ID NO:25)
  • a single clone was picked from the plate and cultured in a 96-well plate containing 250ul volume of 0.05% glucose, 2 ⁇ YT medium (1L containing 16g tryptone, 10g yeast extract, 5g sodium chloride) at 37°C for 4 hours.
  • 2 ⁇ YT medium (1L containing 16g tryptone, 10g yeast extract, 5g sodium chloride) at 37°C for 4 hours.
  • OD 600 value is greater than or equal to 0.6
  • add 1M IPTG to a final concentration of 1mM shake well, and incubate at 30 degrees overnight.
  • Dilute streptavidin to 5 ⁇ g/ml coating add 50ul per well and leave overnight at 4°C. Plates were washed and blocked with 2% skim milk.
  • the plate was washed 4 times with PBST washing solution, and the biotinylated SARS-CoV-2 RBD (self-made by Junshi) antigen was diluted to 3ug/ml for coating, 50ul per well, incubated at room temperature for 0.5 hours, and used after washing.
  • the 96-well plate cultured overnight was centrifuged at 4000 rpm/min for 10 minutes, and the supernatant was collected. 40ul of the supernatant and 10ul of 10% skim milk were added to the antigen-coated plate and incubated for 1 hour at room temperature.
  • the amino acid sequence of CB6-HC is shown in SEQ ID NO: 9
  • the VH sequences of heavy chain CB6-1-HC to CB6-16-HC are respectively shown in SEQ ID NO: 58-73, and the rest are The corresponding part in SEQ ID NO: 9
  • the amino acid sequence of CB6-LC is shown in SEQ ID NO: 10
  • the VL parts of light chain CB6-17-LC to CB6-43-LC are shown in SEQ ID NO: 74-100, respectively The remainder shown is the corresponding portion in SEQ ID NO:10.
  • Recombinant SARS-CoV-2 RBD-His (self-made by Junshi Biotechnology) was diluted to 3.0 ⁇ g/ml for coating, incubated at 37°C for 90 minutes, washed and blocked with 2% skim milk.
  • Different concentrations of CB6 (JS016) and combinatorial library antibodies shown in Table 2 were added, incubated at 37°C for 1 hour and the plate was washed.
  • JS016-17 1.76 JS016-53 1.75 JS016-89 2.13 JS016-18 1.71 JS016-54 2.06 JS016-90 2.24 JS016-19 1.69 JS016-55 2.01 JS016-91 2.22 JS016-20 1.56 JS016-56 1.62 JS016-92 2.31 JS016-21 1.59 JS016-57 1.76 JS016-93 2.25 JS016-22 1.54 JS016-58 1.80 JS016-94 2.23 JS016-23 0.22 JS016-59 1.78 JS016-95 2.27 JS016-24 1.48 JS016-60 1.52 JS016-96 2.24 JS016-25 1.51 JS016-61 1.80 JS016-97 2.29 JS016-26 1.26 JS016-62 1.37 JS016-98 2.13 JS016-27 1.47 JS016-63 1.53 JS016-99 2.14 JS
  • Example 3 In vitro binding activity of the antibodies of the present invention
  • Recombinant SARS-CoV-2-RBD-His (self-made by Junshi Biotechnology) was diluted to 3.0 ⁇ g/ml for coating, incubated at 37°C for 90 minutes, washed and blocked with 2% skim milk. Add different concentrations of JS016 antibody (positive control CB6 (JS016) from 40 ⁇ g/ml to 0.009537ng/ml, candidate antibody from 8 ⁇ g/mL to 0.001907ng/mL, 4-fold serial dilution), incubate at 37°C for 1 hour and wash the plate.
  • JS016 antibody positive control CB6 (JS016) from 40 ⁇ g/ml to 0.009537ng/ml, candidate antibody from 8 ⁇ g/mL to 0.001907ng/mL, 4-fold serial dilution
  • Example 4 In vitro blocking activity of the antibodies of the present invention
  • Recombinant SARS-CoV-2-RBD-His was diluted to 5.0 ⁇ g/ml for plating and incubated at 37°C for 90 minutes. Plates were washed and blocked with 2% skim milk.
  • Recombinant human ACE2, C-terminal mouse Fc tag was diluted to 5.0 ⁇ g/ml with 2% skim milk, and then used to dilute JS016 antibody (positive control CB6 (JS016) from 400 ⁇ g/ml to 2.26 ng/ml, candidate antibody from 80 ⁇ g/ml mL to 0.452ng/ml, 3-fold serial dilution). The mixture was added to the plate and incubated at 37°C for 1 hour and the plate was washed.
  • the antibodies of the present invention can more effectively block the binding of the RBD of the SARS-CoV-2 virus S protein to its receptor ACE2, and the blocking ability is about 2.5% higher. -4.5 times, in which the blocking ability of JS016-38/40/83/106 is slightly higher than that of other antibodies.
  • Example 5 Determination of the affinity of the antibody of the present invention to SARS-CoV-2 RBD with octet Red 96e
  • JS016-38 Kinetic parameters of binding of JS016-38 to the antigen RBD-his (SARS-CoV-2 RBD) were determined using a protein A capture method.
  • JS016-38 at a concentration of 5 ⁇ g/ml was combined with the Protein A probe (Cat No: 18-5010; lot: 2001131), and the antigen RBD-his was used in 1X Fortebio working solution (1X PBS+0.05% Tween 20) 2-fold dilution from 120nM down to 5 concentration gradients for antibody binding and dissociation in 1X Fortebio working solution.
  • JS016-40 at a concentration of 5 ⁇ g/ml was bound to the Protein A probe (Cat No: 18-5010; lot: 2001131), and the antigen RBD-his was treated with 1X Fortebio working solution (1X PBS+0.05% P20) from 120nM 2-fold dilution to set 5 concentration gradients to bind to the antibody and dissociate in 1X Fortebio working solution.
  • the on-rate of JS016-38 and JS016-40 is comparable to that of CB6, the dissociation rate is about 100 times slower than that of CB6, and the affinity constant is about 100 times higher than that of CB6.
  • the improved binding ability of JS016-38 and JS016-40 was mainly contributed by the slower dissociation.
  • KD is the affinity constant
  • kon is the antigen-antibody binding rate
  • kdis is the antigen-antibody dissociation rate
  • KD kdis/kon.
  • Example 6 Live virus neutralization activity of the antibody of the present invention
  • the confluence rate is 80% to 90%
  • take the T75 culture flask as an example, aspirate the medium in the flask, add 5 mL of PBS buffer to wash the cells, and pour off the PBS.
  • Add 3 mL of 0.25% trypsin-EDTA to submerge the cells for 1 minute of digestion, pour off the trypsin, and place them in a cell incubator to digest for 5 minutes.
  • Enzyme pipetting several times and then transferring to a centrifuge tube, centrifuging at 210g for 5 minutes, decanting the supernatant, resuspending the cells with 10 mL DMEM complete medium (Gibco), counting the cells, and diluting the cells with DMEM complete medium to 1.5 ⁇ 10 5 /mL.
  • DMEM complete medium Gibco
  • dilute SARS-CoV-2 with antibody diluent to 2 ⁇ 10 3 TCID 50 /ml (calculate the dilution factor according to the provided virus titer); in 96 wells with antibodies
  • the above 96-well plate was placed in a cell culture incubator (37°C, 5% CO 2 ) and incubated for 1 hour.
  • both monoclonal antibodies can inhibit the infection of Vero E6 cells by SARS-CoV-2 with a concentration gradient dependent effect.
  • the ND 50 of JS016-38 and JS016-40 are 0.47 ⁇ 0.16 ⁇ g/ mL and 0.09 ⁇ 0.02 ⁇ g/mL.
  • affinity maturation mutation the neutralizing activity of JS016-40 was increased 4-fold compared with CB6, while the neutralizing activity of JS016-38 was comparable to CB6.
  • Example 7 Binding activity of the antibody of the present invention to SARS-CoV-2-RBD mutant
  • the recombinant SARS-CoV-2-RBD mutants were: K417N, E484K and N501Y mutations.
  • Example 8 Antibodies of the present invention block the binding of SARS-CoV-2 mutants to ACE2
  • the recombinant SARS-CoV-2-RBD mutants were: K417N, E484K and N501Y mutations.
  • the CB6 antibody required very high concentrations to block the interaction of RBD-N501Y-his and RBD-E484K-his with Binding of ACE2 did not block the binding of RBD-K417N-his to ACE2 (Fig. 7A).
  • the antibodies JS016-38 and JS016-40 of the present invention can block the binding of RBD-his, RBD-N501Y-his, RBD-K417N-his and RBD-E484K-his mutants to ACE2 respectively, and the IC 50 is significantly better than the control Antibody CB6 ( Figure 7B, 7C).
  • Antibodies of the present invention block the binding of SARS-CoV-2 RBD and its mutants to ACE2
  • the JS016-41-YTE and JS016-77-YTE described herein are obtained by replacing the heavy chain constant region with JS016-41 and JS016-77, respectively.
  • the heavy chain amino acid sequence of the antibody JS016-41-YTE is shown in SEQ ID NO: 148 , the light chain amino acid sequence is shown in SEQ ID NO: 149; the heavy chain amino acid sequence of JS016-77-YTE is shown in SEQ ID NO: 148, and the light chain amino acid sequence is shown in SEQ ID NO: 150.
  • the antibody at a concentration of 1 ⁇ g/ml was bound to a Protein A chip (label No: 29139131-AA; lot: 10261132), and the antigen was treated with 1X HBS-EP working solution (purchased from Life science, BR-1006- 69) 2-fold dilution from 72nM or 36nM down to set 6 concentration gradients to bind to the antibody, and dissociate in HBS-EP working solution.
  • 1X HBS-EP working solution purchased from Life science, BR-1006- 69 2-fold dilution from 72nM or 36nM down to set 6 concentration gradients to bind to the antibody, and dissociate in HBS-EP working solution.
  • the kinetic parameters of the binding of each antibody to RBD-his and RBD-Omicron-his are shown in Table 8, and the detection results of kinetic characteristic parameters are shown in Figures 8A, 8B, 8C and 8D, respectively.
  • the results showed that CB6 did not bind to Omicron, while JS016-40, JS016-41-YTE and JS016-77-YTE had better binding to Omicron; the binding rates of JS016-41-YTE and JS016-77-YTE to RBD-his Comparable to CB6, the dissociation rate is about 100 times slower than that of CB6, and the affinity constant with RBD-his is about 100 times higher than that of CB6.
  • Example 10 Binding activity of antibody to S protein of omicron mutant strain of SARS-CoV-2 in vitro (ELISA)
  • the recombinant SARS-CoV-2 omicron mutant S protein (purchased from Acro, product number: SPN-C52Hz) was diluted to 3.0 ⁇ g/mL, placed in a coated plate, incubated at 37°C for 90 minutes, washed and washed with 2% Skim milk in PBS for blocking.
  • Antibodies at different concentrations (from 10 ⁇ g/mL to 0.61 ng/mL, 4-fold serial dilution, 8 concentrations in total) were added, incubated at 37°C for 1 hour and the plate was washed. It was then incubated with 1:5000 (v/v) diluted goat anti-human IgG (Fc-specific) peroxidase antibody (purchased from Sigma, Cat. No.
  • CB6 has no binding activity to S protein of omicron mutant strain, and JS016-40, JS016-41-YTE and JS016-77-YTE are better than CB6.
  • the SARS-CoV-2 Omicron mutant mutant (1 ⁇ L virus/well) pseudovirus was pre-incubated with the test antibody (from 100 ⁇ g/mL to 0.1 pg/mL, 10-fold serial dilution) at 37 °C for 1 h. Then resuspend 293-ACE2 cells with experimental buffer (DMEM medium (1X) + 10v/v% FBS), add 20,000 cells per well to the mixture of pseudovirus and antibody, and incubate at 37°C 24h.
  • DMEM medium (1X) + 10v/v% FBS experimental buffer
  • luciferase reporter gene detection reagent Bright-Lite Luciferase Assay System, purchased from Vazyme, product number: DD1204
  • Plot regression (4PL) model was used to fit the curve to obtain the IC50 value.
  • CB6 has no neutralizing activity against omicron mutants.
  • the IC50 of JS016-40, JS016-41-YTE and JS016-77-YTE are 24.57ng/ml, 17.62ng/ml and 776.4ng/ml, respectively, which are all better than CB6.

Abstract

A SARS-CoV-2 antibody and an application thereof. Provided is an antibody or an antigen-binding fragment thereof that specifically binds to a receptor-binding domain (RBD) of SARS-CoV-2 or a variant thereof. Also provided are a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a vector and a host cell for expressing the antibody or the antigen-binding fragment thereof, and a therapeutic and diagnostic method and an application of the antibody or the antigen-binding fragment thereof.

Description

新型冠状病毒抗体及其用途Novel coronavirus antibodies and their uses 技术领域technical field
本发明属于医药技术领域，具体涉及高中和活性的新型冠状病毒SARS-CoV-2或其变体的抗体或其抗原结合片段及其应用。还提供了编码本发明抗体或其抗原结合片段的核酸分子，用于表达本发明抗体或其抗原结合片段的载体和宿主细胞，以及本发明抗体或其抗原结合片段的治疗和诊断方法和用途。The invention belongs to the field of medical technology, and particularly relates to an antibody or an antigen-binding fragment thereof of a novel coronavirus SARS-CoV-2 or a variant thereof with high neutralizing activity and application thereof. Also provided are nucleic acid molecules encoding the antibodies of the invention or antigen-binding fragments thereof, vectors and host cells for expressing the antibodies of the invention or antigen-binding fragments thereof, and therapeutic and diagnostic methods and uses of the antibodies of the invention or antigen-binding fragments thereof.
背景技术Background technique
截至2020年10月22日，新型冠状病毒2019-nCoV导致的疾病(COVID-19)的全球确诊病例已超4000万例，死亡病例累计已超110万例，对公众的生命和健康造成重大威胁。然而，目前对于该病毒还没有特效药物。As of October 22, 2020, the number of confirmed cases of the disease caused by the new coronavirus 2019-nCoV (COVID-19) has exceeded 40 million globally, and the cumulative number of deaths has exceeded 1.1 million, posing a major threat to the life and health of the public . However, there is currently no specific drug for the virus.
治疗性抗体药物不但在肿瘤和自身免疫疾病方面占有重要地位，在传染性疾病的治疗中也同样有效。目前已经上市的治疗和预防病毒感染的药物有预防小儿呼吸道合胞病毒(RSV)感染的帕利珠单抗(Synagis)，治疗HIV感染的艾巴利珠单抗(Trogarzo)，以及用于狂犬病毒暴露后预防的Rabishield。同时还有针对众多病毒的单克隆抗体处于临床研究的不同阶段。Therapeutic antibody drugs play an important role not only in tumors and autoimmune diseases, but also in the treatment of infectious diseases. Drugs currently on the market for the treatment and prevention of viral infections include palivizumab (Synagis) for the prevention of pediatric respiratory syncytial virus (RSV) infection, ibalizumab (Trogarzo) for HIV infection, and rabies Rabishield for post-exposure prophylaxis. There are also monoclonal antibodies against numerous viruses in various stages of clinical research.
2019-nCoV属于冠状病毒。同属冠状病毒的重症急性呼吸综合征冠状病毒(SARS-CoV)以及中东呼吸综合征冠状病毒(MERS-CoV)也曾在分别在2002-2003年和2012年引发疫情。据世界卫生组织(WHO)统计SARS-CoV共引发8000人感染，794人死亡(https://www.who.int/)。自2012年至今，MERS-CoV感染病毒病例在持续增加，截至2019年底，全球确诊2499例感染病例，861例死亡病例。2020年1月12日，世界卫生组织正式将该新型冠状病毒命名为“2019新型冠状病毒(2019-nCoV)”，其后在2020年2月11-12日国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)宣布，新型冠状病毒(2019-nCoV)的正式分类名为严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)，世界卫生组织(WHO)同日在日内瓦举办全球研究和创新论坛上宣布，由这一病毒导致的疾病的正式名称为“COVID-19”。2019-nCoV is a coronavirus. Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), both of which are coronaviruses, also caused outbreaks in 2002-2003 and 2012, respectively. According to the World Health Organization (WHO), SARS-CoV caused a total of 8,000 infections and 794 deaths (https://www.who.int/). Since 2012, cases of MERS-CoV infection have continued to increase. As of the end of 2019, 2,499 infections and 861 deaths have been confirmed worldwide. On January 12, 2020, the World Health Organization officially named the new coronavirus "2019 novel coronavirus (2019-nCoV)", and later on February 11-12, 2020, the International Committee on Taxonomy of Viruses (International Committee on Taxonomy) of Viruses, ICTV) announced that the official classification of the new coronavirus (2019-nCoV) is called severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), the World Health Organization (WHO) on the same day The official name of the disease caused by the virus is "COVID-19", announced at the Global Research and Innovation Forum in Geneva.
近日，南非又曝出了另一种更具传染性的新冠病毒变体，其导致南非近期新冠确诊病例数激增，南非科学家和卫生官员将其命名为“501.V2”，并认为这种变异病毒可能是引发南非第二波疫情的主要原因。然而，目前的多种新冠疫苗均对南非变异的病毒无效。Recently, South Africa has revealed another more contagious new coronavirus variant, which has led to a recent surge in the number of confirmed new crown cases in South Africa. South African scientists and health officials have named it "501.V2" and believe that this variant The virus may be the main cause of the second wave of outbreaks in South Africa. However, many of the current Covid-19 vaccines are ineffective against the South African mutated virus.
病毒要感染细胞，首先需要通过囊膜蛋白结合宿主的受体。抗体，尤其是中和活性抗体，通过结合到囊膜蛋白上，阻断病毒与细胞受体的结合，从而阻断病毒感染。同时，抗体结合到囊膜蛋白上，从而对游离的病毒或是被感染的细胞进行标记，通过抗体的Fc区募集巨噬细胞或是补体等免疫细胞和免疫分子，从而清除游离的病毒以及被感染的细胞。因此，靶向受体结合区(receptor binding domain，RBD)的抗体，不但具有中和病毒感染的活性，还可以通过Fc区发挥作用，促进病毒以及被感染细胞的清除。For a virus to infect a cell, it first needs to bind to the host's receptor through an envelope protein. Antibodies, especially neutralizing antibodies, block viral infection by binding to envelope proteins and blocking the binding of viruses to cellular receptors. At the same time, the antibody binds to the envelope protein to label free virus or infected cells, and recruits immune cells and immune molecules such as macrophages or complement through the Fc region of the antibody, thereby removing free virus and infected cells. infected cells. Therefore, antibodies targeting the receptor binding domain (RBD) not only have the activity of neutralizing virus infection, but can also act through the Fc region to promote the clearance of viruses and infected cells.
与受体结合的重要囊膜蛋白是刺突蛋白(S)，S可进一步分为S1和S2两部分。S2的作用是介导膜融合。S1的N端(NTD)和C端(CTD)都可能是RBD，CTD是此冠状病毒的RBD，结合受体ACE2。因此靶向RBD的抗体，并且是阻断S与ACE2结合的抗体，可能成为抑制病毒感染的中和抗体。An important envelope protein that binds to the receptor is the spike protein (S), which can be further divided into two parts, S1 and S2. The role of S2 is to mediate membrane fusion. Both the N-terminal (NTD) and C-terminal (CTD) of S1 may be RBD, and CTD is the RBD of this coronavirus, which binds to the receptor ACE2. Therefore, an antibody targeting RBD, which is an antibody that blocks the binding of S to ACE2, may become a neutralizing antibody that inhibits viral infection.
发明内容SUMMARY OF THE INVENTION
本发明提供了特异性结合SARS-CoV-2或其变体RBD的抗体或其抗原结合片段，其具有针对SARS-CoV-2或其变体的高中和活性等优势。本发明提供的中和SARS-CoV-2或其变体的抗体或其抗原结合片段可作为独立的疗法或与其它疗法/或其他药物联合。The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to SARS-CoV-2 or its variant RBD, which has advantages such as high neutralization activity against SARS-CoV-2 or its variant. Antibodies or antigen-binding fragments thereof that neutralize SARS-CoV-2 or variants thereof provided by the present invention can be used as stand-alone therapy or in combination with other therapies/or other drugs.
在一个方面，本发明提供了一种抗体或其抗原结合片段，其特异性结合SARS-CoV-2或其变体的受体结合结构域(RBD)，其中所述抗体或其抗原结合片段包含如下所示的重链可变区的CDR序列HCDR1、HCDR2与HCDR3和/或轻链可变区的CDR序列LCDR1、LCDR2和LCDR3：In one aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain (RBD) of SARS-CoV-2 or a variant thereof, wherein the antibody or antigen-binding fragment thereof comprises The CDR sequences HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and/or the CDR sequences LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown below:
HCDR1：GFX 1VX 2X 3NY(SEQ ID NO:144)，其中，X 1选自L、T、E、R、Q、V、W、I或S，优选地，X 1选自L、T、E、R、Q、V或I；X 2选自Q、G、D、R、P、M、K、V、A、N或Y，优选地，X 2选自Q、G、D、R、P、N或Y；X 3选自R、W、Y、A、F、V或H，优选地，X 3选自W、R、A、F或V； HCDR1: GFX 1 VX 2 X 3 NY (SEQ ID NO: 144), wherein X 1 is selected from L, T, E, R, Q, V, W, I or S, preferably, X 1 is selected from L, T, E, R, Q, V or I; X 2 is selected from Q, G, D, R, P, M, K, V, A, N or Y, preferably, X 2 is selected from Q, G, D , R, P, N or Y; X 3 is selected from R, W, Y, A, F, V or H, preferably, X 3 is selected from W, R, A, F or V;
HCDR2：IYPGGX 4T(SEQ ID NO:145)，其中，X 4为T或S； HCDR2: IYPGGX 4 T (SEQ ID NO: 145), wherein X 4 is T or S;
HCDR3：ARVLPMYGDYLDY(SEQ ID NO:3)；HCDR3: ARVLPMYGDYLDY (SEQ ID NO: 3);
LCDR1：QX 5IX 6X 7Y(SEQ ID NO:146)，其中，X 5选自V、D、Q、A、W、R、N、S、D、M、K或P，优选地，X 5选自Q、A、R、N、S、D或M；X 6选自N、H、L、G、P、S、M、E、V、R、D、A或I，优选地，X 6选自E、L、V、R、D、E或A；X 7选自H、 V、F、P、N、S、R、Q、G、Y或T，优选地，X 7选自Q、P、S、G、P、R或Y； LCDR1: QX 5 IX 6 X 7 Y (SEQ ID NO: 146), wherein X 5 is selected from V, D, Q, A, W, R, N, S, D, M, K or P, preferably, X 5 is selected from Q, A, R, N, S, D or M; X 6 is selected from N, H, L, G, P, S, M, E, V, R, D, A or I, preferably , X 6 is selected from E, L, V, R, D, E or A; X 7 is selected from H, V, F, P, N, S, R, Q, G, Y or T, preferably, X 7 selected from Q, P, S, G, P, R or Y;
LCDR2：AAS(SEQ ID NO:5)；LCDR2: AAS (SEQ ID NO: 5);
LCDR3：QQSX 8SX 9X 10PEYT(SEQ ID NO:147)，其中，X 8选自G、Y、T、S、K、A、N、E或P，优选地，X 8选自A、N、S或P；X 9选自P、S、I、N、A、W或F，优选地，X 9选自S、P或A；X 10选自T、V、L、I、R、K、S、M或F，优选地，X 10选自S、R、T、K、V、L或F。 LCDR3: QQSX 8 SX 9 X 10 PEYT (SEQ ID NO: 147), wherein X 8 is selected from G, Y, T, S, K, A, N, E or P, preferably, X 8 is selected from A, N, S or P; X 9 is selected from P, S, I, N, A, W or F, preferably, X 9 is selected from S, P or A; X 10 is selected from T, V, L, I, R , K, S, M or F, preferably X 10 is selected from S, R, T, K, V, L or F.
在一些实施方式中，SEQ ID NO:146所示的LCDR1不包括SEQ ID NO:4所示的LCDR1。In some embodiments, the LCDR1 shown in SEQ ID NO:146 does not include the LCDR1 shown in SEQ ID NO:4.
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:144、145和3所示的HCDR1、HCDR2与HCDR3和SEQ ID NO:146、5和147所示的LCDR1、LCDR2和LCDR3。In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 144, 145, and 3 and LCDRl, LCDR2, and LCDR3 set forth in SEQ ID NOs: 146, 5, and 147 .
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:144、145和3所示的HCDR1、HCDR2与HCDR3和SEQ ID NO:4、5和6所示的LCDR1、LCDR2和LCDR3。In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 144, 145, and 3 and LCDRl, LCDR2, and LCDR3 set forth in SEQ ID NOs: 4, 5, and 6 .
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:1、2和3所示的HCDR1、HCDR2与HCDR3和SEQ ID NO:146、5和147所示的LCDR1、LCDR2和LCDR3。In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 1, 2, and 3 and LCDRl, LCDR2, and LCDR3 set forth in SEQ ID NOs: 146, 5, and 147 .
优选地，本文任一实施方式所述的SEQ ID NO:144所示的HCDR1选自SEQ ID NO:26、28、29、31、32、33、34、35、104、105、106、107、108、109、110和111中任一所示的HCDR1，优选选自SEQ ID NO:26、28、29、31、32、33、34和35中任一所示的HCDR1。Preferably, the HCDR1 shown in SEQ ID NO: 144 described in any of the embodiments herein is selected from SEQ ID NO: 26, 28, 29, 31, 32, 33, 34, 35, 104, 105, 106, 107, The HCDR1 shown in any one of 108, 109, 110 and 111 is preferably selected from the HCDR1 shown in any one of SEQ ID NOs: 26, 28, 29, 31, 32, 33, 34 and 35.
优选地，本文任一实施方式所述的SEQ ID NO:145所示的HCDR2选自SEQ ID NO:27和30任一所示的HCDR2。Preferably, the HCDR2 set forth in SEQ ID NO: 145 according to any of the embodiments herein is selected from the HCDR2 set forth in any one of SEQ ID NO: 27 and 30.
优选地，本文任一实施方式所述的SEQ ID NO:146所示的LCDR1选自SEQ ID NO:36、38、40、42、44、46、48、50、52、54、56、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140和142中任一所示的LCDR1，优选选自SEQ ID NO:36、38、40、42、44、46、48、50、52、54和56中任一所示的LCDR1。Preferably, the LCDR1 shown in SEQ ID NO: 146 according to any embodiment herein is selected from SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 112, LCDR1 shown in any one of 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140 and 142, preferably selected from SEQ ID NOs: 36, 38, 40, LCDR1 shown in any of 42, 44, 46, 48, 50, 52, 54 and 56.
优选地，本文任一实施方式所述的SEQ ID NO:147所示的LCDR3选自SEQ ID NO:37、39、41、43、45、47、49、51、53、55、57、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141和143中任一所示的LCDR3，优选选自SEQ ID NO:37、39、41、43、45、47、49、51、53、55和57中任一所示的LCDR3。Preferably, the LCDR3 shown in SEQ ID NO: 147 according to any embodiment herein is selected from SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 113, LCDR3 shown in any one of 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 and 143, preferably selected from SEQ ID NOs: 37, 39, 41, LCDR3 shown in any of 43, 45, 47, 49, 51, 53, 55 and 57.
在一些实施方式中，本发明所述的抗体或其抗原结合片段的重链可变区含有选自以下组的HCDR1、HCDR2和HCDR3：SEQ ID NO:104、27和3；SEQ ID NO:26、27和3；SEQ ID NO:28、27和3；SEQ ID NO:29、30和3；SEQ ID NO:105、27和3；SEQ ID NO:31、 30和3；SEQ ID NO:32、30和3；SEQ ID NO:106、30和3；SEQ ID NO:107、30和3；SEQ ID NO:108、27和3；SEQ ID NO:33、30和3；SEQ ID NO:109、27和3；SEQ ID NO:110、30和3；SEQ ID NO:111、30和3；SEQ ID NO:34、30和3；和SEQ ID NO:35、30和3。在一些实施方式中，本发明所述的抗体或其抗原结合片段的重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:31、30和3所示。优选地，在这些实施方式的一部分实施方式中，所述抗体或其抗原结合片段的轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:4、5和6所示。In some embodiments, the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention contains HCDR1, HCDR2, and HCDR3 selected from the group consisting of: SEQ ID NOs: 104, 27, and 3; SEQ ID NO: 26 , 27 and 3; SEQ ID NO: 28, 27 and 3; SEQ ID NO: 29, 30 and 3; SEQ ID NO: 105, 27 and 3; SEQ ID NO: 31, 30 and 3; SEQ ID NO: 32 , 30 and 3; SEQ ID NO: 106, 30 and 3; SEQ ID NO: 107, 30 and 3; SEQ ID NO: 108, 27 and 3; SEQ ID NO: 33, 30 and 3; SEQ ID NO: 109 , 27 and 3; SEQ ID NO: 110, 30 and 3; SEQ ID NO: 111, 30 and 3; SEQ ID NO: 34, 30 and 3; and SEQ ID NO: 35, 30 and 3. In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention are shown in SEQ ID NOs: 31, 30 and 3, respectively. Preferably, in some of these embodiments, the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 4, 5 and 6, respectively.
在一些实施方式中，本发明所述的抗体或其抗原结合片段的轻链可变区含有选自以下组的LCDR1、LCDR2和LCDR3：SEQ ID NO:112、5、113；SEQ ID NO:114、5、115；SEQ ID NO:116、5、117；SEQ ID NO:118、5、119；SEQ ID NO:120、5、121；SEQ ID NO:122、5、123；SEQ ID NO:124、5、125；SEQ ID NO:126、5、127；SEQ ID NO:128、5、129；SEQ ID NO:130、5、131；SEQ ID NO:132、5、133；SEQ ID NO:134、5、135；SEQ ID NO:136、5、137；SEQ ID NO:138、5、139；SEQ ID NO:140、5、141；SEQ ID NO:142、5、143；SEQ ID NO:36、5、37；SEQ ID NO:38、5、39；SEQ ID NO:40、5、41；SEQ ID NO:42、5、43；SEQ ID NO:44、5、45；SEQ ID NO:46、5、47；SEQ ID NO:48、5、49；SEQ ID NO:50、5、51；SEQ ID NO:52、5、53；SEQ ID NO:54、5、55；和SEQ ID NO:56、5、57。在一些实施方式中，本发明所述的抗体或其抗原结合片段的轻链可变区含有选自以下组的LCDR1、LCDR2和LCDR3：SEQ ID NO:48、5、49；和SEQ ID NO:54、5、55。优选地，在这些实施方式的一部分实施方式中，所述抗体或其抗原结合片段的重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:1、2和3所示。In some embodiments, the light chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises LCDR1, LCDR2, and LCDR3 selected from the group consisting of: SEQ ID NO: 112, 5, 113; SEQ ID NO: 114 , 5, 115; SEQ ID NO: 116, 5, 117; SEQ ID NO: 118, 5, 119; SEQ ID NO: 120, 5, 121; SEQ ID NO: 122, 5, 123; SEQ ID NO: 124 , 5, 125; SEQ ID NO: 126, 5, 127; SEQ ID NO: 128, 5, 129; SEQ ID NO: 130, 5, 131; SEQ ID NO: 132, 5, 133; SEQ ID NO: 134 , 5, 135; SEQ ID NO: 136, 5, 137; SEQ ID NO: 138, 5, 139; SEQ ID NO: 140, 5, 141; SEQ ID NO: 142, 5, 143; SEQ ID NO: 36 , 5, 37; SEQ ID NO: 38, 5, 39; SEQ ID NO: 40, 5, 41; SEQ ID NO: 42, 5, 43; SEQ ID NO: 44, 5, 45; SEQ ID NO: 46 , 5, 47; SEQ ID NO: 48, 5, 49; SEQ ID NO: 50, 5, 51; SEQ ID NO: 52, 5, 53; SEQ ID NO: 54, 5, 55; and SEQ ID NO: 56, 5, 57. In some embodiments, the light chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises LCDR1, LCDR2, and LCDR3 selected from the group consisting of: SEQ ID NO: 48, 5, 49; and SEQ ID NO: 54, 5, 55. Preferably, in some of these embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 1, 2 and 3, respectively.
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和/或轻链可变区，其中：In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and/or a light chain variable region, wherein:
所述重链可变区包含：The heavy chain variable region comprises:
(Ⅰ)氨基酸序列分别如SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(I) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 3 respectively; or with SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(Ⅱ)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:28、SEQ ID NO:27和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(II) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 28, SEQ ID NO: 27 and SEQ ID NO: 3 respectively; or with SEQ ID NO: 28, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(Ⅲ)氨基酸序列分别如SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序 列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(III) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3 respectively; : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(Ⅳ)氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:31、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(IV) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(Ⅴ)氨基酸序列分别如SEQ ID NO32、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(V) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO32, SEQ ID NO:30 and SEQ ID NO:3, respectively; or with SEQ ID NO:32, SEQ ID NO:30 and SEQ ID NO:3 HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in the indicated amino acid sequences, respectively; or
(Ⅵ)氨基酸序列分别如SEQ ID NO:33、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:33、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(VI) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(Ⅶ)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:34、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(VII) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 34, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 34, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(Ⅷ)氨基酸序列分别如SEQ ID NO:35、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:35、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(VIII) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:35, SEQ ID NO:30 and SEQ ID NO:3, respectively; or with SEQ ID NO:35, SEQ ID NO:30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(IX)氨基酸序列分别如SEQ ID NO:104、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:104、SEQ ID NO:27和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(IX) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 104, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 104, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(X)氨基酸序列分别如SEQ ID NO:105、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:105、SEQ ID NO:27和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(X) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 105, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 105, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(XI)氨基酸序列分别如SEQ ID NO:106、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:106、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(XI) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 106, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 106, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(XII)氨基酸序列分别如SEQ ID NO:107、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:107、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(XII) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 107, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 107, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(XIII)氨基酸序列分别如SEQ ID NO:108、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:108、SEQ ID NO:27和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(XIII) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 108, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 108, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(XIV)氨基酸序列分别如SEQ ID NO:109、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:109、SEQ ID NO:27和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3(XIV) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 109, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 109, SEQ ID NO: 27 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 whose amino acid sequences shown in 3 have 1, 2 or 3 amino acid differences, respectively
(XV)氨基酸序列分别如SEQ ID NO:110、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:110、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(XV) HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO: 110, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 110, SEQ ID NO: 30 and SEQ ID NO : HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in their amino acid sequences respectively; or
(XVI)氨基酸序列分别如SEQ ID NO:111、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:111、SEQ ID NO:30和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；(XVI) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 111, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or with SEQ ID NO: 111, SEQ ID NO: 30 and SEQ ID NO HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 3;
所述轻链可变区包含：The light chain variable region comprises:
(Ⅰ)氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(I) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:36, SEQ ID NO:5 and SEQ ID NO:37 respectively; or with SEQ ID NO:36, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 37; or
(Ⅱ)氨基酸序列分别如SEQ ID NO:38、SEQ ID NO:5和SEQ ID NO:39所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:38、SEQ ID NO:5和SEQ ID NO:39所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(II) the amino acid sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:38, SEQ ID NO:5 and SEQ ID NO:39 respectively; or with SEQ ID NO:38, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 39; or
(Ⅲ)氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:5和SEQ ID NO:41所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:40、SEQ ID NO:5和SEQ ID NO:41所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(III) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 40, SEQ ID NO: 5 and SEQ ID NO: 41 respectively; or with SEQ ID NO: 40, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 41; or
(Ⅳ)氨基酸序列分别如SEQ ID NO:42、SEQ ID NO:5和SEQ ID NO:43所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:42、SEQ ID NO:5和SEQ ID NO:43所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(IV) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 42, SEQ ID NO: 5 and SEQ ID NO: 43, respectively; or with SEQ ID NO: 42, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 43; or
(Ⅴ)氨基酸序列分别如SEQ ID NO:44、SEQ ID NO:5和SEQ ID NO:45所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:44、SEQ ID NO:5和SEQ ID NO:45所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(V) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:44, SEQ ID NO:5 and SEQ ID NO:45, respectively; or with SEQ ID NO:44, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 45; or
(Ⅵ)氨基酸序列分别如SEQ ID NO:46、SEQ ID NO:5和SEQ ID NO:47所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:46、SEQ ID NO:5和SEQ ID NO:47所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(VI) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 46, SEQ ID NO: 5 and SEQ ID NO: 47, respectively; or with SEQ ID NO: 46, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 47; or
(Ⅶ)氨基酸序列分别如SEQ ID NO:48、SEQ ID NO:5和SEQ ID NO:49所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:48、SEQ ID NO:5和SEQ ID NO:49所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(VII) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 48, SEQ ID NO: 5 and SEQ ID NO: 49, respectively; or with SEQ ID NO: 48, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 49; or
(Ⅷ)氨基酸序列分别如SEQ ID NO:50、SEQ ID NO:5和SEQ ID NO:51所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:50、SEQ ID NO:5和SEQ ID NO:51所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(VIII) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:50, SEQ ID NO:5 and SEQ ID NO:51, respectively; or with SEQ ID NO:50, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 51; or
(Ⅸ)氨基酸序列分别如SEQ ID NO:52、SEQ ID NO:5和SEQ ID NO:53所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:52、SEQ ID NO:5和SEQ ID NO:53所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(IX) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:52, SEQ ID NO:5 and SEQ ID NO:53, respectively; or with SEQ ID NO:52, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 53; or
(Ⅹ)氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(X) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or with SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 55; or
(Ⅺ)氨基酸序列分别如SEQ ID NO:56、SEQ ID NO:5和SEQ ID NO:57所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:56、SEQ ID NO:5和SEQ ID NO:57所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XI) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:56, SEQ ID NO:5 and SEQ ID NO:57, respectively; or with SEQ ID NO:56, SEQ ID NO:5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 57; or
(XII)氨基酸序列分别如SEQ ID NO:112、SEQ ID NO:5和SEQ ID NO:113所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:112、SEQ ID NO:5和SEQ ID NO:113所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XII) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 112, SEQ ID NO: 5 and SEQ ID NO: 113, respectively; or with SEQ ID NO: 112, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 113; or
(XIII)氨基酸序列分别如SEQ ID NO:114、SEQ ID NO:5和SEQ ID NO:115所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:114、SEQ ID NO:5和SEQ ID NO:115所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XIII) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 114, SEQ ID NO: 5 and SEQ ID NO: 115, respectively; or with SEQ ID NO: 114, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 115; or
(XIV)氨基酸序列分别如SEQ ID NO:116、SEQ ID NO:5和SEQ ID NO:117所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:116、SEQ ID NO:5和SEQ ID NO:117所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XIV) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 116, SEQ ID NO: 5 and SEQ ID NO: 117, respectively; or with SEQ ID NO: 116, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 117; or
(XV)氨基酸序列分别如SEQ ID NO:118、SEQ ID NO:5和SEQ ID NO:119所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:118、SEQ ID NO:5和SEQ ID NO:119所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XV) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 118, SEQ ID NO: 5 and SEQ ID NO: 119, respectively; or with SEQ ID NO: 118, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 119; or
(XVI)氨基酸序列分别如SEQ ID NO:120、SEQ ID NO:5和SEQ ID NO:121所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:120、SEQ ID NO:5和SEQ ID NO:121所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XVI) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 120, SEQ ID NO: 5 and SEQ ID NO: 121, respectively; or with SEQ ID NO: 120, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 121; or
(XVII)氨基酸序列分别如SEQ ID NO:122、SEQ ID NO:5和SEQ ID NO:123所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:122、SEQ ID NO:5和SEQ ID NO:123所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XVII) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 122, SEQ ID NO: 5 and SEQ ID NO: 123, respectively; or with SEQ ID NO: 122, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 123; or
(XVIII)氨基酸序列分别如SEQ ID NO:124、SEQ ID NO:5和SEQ ID NO:125所示的 LCDR1、LCDR2和LCDR3；或与SEQ ID NO:124、SEQ ID NO:5和SEQ ID NO:125所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XVIII) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 124, SEQ ID NO: 5 and SEQ ID NO: 125, respectively; or with SEQ ID NO: 124, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 125; or
(XIX)氨基酸序列分别如SEQ ID NO:126、SEQ ID NO:5和SEQ ID NO:127所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:126、SEQ ID NO:5和SEQ ID NO:127所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XIX) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 126, SEQ ID NO: 5 and SEQ ID NO: 127, respectively; or with SEQ ID NO: 126, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 127; or
(XX)氨基酸序列分别如SEQ ID NO:128、SEQ ID NO:5和SEQ ID NO:129所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:128、SEQ ID NO:5和SEQ ID NO:129所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XX) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 128, SEQ ID NO: 5 and SEQ ID NO: 129, respectively; or with SEQ ID NO: 128, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 129; or
(XXI)氨基酸序列分别如SEQ ID NO:130、SEQ ID NO:5和SEQ ID NO:131所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:130、SEQ ID NO:5和SEQ ID NO:131所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XXI) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 130, SEQ ID NO: 5 and SEQ ID NO: 131, respectively; or with SEQ ID NO: 130, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 131; or
(XXII)氨基酸序列分别如SEQ ID NO:132、SEQ ID NO:5和SEQ ID NO:133所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:132、SEQ ID NO:5和SEQ ID NO:133所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XXII) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 132, SEQ ID NO: 5 and SEQ ID NO: 133, respectively; or with SEQ ID NO: 132, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 133; or
(XXIII)氨基酸序列分别如SEQ ID NO:134、SEQ ID NO:5和SEQ ID NO:135所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:134、SEQ ID NO:5和SEQ ID NO:135所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XXIII) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 134, SEQ ID NO: 5 and SEQ ID NO: 135, respectively; or with SEQ ID NO: 134, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 135; or
(XXIV)氨基酸序列分别如SEQ ID NO:136、SEQ ID NO:5和SEQ ID NO:137所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:136、SEQ ID NO:5和SEQ ID NO:137所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XXIV) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 136, SEQ ID NO: 5 and SEQ ID NO: 137, respectively; or with SEQ ID NO: 136, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 137; or
(XXV)氨基酸序列分别如SEQ ID NO:138、SEQ ID NO:5和SEQ ID NO:139所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:138、SEQ ID NO:5和SEQ ID NO:139所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XXV) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 138, SEQ ID NO: 5 and SEQ ID NO: 139, respectively; or with SEQ ID NO: 138, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 139; or
(XXVI)氨基酸序列分别如SEQ ID NO:140、SEQ ID NO:5和SEQ ID NO:141所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:140、SEQ ID NO:5和SEQ ID NO:141所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(XXVI) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 140, SEQ ID NO: 5 and SEQ ID NO: 141, respectively; or with SEQ ID NO: 140, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences respectively in the amino acid sequences shown in 141; or
(XXVII)氨基酸序列分别如SEQ ID NO:142、SEQ ID NO:5和SEQ ID NO:143所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:142、SEQ ID NO:5和SEQ ID NO:143所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3。(XXVII) the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 142, SEQ ID NO: 5 and SEQ ID NO: 143, respectively; or with SEQ ID NO: 142, SEQ ID NO: 5 and SEQ ID NO : LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences in the amino acid sequence shown at 143, respectively.
在一些实施方式中，本发明所述抗体或其抗原结合片段不包括HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:35、30和3所示，LCDR1、LCDR2和LCDR3 的氨基酸序列分别如SEQ ID NO:54、5和55所示的抗体或其抗原结合片段。在一些实施方式中，本发明所述抗体或其抗原结合片段也不包括HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:1、2和3所示，LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:38、5和39所示的抗体或其抗原结合片段。In some embodiments, the antibody or antigen-binding fragment thereof of the present invention does not include the amino acid sequences of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 35, 30 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 as shown in The antibodies or antigen-binding fragments thereof set forth in SEQ ID NOs: 54, 5 and 55. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention also does not include the amino acid sequences of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively The antibodies or antigen-binding fragments thereof set forth in SEQ ID NOs: 38, 5 and 39.
在一些实施方式中，本发明所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
(Ⅰ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(I) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
(Ⅱ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(II) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 32, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
(Ⅲ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(III) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
(Ⅳ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:35、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:48、SEQ ID NO:5和SEQ ID NO:49所示的LCDR1、LCDR2和LCDR3；或(IV) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 35, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:48, SEQ ID NO:5 and SEQ ID NO:49, respectively; or
(Ⅴ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；或(v) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
(Ⅵ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；或(VI) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:32, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
(Ⅶ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:33、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；或(VII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
(Ⅷ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:33、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或(VIII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or
(Ⅸ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ  ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或(IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:34, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or
(Ⅹ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(X) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
(Ⅺ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:48、SEQ ID NO:5和SEQ ID NO:49所示的LCDR1、LCDR2和LCDR3。(XI) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:31, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:48, SEQ ID NO:5 and SEQ ID NO:49, respectively.
在一些实施方式中，本发明所述抗体或其抗原结合片段的重链可变区选自SEQ ID NO:58-73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区，优选选自SEQ ID NO:59、60、61、63、64、68、72和73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区，更优选选自SEQ ID NO:59、61、64、72和73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区。在这些方案的一些优选实施方式中，所述抗体或其抗原结合片段的轻链可变区如SEQ ID NO:8所示。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is selected from the heavy chain variable region shown in any of SEQ ID NOs: 58-73 or can be combined with any of the heavy chain variable regions. Variable region heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity, preferably selected from SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 and 73 Any of the indicated heavy chain variable regions or heavy chain variable regions having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said heavy chain variable regions, more preferably Has at least 95%, 96%, 97%, 98% or 99% from or with any of the heavy chain variable regions set forth in SEQ ID NOs: 59, 61, 64, 72 and 73 % sequence identity of heavy chain variable regions. In some preferred embodiments of these schemes, the light chain variable region of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO:8.
在一些实施方式中，本发明所述抗体或其抗原结合片段的轻链可变区选自SEQ ID NO:74-100中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变区，优选选自SEQ ID NO:83、84、85、86、88、89、90、93、94、95和98中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变区，更优选选自SEQ ID NO:83、90和95中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变区。在这些方案的一些优选实施方式中，所述抗体或其抗原结合片段的重链可变区如SEQ ID NO:7所示。In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention is selected from the light chain variable region shown in any one of SEQ ID NOs: 74-100 or the light chain variable region of any of the light chain A light chain variable region having a variable region of at least 95%, 96%, 97%, 98% or 99% sequence identity, preferably selected from SEQ ID NOs: 83, 84, 85, 86, 88, 89, 90, A light chain variable region shown in any one of 93, 94, 95 and 98 or a light chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said light chain variable regions A chain variable region, more preferably selected from the light chain variable region shown in any of SEQ ID NOs: 83, 90 and 95 or having at least 95%, 96%, 97% with any of said light chain variable regions , 98% or 99% sequence identity of the light chain variable region. In some preferred embodiments of these schemes, the heavy chain variable region of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO:7.
在一些实施方式中，本发明所述抗体或其抗原结合片段的重链可变区选自SEQ ID NO:58-73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区，优选选自SEQ ID NO:59、60、61、63、64、68、72和73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区，更优选选自SEQ ID NO:59、61、64、72和73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区；轻链可变区选自SEQ ID NO:74-100中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变 区，优选选自SEQ ID NO:83、84、85、86、88、89、90、93、94、95和98中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变区，更优选选自SEQ ID NO:83、90和95中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变区。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is selected from the heavy chain variable region shown in any of SEQ ID NOs: 58-73 or can be combined with any of the heavy chain variable regions. Variable region heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity, preferably selected from SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 and 73 Any of the indicated heavy chain variable regions or heavy chain variable regions having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said heavy chain variable regions, more preferably Has at least 95%, 96%, 97%, 98% or 99% from or with any of the heavy chain variable regions set forth in SEQ ID NOs: 59, 61, 64, 72 and 73 % heavy chain variable regions of sequence identity; light chain variable regions are selected from or have at least 95% of the light chain variable regions shown in any of SEQ ID NOs: 74-100 A light chain variable region of %, 96%, 97%, 98% or 99% sequence identity, preferably selected from SEQ ID NOs: 83, 84, 85, 86, 88, 89, 90, 93, 94, 95 and A light chain variable region shown in any one of 98 or a light chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity with said any light chain variable region, more Preferably selected from or at least 95%, 96%, 97%, 98% or 99% of the light chain variable region shown in any one of SEQ ID NOs: 83, 90 and 95 Sequence identity of light chain variable regions.
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和轻链可变区：In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region:
(Ⅰ)所述重链可变区包含如SEQ ID NO:59、60、61、63、64、68、72或73中任一项所示的氨基酸序列，或与SEQ ID NO:59、60、61、63、64、68、72或73中任一项所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83、84、85、86、88、89、90、93、94、95或98中任一项所示的氨基酸序列，或与SEQ ID NO:83、84、85、86、88、89、90、93、94、95或98中任一项所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(I) The heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 or 73, or the amino acid sequence shown in any one of SEQ ID NOs: 59, 60 , 61, 63, 64, 68, 72 or 73 amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity; and the light chain can be The variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, 85, 86, 88, 89, 90, 93, 94, 95, or 98, or the same as SEQ ID NOs: 83, 84, 85 , 86, 88, 89, 90, 93, 94, 95, or 98 amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity; or
(Ⅱ)所述重链可变区包含如SEQ ID NO:61所示的氨基酸序列，或与SEQ ID NO:61所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(II) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or has at least 95%, 96%, 97%, 98% or 99% with the amino acid sequence shown in SEQ ID NO: 61 an amino acid sequence of % sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 95, or has at least 95%, 96%, 97%, or at least 95%, 96%, 97 amino acid sequences of %, 98% or 99% sequence identity; or
(Ⅲ)所述重链可变区包含如SEQ ID NO:64所示的氨基酸序列，或包含与SEQ ID NO:64所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或包含与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(III) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:64, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:64 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅳ)所述重链可变区包含如SEQ ID NO:59所示的氨基酸序列，或包含与SEQ ID NO:59所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或包含与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(IV) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:59, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:59 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅴ)所述重链可变区包含如SEQ ID NO:73所示的氨基酸序列，或包含与SEQ ID NO:73所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:90所示的氨基酸序列，或包含与SEQ ID NO:90所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(V) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:73 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 90 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅵ)所述重链可变区包含如SEQ ID NO:61所示的氨基酸序列，或包含与SEQ ID NO:61所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列，或包含与SEQ ID NO:83所 示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(VI) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:61 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅶ)所述重链可变区包含如SEQ ID NO:64所示的氨基酸序列，或包含与SEQ ID NO:64所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列，或包含与SEQ ID NO:83所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(VII) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:64, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:64 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅷ)所述重链可变区包含如SEQ ID NO:68所示的氨基酸序列，或包含与SEQ ID NO:68所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列，或包含与SEQ ID NO:83所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(VIII) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 68 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅸ)所述重链可变区包含如SEQ ID NO:68所示的氨基酸序列，或包含与SEQ ID NO:68所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列，或包含与SEQ ID NO:8所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(IX) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 68 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 8 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅹ)所述重链可变区包含如SEQ ID NO:72所示的氨基酸序列，或包含与SEQ ID NO:72所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列，或包含与SEQ ID NO:8所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(X) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:72, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:72 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 8 , 97%, 98% or 99% amino acid sequence identity; or
(Ⅺ)所述重链可变区包含如SEQ ID NO:63所示的氨基酸序列，或包含与SEQ ID NO:63所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或包含与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(XI) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:63 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
(XII)所述重链可变区包含如SEQ ID NO:63所示的氨基酸序列，或包含与SEQ ID NO:63所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:90所示的氨基酸序列，或包含与SEQ ID NO:90所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(XII) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:63 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 90 , 97%, 98% or 99% amino acid sequence identity; or
(XIII)所述重链可变区包含如SEQ ID NO:61、64或59所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列；或所述重链可变区包含如SEQ ID NO:73所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:90所示的氨基酸序列；或所述重链可变区包含如SEQ ID NO:61、64或68所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列；或所述重链可变区包含如SEQ ID NO:68或72所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列。(XIII) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, 64 or 59, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 95; or the The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:90; or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:90; The amino acid sequence set forth in SEQ ID NO: 61, 64 or 68, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 or 72, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.
在一些实施方式中，本发明的抗体或其抗原结合片段不包括重链可变区的氨基酸序列如SEQ ID NO:73所示、轻链可变区的氨基酸序列如SEQ ID NO:95所示的抗体或其抗原结合片段。在进一步的实施方式中，本发明的抗体或其抗原结合片段也不包括重链可变区的氨基酸序列如SEQ ID NO:7所示、轻链可变区的氨基酸序列如SEQ ID NO:84所示的抗体或其抗原结合片段。In some embodiments, the antibody or antigen-binding fragment thereof of the present invention does not include the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:73, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:95 antibodies or antigen-binding fragments thereof. In a further embodiment, the antibody or antigen-binding fragment thereof of the present invention also does not include the amino acid sequence of the variable region of the heavy chain as shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the light chain as shown in SEQ ID NO:84 The indicated antibody or antigen-binding fragment thereof.
在一些实施方式中，本发明所述抗体包含重链和轻链：In some embodiments, the antibody of the invention comprises a heavy chain and a light chain:
(Ⅰ)所述重链包含如SEQ ID NO:101所示的氨基酸序列，或与SEQ ID NO:101所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:102所示的氨基酸序列，或与SEQ ID NO:102所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(I) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 101, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 101 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 90%, 92 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
(Ⅱ)所述重链包含如SEQ ID NO:103所示的氨基酸序列，或与SEQ ID NO:103所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:102所示的氨基酸序列，或与SEQ ID NO:102所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(II) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 103, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 103 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 90%, 92 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
(III)所述重链包含如SEQ ID NO:101或103所示的氨基酸序列，和所述轻链包含如SEQ ID NO:102所示的氨基酸序列；或(III) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 101 or 103, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102; or
(Ⅳ)所述重链包含如SEQ ID NO:148所示的氨基酸序列，或与SEQ ID NO:148所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:149所示的氨基酸序列，或与SEQ ID NO:149所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(IV) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 148, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 148 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 149, or has at least 90%, 92% with the amino acid sequence shown in SEQ ID NO: 149 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
(Ⅴ)所述重链包含如SEQ ID NO:148所示的氨基酸序列，或与SEQ ID NO:148所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:150所示的氨基酸序列，或与SEQ ID NO:150所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。(V) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 148, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 148 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 150, or has at least 90%, 92% with the amino acid sequence shown in SEQ ID NO: 150 Amino acid sequences of %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
在一些实施方式中，本发明所述抗体为全人抗体或人源化抗体。In some embodiments, the antibodies of the invention are fully human antibodies or humanized antibodies.
在一些实施方式中，本发明所述抗原结合片段选自Fab、Fab'、Fab'-SH、Fv、scFv、F(ab')2、sdAb或双抗体。In some embodiments, the antigen-binding fragments of the present invention are selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, sdAb, or diabodies.
在一些实施方式中，本发明所述抗体或其抗原结合片段是任何IgG亚型，如IgG1、IgG2、IgG3或IgG4。在一些实施方式中，本发明所述抗体或其抗原结合片段是IgG1型。In some embodiments, the antibodies or antigen-binding fragments thereof of the invention are of any IgG subtype, such as IgGl, IgG2, IgG3, or IgG4. In some embodiments, the antibody or antigen-binding fragment thereof of the invention is of the IgGl type.
在一些实施方式中，本发明所述的抗体是单克隆抗体。In some embodiments, the antibodies of the invention are monoclonal antibodies.
本发明还提供了一种多特异性抗体，包含本文所述抗体或其抗原结合片段的轻链可变区和重链可变区。The present invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
本发明还提供了一种单链抗体，包含本文所述抗体或其抗原结合片段的轻链可变区和重链可变区。The present invention also provides a single chain antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
本发明还提供了一种免疫缀合物，其包含与治疗剂或诊断剂缀合的本文所述抗体或其抗原结合片段。The invention also provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof as described herein conjugated to a therapeutic or diagnostic agent.
在又一个方面，本发明提供了多核苷酸分子，其编码权利要求本文所述的抗体或其抗原结合片段。In yet another aspect, the present invention provides a polynucleotide molecule encoding an antibody or antigen-binding fragment thereof as claimed herein.
在又一个方面，本发明提供了表达载体，其包含本文所述的多核苷酸分子，优选地，所述载体为真核表达载体。In yet another aspect, the present invention provides an expression vector comprising the polynucleotide molecule described herein, preferably, the vector is a eukaryotic expression vector.
在又一个方面，本发明提供了宿主细胞，其包含本文所述的多核苷酸或表达载体，或表达本文所述的抗体或其抗原结合片段。优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。In yet another aspect, the present invention provides host cells comprising a polynucleotide or expression vector described herein, or expressing an antibody or antigen-binding fragment thereof described herein. Preferably, the host cells are eukaryotic cells, more preferably mammalian cells.
在又一个方面，本发明提供了一种制备本文所述的抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞，以使其表达所述抗体或其抗原结合片段，并回收所表达的抗体或其抗原结合片段。In yet another aspect, the present invention provides a method of making an antibody or antigen-binding fragment thereof described herein, the method comprising culturing a host described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof cells to express the antibody or antigen-binding fragment thereof, and recover the expressed antibody or antigen-binding fragment thereof.
在又一个方面，本发明提供了药物组合物，其含有本文所述的抗体或其抗原结合片段、多核苷酸分子、所述表达载体和/或所述宿主细胞，以及药学上可接受的载体或赋形剂。In yet another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule, the expression vector and/or the host cell, and a pharmaceutically acceptable carrier or excipients.
在又一个方面，本发明提供了药物组合，其包含如本文所述的抗体或其抗原结合片段或药物组合物，以及一种或多种另外的治疗剂。In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment or pharmaceutical composition thereof as described herein, and one or more additional therapeutic agents.
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物和/或本文所述的药物组合在制备治疗和/或预防SARS-CoV-2或其变体感染的药物中的用途。In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, a pharmaceutical combination as described herein Use of the drug and/or the pharmaceutical combination described herein in the manufacture of a medicament for the treatment and/or prevention of infection by SARS-CoV-2 or a variant thereof.
在又一个方面，本发明提供了一种治疗和/或预防SARS-CoV-2或其变体感染的方法，其包括向有需要的受试者施用本文所述的抗体或其抗原结合片段、所述多核苷酸分子、所述表达载体、所述宿主细胞、所述药物组合物和/或所述的药物组合。In yet another aspect, the present invention provides a method of treating and/or preventing infection by SARS-CoV-2 or a variant thereof, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, The polynucleotide molecule, the expression vector, the host cell, the pharmaceutical composition and/or the pharmaceutical combination.
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物或本文所述 的药物组合，其用于治疗和/或预防SARS-CoV-2或其变体感染的疾病；优选地，所述SARS-CoV-2变体包括阿尔法突变株(Alpha突变株)、贝塔突变株(Beta突变株)、伽马突变株(Gamma突变株)、德尔塔突变株(Delta突变株)、Epsilon突变株、Zeta突变株、Eta突变株、Theta突变株、Iota突变株、卡帕突变株(Kappa突变株)、缪突变株(Mu突变株)和奥密克戎突变株(omicron突变株)中的至少一种；优选为奥密克戎突变株。In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein Or a pharmaceutical combination as described herein for the treatment and/or prevention of a disease infected by SARS-CoV-2 or a variant thereof; preferably, the SARS-CoV-2 variant comprises an alpha mutant (Alpha mutant) , Beta mutants (Beta mutants), Gamma mutants (Gamma mutants), Delta mutants (Delta mutants), Epsilon mutants, Zeta mutants, Eta mutants, Theta mutants, Iota mutants, At least one of a Kappa mutant (Kappa mutant), a Mu mutant (Mu mutant), and an Omicron mutant (omicron mutant); preferably an Omicron mutant.
在又一个方面，本发明提供了一种试剂盒，其包括本文所述的抗体或其抗原结合片段、所述多核苷酸分子、所述表达载体、所述宿主细胞和/或所述药物组合物。In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule, the expression vector, the host cell and/or the pharmaceutical combination thing.
在一些实施方式中，本发明提供了所述的试剂盒在制备诊断SARS-CoV-2或其变体感染的药物中的用途。In some embodiments, the present invention provides the use of the kit in the manufacture of a medicament for diagnosing infection by SARS-CoV-2 or a variant thereof.
在又一个方面，本发明提供了一种使用本文所述的抗体或其抗原结合片段或所述多肽检测SARS-CoV-2或其变体在样品中的存在的方法，所述方法包括使本文所述的抗体或其抗原结合片段与所述样品接触，并检测是否存在所述抗体或其抗原结合片段与SARS-CoV-2或其变体CBD结合产生的结合物或结合信号。In yet another aspect, the present invention provides a method of detecting the presence of SARS-CoV-2 or a variant thereof in a sample using the antibody or antigen-binding fragment thereof or the polypeptide described herein, the method comprising causing the The antibody or antigen-binding fragment thereof is contacted with the sample, and the presence of a conjugate or a binding signal produced by the binding of the antibody or antigen-binding fragment thereof to SARS-CoV-2 or its variant CBD is detected.
附图说明Description of drawings
图1：JS016轻重链CDR序列。Figure 1: JS016 light and heavy chain CDR sequences.
图2：突变文库构建流程。Figure 2: Mutation library construction workflow.
图3A-3D：结合ELISA检测JS016亲和力成熟抗体的结合作用。Figures 3A-3D: Binding ELISA to detect binding of JS016 affinity matured antibody.
图4A-4D：阻断ELISA检测JS016亲和力成熟抗体的阻断作用。Figures 4A-4D: Blocking ELISA to detect blocking effect of JS016 affinity matured antibody.
图5：JS016-38和JS016-40两株抗体体外对SARS-CoV-2活病毒中和活性。Figure 5: In vitro neutralizing activity of JS016-38 and JS016-40 antibodies against live SARS-CoV-2 virus.
图6A：JS016与RBD和RBD突变蛋白结合ELISA图谱。Figure 6A: ELISA profile of JS016 binding to RBD and RBD muteins.
图6B：JS016-38与RBD和RBD突变蛋白结合ELISA图谱。Figure 6B: ELISA profile of JS016-38 binding to RBD and RBD muteins.
图6C：JS016-40与RBD和RBD突变蛋白结合ELISA图谱。Figure 6C: ELISA profile of JS016-40 binding to RBD and RBD muteins.
图7A：JS016抑制RBD和RBD突变蛋白与ACE2结合。Figure 7A: JS016 inhibits binding of RBD and RBD muteins to ACE2.
图7B：JS016-38抑制RBD和RBD突变蛋白与ACE2结合。Figure 7B: JS016-38 inhibits binding of RBD and RBD muteins to ACE2.
图7C：JS016-40抑制RBD和RBD突变蛋白与ACE2结合。Figure 7C: JS016-40 inhibits binding of RBD and RBD muteins to ACE2.
图8A：CB6和RBD-his、RBD-Omicron-his结合的动力学参数。Figure 8A: Kinetic parameters of CB6 binding to RBD-his, RBD-Omicron-his.
图8B：JS016-40和RBD-his、RBD-Omicron-his结合的动力学参数。Figure 8B: Kinetic parameters of JS016-40 binding to RBD-his, RBD-Omicron-his.
图8C：JS016-41-YTE和RBD-his、RBD-Omicron-his结合的动力学参数。Figure 8C: Kinetic parameters of binding of JS016-41-YTE to RBD-his, RBD-Omicron-his.
图8D：JS016-77-YTE和RBD-his、RBD-Omicron-his结合的动力学参数。Figure 8D: Kinetic parameters of JS016-77-YTE binding to RBD-his, RBD-Omicron-his.
图9：抗体与SARS-CoV-2的omicron突变株S蛋白体外结合活性。Figure 9: In vitro binding activity of the antibody to the S protein of the omicron mutant strain of SARS-CoV-2.
图10A：CB6和JS016-40的假病毒中和活性。Figure 10A: Pseudovirus neutralizing activity of CB6 and JS016-40.
图10B：JS016-41-YTE的假病毒中和活性。Figure 10B: Pseudovirus neutralizing activity of JS016-41-YTE.
图10C：JS016-77-YTE的假病毒中和活性。Figure 10C: Pseudovirus neutralizing activity of JS016-77-YTE.
具体实施方式Detailed ways
定义definition
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill in the art.
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。For easier understanding of the present invention, certain scientific and technical terms are specifically defined as follows. Unless explicitly defined otherwise elsewhere herein, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For definitions and terms in the art, professionals can refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including in the claims), the singular includes its corresponding plural unless the context clearly dictates otherwise.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值，包括但不限于±5％、±2％、±1％和±0.1％，因为这些变化适于进行所公开的方法。The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ± 2%, ±1% and ±0.1% as these variations are suitable for carrying out the disclosed method.
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。The term "and/or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.
如本文所用，术语“或”应被理解为与如上定义的“和/或”具有相同的含义。例如，当分离列表中的项目时，“或”或“和/或”应被解释为包容性的，即，包括数量或元素列表中的至少一个，但也包括多于一个，以及任选地，额外的未列出的项目。只有明确指出相反的术语下，例如“只有一个”或“的确一个”或者在权利要求中使用“由...组成”时，将指的是仅列出的一个数字或列表的一个元素。As used herein, the term "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be construed as inclusive, that is, including at least one of the number or list of elements, but also including more than one, and optionally , additional unlisted items. Only under terms expressly stated to the contrary, such as "only one" or "indeed one" or when "consisting of" is used in a claim, will refer to only one of the listed numbers or one element of the list.
术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the maximum percent sequence identity obtained when amino acid sequences are aligned (and where necessary gaps are introduced), and no conservative substitutions are considered to be Following the portion of sequence identity, the percentage of amino acid residues in the candidate amino acid sequence that are identical to those in the reference amino acid sequence. Sequence alignments to determine percent amino acid sequence identity can be performed using various methods in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
术语“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细 胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。The term "immune response" refers to the action by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines and complement) by these cells or by the liver, which results in selective Damage, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
术语“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of signals from one part of a cell to another of the cell. part. Typically, delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions leading to signal transduction. The penultimate process usually involves nuclear events that lead to changes in gene expression.
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗SARS-CoV-2活性的能力、IC50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。The terms "activity" or "biological activity", or the terms "biological property" or "biological signature" are used interchangeably herein, including but not limited to epitope/antigen affinity and specificity, neutralization or antagonism of SARS in vivo or in vitro - Capacity for CoV-2 activity, IC50, in vivo stability of antibodies and immunogenic properties of antibodies. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (ie, generally with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of The ability of proteins to be expressed at high levels in mammalian cells. The aforementioned properties or characteristics are observed, measured or assessed using techniques known in the art, including but not limited to ELISA, FACS or BIACORE plasmon resonance analysis, non-limiting in vitro or in vivo neutralization assays, receptors Binding, cytokine or growth factor production and/or secretion, signal transduction and immunohistochemistry of tissue sections of different origins including human, primate or any other origin.
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。The term "antibody" refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric and camelized single domain antibodies.
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media . The term "isolated" does not mean the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的 至少一些结合特异性。抗原结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody that retains the parental At least some binding specificity of an antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies. A binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of an antibody). The term "binding compound" refers to both antibodies and binding fragments thereof.
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。A "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain.
“Fab′片段”含有一条轻链和一条包含VH结构域、CH1结构域以及CH1和CH2结构域之间的恒定区的部分的重链的部分，两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′) 2分子。 A "Fab'fragment" contains a light chain and a portion of a heavy chain comprising a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F(ab') 2 molecules.
“F(ab′) 2片段”含有两条轻链和两条包含VH结构域、CH1结构域以及CH1和CH2结构域之间的恒定区的部分的重链的部分，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。 An "F(ab') 2 fragment" contains two light chains and two parts of a heavy chain comprising a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby the two heavy chains are Interchain disulfide bonds are formed between chains. Thus, an F(ab') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。"Fv regions" comprise variable regions from both heavy and light chains, but lack constant regions.
“单链Fv抗体(scFv抗体)”是指包含抗体的VH和VL结构域的抗原结合片段，这些结构域包含于单个多肽链中。一般而言，scFv多肽在VH和VL结构域之间包含多肽接头，该接头使得scFv能形成用于抗原结合的所需结构。"Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, these domains being contained in a single polypeptide chain. In general, scFv polypeptides contain a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
“Fc区”用于定义包含至少一部分恒定区的免疫球蛋白重链的C端区域。该术语包括天然序列Fc区和变异Fc区。在一些实施方式中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羧基末端。但是，Fc区的C端赖氨酸(Lys447)可能存在或不存在(此段中的编号是根据EU编号系统，也称为EU索引，如Rabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991)。"Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. .Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
术语“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。The term "domain antibody" is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.
术语“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短 得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。The term "diabody" refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites.
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。The term "humanized antibody" refers to a form of antibody that contains sequences from human and non-human (eg, mouse, rat) antibodies. In general, humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops Framework (FR) regions are the framework regions of human immunoglobulin sequences. A humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。The term "fully human antibody" refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, "mouse antibody" refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, "rat antibody" refers to an antibody comprising only rat immunoglobulin sequences.
当提及配体/受体、抗体/抗原或其它结合对时，“特异性”结合是指在蛋白和/或其它生物试剂的异质群体中确定是否存在所述蛋白例如本发明的抗体与2019-nCoV RBD蛋白的结合反应。因此，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著量与样品中存在的其它蛋白结合。When referring to a ligand/receptor, antibody/antigen, or other binding pair, "specific" binding refers to determining the presence or absence of a protein, eg, an antibody of the invention, in a heterogeneous population of proteins and/or other biological agents that binds to Binding response of 2019-nCoV RBD protein. Thus, under the specified conditions, a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in significant amounts.
“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4). Isotypes also include modified forms of one of these species, wherein modifications have been made to alter Fc function, eg, to enhance or reduce effector function or binding to Fc receptors.
术语“表位”指能够与抗体特异性结合的蛋白质决定簇。表位通常由各种化学活性表面分子诸如氨基酸或糖侧链组成，并且通常具有特定三维结构特征以及特定电荷特征。构象性表位和非构象性表位的区别在于在变性溶剂存在下，与前者而非与后者的结合丧失。The term "epitope" refers to a protein determinant capable of specific binding by an antibody. Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is the loss of binding to the former but not to the latter in the presence of a denaturing solvent.
本文中所描述的术语“交叉反应”指的是对人类、猴、和/或鼠源(小鼠或大鼠)相同靶分子的抗原片段的结合。因此，“交叉反应”应被理解为与在不同物种中表达的相同分子X的种属间反应。The term "cross-reactivity" as described herein refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Thus, "cross-reactivity" should be understood as an inter-species reaction with the same molecule X expressed in different species.
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。"Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的KD为1.0×10 -6M或更高，更优选1.0×10 -5M或更高，更优选1.0×10 -4M或更高、1.0×10 -3M或更高，更优选1.0×10 -2M或更高。 The term "does not bind" to a protein or cell means that it does not bind to a protein or cell, or does not bind to it with high affinity, i.e. binds the protein or cell with a KD of 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 - 5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.
术语“高亲和性”对于IgG抗体而言，是指对于抗原的KD为1.0×10 -6M或更低，优选5.0×10 -8M或更低，更优选1.0×10 -8M或更低、5.0×10 -9M或更低，更优选1.0×10 -9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指KD为10 -6M或更低，优选10 -7M或更低，更优选10 -8M或更低。 The term "high affinity" for IgG antibodies means a KD for antigen of 1.0x10-6 M or less, preferably 5.0x10-8 M or less, more preferably 1.0x10-8 M or lower, 5.0×10 −9 M or lower, more preferably 1.0×10 −9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of an IgM subtype refers to a KD of 10-6 M or lower, preferably 10-7 M or lower, more preferably 10-8 M or lower.
术语“核酸”、“多核苷酸”、“核酸分子”以及“多核苷酸分子”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。The terms "nucleic acid", "polynucleotide", "nucleic acid molecule" and "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Human US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements thereof, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。"Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from Any source, capable of integrating with the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked). The recombinant construct will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be directed using both heterologous and non-heterologous (ie, endogenous) promoters.
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。"Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell). 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). After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors".
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。The term "expression vector" as used herein refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。"Activation," "stimulation," and "treatment" of a cell or receptor can have the same meaning, eg, activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context otherwise or clearly dictates. "Ligand" includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies. "Activation" can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors. A "response/response", eg, the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (eg, concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), wherein changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方式中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方式中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方式中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。As used herein, the terms "treating" or "treating" of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treating" refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernible by a patient. In another embodiment, "treating" or "treating" refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。"Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey.
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。Administration "in combination with" one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的新型冠状病毒抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。"Therapeutically effective amount", "therapeutically effective dose" and "effective amount" mean that the novel coronavirus antibody or antigen-binding fragment thereof of the present invention, when administered to cells, tissues or subjects alone or in combination with other therapeutic drugs, is effective in preventing Or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition. A therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition. When an active ingredient is administered alone to an individual, the therapeutically effective dose refers to that ingredient only. When administered in combination, a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of the therapeutic agent will result in an improvement in the diagnostic criterion or parameter by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.
“药学上可接受的载体”是指药物制剂或组合物中除活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲剂，赋形剂，稳定剂或防腐剂。"Pharmaceutically acceptable carrier" refers to ingredients other than the active ingredient in a pharmaceutical formulation or composition that are not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
术语“癌症”在本文中用于指表现出异常高水平的增殖和生长的一组细胞。癌症可能是良性的(也称为良性肿瘤)，恶性前或恶性。癌细胞可以是实体癌细胞或白血病癌细胞。本文使用的术语“肿瘤”是指包含癌症的一个或多个细胞。术语“肿瘤生长”在本文中用于指代包含癌症的一种或多种细胞的增殖或生长，其导致癌症的大小或程度的相应增加。The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers can be benign (also called benign tumors), premalignant, or malignant. The cancer cells can be solid cancer cells or leukemia cancer cells. The term "tumor" as used herein refers to one or more cells comprising cancer. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells comprising the cancer, which results in a corresponding increase in the size or extent of the cancer.
抗体或其抗原结合片段Antibody or antigen-binding fragment thereof
在一个方面，本发明提供了特异性结合SARS-CoV-2或其变体的RBD的抗体或其抗原结合片段。In one aspect, the invention provides antibodies or antigen-binding fragments thereof that specifically bind to the RBD of SARS-CoV-2 or a variant thereof.
本文中，SARS-CoV-2的变体包括但不限于阿尔法突变株(Alpha突变株)、贝塔突变株(Beta突变株)、伽马突变株(Gamma突变株)、德尔塔突变株(Delta突变株)、Epsilon突变株、Zeta突变株、Eta突变株、Theta突变株、Iota突变株、卡帕突变株(Kappa突变株)、缪突变株(Mu突变株)和奥密克戎突变株(omicron突变株)中的至少一种。在一些实施方案中，SARS-CoV-2的变体为奥密克戎突变株。Herein, variants of SARS-CoV-2 include, but are not limited to, alpha mutants (Alpha mutants), beta mutants (Beta mutants), gamma mutants (Gamma mutants), delta mutants (Delta mutants) strains), Epsilon mutants, Zeta mutants, Eta mutants, Theta mutants, Iota mutants, Kappa mutants (Kappa mutants), Mu mutants (Mu mutants) and Omicron mutants (omicron mutants) at least one of the mutants). In some embodiments, the variant of SARS-CoV-2 is an Omicron mutant.
在一些实施方式中，SARS-CoV-2变体为具有选自K417N、E484K和N501Y中的任意一个、任意两个或全部三个的突变体。在一些实施方式中，SARS-CoV-2变体为SARS-CoV-2南非突变株501Y.V2，其包含三个主要的特征性突变位点：K417N、E484K和N501Y。南非突变株501Y.V2除了与英国突变株B.1.1.7亚型有相同的N501Y突变外，不同之处在于还包含了对病毒感染能力有潜在重要影响的S蛋白E484K和K417N两个关键位点的突变。这两个位点可能会提高S蛋白与人表皮细胞受体的结合能力。In some embodiments, the SARS-CoV-2 variant is a mutant having any one, any two, or all three selected from K417N, E484K, and N501Y. In some embodiments, the SARS-CoV-2 variant is SARS-CoV-2 South African mutant 501Y.V2, which contains three major characteristic mutation sites: K417N, E484K, and N501Y. The South African mutant 501Y.V2 has the same N501Y mutation as the British mutant B.1.1.7 subtype, the difference is that it also contains two key loci of S protein E484K and K417N which have a potentially important influence on the virus infectivity. point mutation. These two sites may enhance the binding ability of S protein to human epidermal cell receptor.
在一些实施方式中，本发明提供了结合SARS-CoV-2或其变体RBD的抗体或其抗原结合片段。在一些实施方式中，本发明提供了阻断SARS-CoV-2或其变体RBD与ACE2结合的抗体。In some embodiments, the present invention provides antibodies or antigen-binding fragments thereof that bind to SARS-CoV-2 or a variant RBD thereof. In some embodiments, the present invention provides antibodies that block the binding of SARS-CoV-2 or its variant RBD to ACE2.
在一些实施方式中，本发明提供了一种抗体或其抗原结合片段，其特异性结合SARS-CoV-2或其变体的受体结合结构域(RBD)，其中所述抗体或其抗原结合片段包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds the receptor binding domain (RBD) of SARS-CoV-2 or a variant thereof, wherein the antibody or antigen thereof binds The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the fragment are:
HCDR1：GFX 1VX 2X 3NY，其中，X 1选自L、T、E、R、Q、V、W、I或S，优选地，X 1选自L、T、E、R、Q、V或I；X 2选自Q、G、D、R、P、M、K、V、A、N或Y，优选地，X 2选自Q、G、D、R、P、N或Y；X 3选自R、W、Y、A、F、V或H，优选地，X3选自W、R、A、F或V； HCDR1: GFX 1 VX 2 X 3 NY, wherein X 1 is selected from L, T, E, R, Q, V, W, I or S, preferably, X 1 is selected from L, T, E, R, Q , V or I; X 2 is selected from Q, G, D, R, P, M, K, V, A, N or Y, preferably, X 2 is selected from Q, G, D, R, P, N or Y; X is selected from R , W, Y, A, F, V or H, preferably, X is selected from W, R, A, F or V;
HCDR2：IYPGGX 4T，其中，X 4为T或S； HCDR2: IYPGGX 4 T, where X 4 is T or S;
HCDR3：ARVLPMYGDYLDY；HCDR3:ARVLPMYGDYLDY;
LCDR1：QX 5IX 6X 7Y，其中，X 5选自V、D、Q、A、W、R、N、S、D、M、K或P，优选地，X 5选自Q、A、R、N、S、D或M；X 6选自N、H、L、G、P、S、M、E、V、R、D、A或I，优选地，X 6选自E、L、V、R、D、E或A；X 7选自H、V、F、P、N、S、R、Q、G、Y或T，优选地，X 7选自Q、P、S、G、P、R或Y； LCDR1: QX 5 IX 6 X 7 Y, wherein X 5 is selected from V, D, Q, A, W, R, N, S, D, M, K or P, preferably, X 5 is selected from Q, A , R, N, S, D or M; X 6 is selected from N, H, L, G, P, S, M, E, V, R, D, A or I, preferably, X 6 is selected from E, L, V, R, D, E or A ; X is selected from H, V, F, P, N, S, R, Q, G, Y or T, preferably, X is selected from Q, P, S , G, P, R or Y;
LCDR2：AAS；LCDR2: AAS;
LCDR3：QQSX 8SX 9X 10PEYT，其中，X 8选自G、Y、T、S、K、A、N、E或P，优选地，X 8选自A、N、S或P；X 9选自P、S、I、N、A、W或F，优选地，X 9选自S、P或A；X 10选自T、V、L、I、R、K、S、M或F，优选地，X 10选自S、R、T、K、V、L或F。 LCDR3: QQSX 8 SX 9 X 10 PEYT, wherein X 8 is selected from G, Y, T, S, K, A, N, E or P, preferably, X 8 is selected from A, N, S or P; X 9 is selected from P, S, I, N, A, W or F, preferably, X 9 is selected from S, P or A; X 10 is selected from T, V, L, I, R, K, S, M or F, preferably, X 10 is selected from S, R, T, K, V, L or F.
上述取代中，N、T和S等均为极性不带电荷的脂肪族氨基酸，S、R、N和D等为极性的脂肪族氨基酸，D、G、V和I等均为脂肪族氨基酸，L和P等均为非极性氨基酸，Y和F等均为芳香族氨基酸，Y和S等均为极性不带电荷的氨基酸，N、Q和H等均为极性氨基酸，H和R等均为极性带电荷的氨基酸。因此，可用这些氨基酸相互替换，所得CDR在用于构建结合SARS-CoV-2或其变体RBD的抗体或其抗原结合片段时仍保留本文所述的结合活性。In the above-mentioned substitutions, N, T and S are polar uncharged aliphatic amino acids, S, R, N and D are polar aliphatic amino acids, and D, G, V and I are all aliphatic Amino acids, L and P are non-polar amino acids, Y and F are aromatic amino acids, Y and S are polar uncharged amino acids, N, Q and H are polar amino acids, H and R, etc. are polar charged amino acids. Thus, these amino acids can be substituted for each other and the resulting CDRs retain the binding activity described herein when used to construct antibodies or antigen-binding fragments thereof that bind SARS-CoV-2 or its variant RBD.
在本发明所述CB6抗体，描述于PCT/CN2021/077392及其同族专利申请中。上述专利申请/专利以引用的方式并入本文中，用于所有目的。The CB6 antibody of the present invention is described in PCT/CN2021/077392 and its related patent applications. The aforementioned patent applications/patents are incorporated herein by reference for all purposes.
抗体CB6的重链可变区(CB6-VH)的氨基酸序列(SEQ ID NO:7)：Amino acid sequence of heavy chain variable region (CB6-VH) of antibody CB6 (SEQ ID NO: 7):
Figure PCTCN2022080100-appb-000001
Figure PCTCN2022080100-appb-000001
抗体CB6的轻链可变区(CB6-VL)的氨基酸序列(SEQ ID NO:8)：Amino acid sequence of the light chain variable region (CB6-VL) of antibody CB6 (SEQ ID NO: 8):
Figure PCTCN2022080100-appb-000002
Figure PCTCN2022080100-appb-000002
CB6的重链(CB6-HC)的氨基酸序列(SEQ ID NO:9)：Amino acid sequence of the heavy chain (CB6-HC) of CB6 (SEQ ID NO: 9):
Figure PCTCN2022080100-appb-000003
Figure PCTCN2022080100-appb-000003
CB6的轻链/VL-Cκ(CB6-LC)的氨基酸序列(SEQ ID NO:10)：Amino acid sequence of the light chain/VL-CK of CB6 (CB6-LC) (SEQ ID NO: 10):
Figure PCTCN2022080100-appb-000004
Figure PCTCN2022080100-appb-000004
CB6的VH-CH1的氨基酸序列(SEQ ID NO:11)：Amino acid sequence of VH-CH1 of CB6 (SEQ ID NO: 11):
Figure PCTCN2022080100-appb-000005
Figure PCTCN2022080100-appb-000005
本发明在上述CB6抗体的基础上，通过噬菌体展示技术，获得如下43个克隆：On the basis of the above-mentioned CB6 antibody, the present invention obtains the following 43 clones through phage display technology:
重链突变库克隆氨基酸序列：Amino acid sequence of heavy chain mutant library clone:
CB6-1-VH SEQ ID NO:58CB6-1-VH SEQ ID NO:58
Figure PCTCN2022080100-appb-000006
Figure PCTCN2022080100-appb-000006
CB6-2-VH SEQ ID NO:59CB6-2-VH SEQ ID NO:59
Figure PCTCN2022080100-appb-000007
Figure PCTCN2022080100-appb-000007
CB6-3-VH SEQ ID NO:60CB6-3-VH SEQ ID NO:60
Figure PCTCN2022080100-appb-000008
Figure PCTCN2022080100-appb-000008
CB6-4-VH SEQ ID NO:61CB6-4-VH SEQ ID NO:61
Figure PCTCN2022080100-appb-000009
Figure PCTCN2022080100-appb-000009
CB6-5-VH SEQ ID NO:62CB6-5-VH SEQ ID NO:62
Figure PCTCN2022080100-appb-000010
Figure PCTCN2022080100-appb-000010
CB6-6-VH SEQ ID NO:63CB6-6-VH SEQ ID NO:63
Figure PCTCN2022080100-appb-000011
Figure PCTCN2022080100-appb-000011
CB6-7-VH SEQ ID NO:64CB6-7-VH SEQ ID NO:64
Figure PCTCN2022080100-appb-000012
Figure PCTCN2022080100-appb-000012
CB6-8-VH SEQ ID NO:65CB6-8-VH SEQ ID NO:65
Figure PCTCN2022080100-appb-000013
Figure PCTCN2022080100-appb-000013
CB6-9-VH SEQ ID NO:66CB6-9-VH SEQ ID NO:66
Figure PCTCN2022080100-appb-000014
Figure PCTCN2022080100-appb-000014
CB6-10-VH SEQ ID NO:67CB6-10-VH SEQ ID NO:67
Figure PCTCN2022080100-appb-000015
Figure PCTCN2022080100-appb-000015
CB6-11-VH SEQ ID NO:68CB6-11-VH SEQ ID NO:68
Figure PCTCN2022080100-appb-000016
Figure PCTCN2022080100-appb-000016
CB6-12-VH SEQ ID NO:69CB6-12-VH SEQ ID NO:69
Figure PCTCN2022080100-appb-000017
Figure PCTCN2022080100-appb-000017
CB6-13-VH SEQ ID NO:70CB6-13-VH SEQ ID NO:70
Figure PCTCN2022080100-appb-000018
Figure PCTCN2022080100-appb-000018
CB6-14-VH SEQ ID NO:71CB6-14-VH SEQ ID NO:71
Figure PCTCN2022080100-appb-000019
Figure PCTCN2022080100-appb-000019
CB6-15-VH SEQ ID NO:72CB6-15-VH SEQ ID NO:72
Figure PCTCN2022080100-appb-000020
Figure PCTCN2022080100-appb-000020
CB6-16-VH SEQ ID NO:73CB6-16-VH SEQ ID NO:73
Figure PCTCN2022080100-appb-000021
Figure PCTCN2022080100-appb-000021
各重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列及其序列编号如下表A所示：The amino acid sequences of HCDR1, HCDR2 and HCDR3 of each heavy chain variable region and their sequence numbers are shown in Table A below:
表ATable A
Figure PCTCN2022080100-appb-000022
Figure PCTCN2022080100-appb-000022
Figure PCTCN2022080100-appb-000023
Figure PCTCN2022080100-appb-000023
轻链突变库克隆氨基酸序列Light chain mutant library cloned amino acid sequence
CB6-17-VL SEQ ID NO:74CB6-17-VL SEQ ID NO: 74
Figure PCTCN2022080100-appb-000024
Figure PCTCN2022080100-appb-000024
CB6-18-VL SEQ ID NO:75CB6-18-VL SEQ ID NO:75
Figure PCTCN2022080100-appb-000025
Figure PCTCN2022080100-appb-000025
CB6-19-VL SEQ ID NO:76CB6-19-VL SEQ ID NO:76
Figure PCTCN2022080100-appb-000026
Figure PCTCN2022080100-appb-000026
CB6-20-VL SEQ ID NO:77CB6-20-VL SEQ ID NO: 77
Figure PCTCN2022080100-appb-000027
Figure PCTCN2022080100-appb-000027
CB6-21-VL SEQ ID NO:78CB6-21-VL SEQ ID NO:78
Figure PCTCN2022080100-appb-000028
Figure PCTCN2022080100-appb-000028
CB6-22-VL SEQ ID NO:79CB6-22-VL SEQ ID NO:79
Figure PCTCN2022080100-appb-000029
Figure PCTCN2022080100-appb-000029
CB6-23-VL SEQ ID NO:80CB6-23-VL SEQ ID NO:80
Figure PCTCN2022080100-appb-000030
Figure PCTCN2022080100-appb-000030
CB6-24-VL SEQ ID NO:81CB6-24-VL SEQ ID NO: 81
Figure PCTCN2022080100-appb-000031
Figure PCTCN2022080100-appb-000031
CB6-25-VL SEQ ID NO:82CB6-25-VL SEQ ID NO:82
Figure PCTCN2022080100-appb-000032
Figure PCTCN2022080100-appb-000032
CB6-26-VL SEQ ID NO:83CB6-26-VL SEQ ID NO:83
Figure PCTCN2022080100-appb-000033
Figure PCTCN2022080100-appb-000033
CB6-27-VL SEQ ID NO:84CB6-27-VL SEQ ID NO:84
Figure PCTCN2022080100-appb-000034
Figure PCTCN2022080100-appb-000034
CB6-28-VL SEQ ID NO:85CB6-28-VL SEQ ID NO:85
Figure PCTCN2022080100-appb-000035
Figure PCTCN2022080100-appb-000035
CB6-29-VL SEQ ID NO:86CB6-29-VL SEQ ID NO:86
Figure PCTCN2022080100-appb-000036
Figure PCTCN2022080100-appb-000036
CB6-30-VL SEQ ID NO:87CB6-30-VL SEQ ID NO:87
Figure PCTCN2022080100-appb-000037
Figure PCTCN2022080100-appb-000037
CB6-31-VL SEQ ID NO:88CB6-31-VL SEQ ID NO:88
Figure PCTCN2022080100-appb-000038
Figure PCTCN2022080100-appb-000038
CB6-32-VL SEQ ID NO:89CB6-32-VL SEQ ID NO:89
Figure PCTCN2022080100-appb-000039
Figure PCTCN2022080100-appb-000039
CB6-33-VL SEQ ID NO:90CB6-33-VL SEQ ID NO:90
Figure PCTCN2022080100-appb-000040
Figure PCTCN2022080100-appb-000040
CB6-34-VL SEQ ID NO:91CB6-34-VL SEQ ID NO: 91
Figure PCTCN2022080100-appb-000041
Figure PCTCN2022080100-appb-000041
CB6-35-VL SEQ ID NO:92CB6-35-VL SEQ ID NO:92
Figure PCTCN2022080100-appb-000042
Figure PCTCN2022080100-appb-000042
CB6-36-VL SEQ ID NO:93CB6-36-VL SEQ ID NO:93
Figure PCTCN2022080100-appb-000043
Figure PCTCN2022080100-appb-000043
CB6-37-VL SEQ ID NO:94CB6-37-VL SEQ ID NO:94
Figure PCTCN2022080100-appb-000044
Figure PCTCN2022080100-appb-000044
CB6-38-VL SEQ ID NO:95CB6-38-VL SEQ ID NO:95
Figure PCTCN2022080100-appb-000045
Figure PCTCN2022080100-appb-000045
CB6-39-VL SEQ ID NO:96CB6-39-VL SEQ ID NO:96
Figure PCTCN2022080100-appb-000046
Figure PCTCN2022080100-appb-000046
CB6-40-VL SEQ ID NO:97CB6-40-VL SEQ ID NO: 97
Figure PCTCN2022080100-appb-000047
Figure PCTCN2022080100-appb-000047
CB6-41-VL SEQ ID NO:98CB6-41-VL SEQ ID NO:98
Figure PCTCN2022080100-appb-000048
Figure PCTCN2022080100-appb-000048
CB6-42-VL SEQ ID NO:99CB6-42-VL SEQ ID NO:99
Figure PCTCN2022080100-appb-000049
Figure PCTCN2022080100-appb-000049
CB6-43-VL SEQ ID NO:100CB6-43-VL SEQ ID NO: 100
Figure PCTCN2022080100-appb-000050
Figure PCTCN2022080100-appb-000050
各轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列和相应的序列编号如下表B所示：The amino acid sequences and corresponding sequence numbers of LCDR1, LCDR2 and LCDR3 of each light chain variable region are shown in Table B below:
表BForm B
Figure PCTCN2022080100-appb-000051
Figure PCTCN2022080100-appb-000051
Figure PCTCN2022080100-appb-000052
Figure PCTCN2022080100-appb-000052
JS016-38-HC SEQ ID NO:101JS016-38-HC SEQ ID NO: 101
Figure PCTCN2022080100-appb-000053
Figure PCTCN2022080100-appb-000053
JS016-38-LC/JS016-40-LC SEQ ID NO:102JS016-38-LC/JS016-40-LC SEQ ID NO: 102
Figure PCTCN2022080100-appb-000054
Figure PCTCN2022080100-appb-000054
JS016-40-HC SEQ ID NO:103JS016-40-HC SEQ ID NO: 103
Figure PCTCN2022080100-appb-000055
Figure PCTCN2022080100-appb-000055
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883；Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))、基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services,National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia based on the three-dimensional structure of the antibody and topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al, Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (1999Nucleic Acids) Research, 27, 209-212), and a North CDR definition based on affinity propagation clustering using a large number of crystal structures.
除非另有说明，否则本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。Unless otherwise stated, the CDRs of the antibodies of the invention can be bounded by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。It should be noted that the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Thus, when referring to antibodies defined by specific CDR sequences as defined in the present invention, the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. different assignment systems or combinations), resulting in their claimed CDR boundaries being different from the specific CDR boundaries defined by the present invention.
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而， 尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia、AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the region of minimum overlap can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimal binding unit can be a sub-portion of a CDR. The residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
在一些实施方式中，本发明抗体或抗原结合片段的CDR的边界采用IMGT方案界定。In some embodiments, the boundaries of the CDRs of the antibodies or antigen-binding fragments of the invention are defined using the IMGT scheme.
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗新型冠状病毒抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的(特别地在上述序列中描绘的CDR区中)但仍与上述抗体有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。在一些实施方式中，本发明的抗体与具体序列中框架区相比较时，在框架区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。In some embodiments, amino acid changes include amino acid deletions, insertions or substitutions. In some embodiments, the anti-novel coronavirus antibodies or antigen-binding fragments thereof of the present invention include those that have been mutated by amino acid deletions, insertions or substitutions (particularly in the CDR regions depicted in the above sequences) but still have the same Those antibodies having amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions that have been mutated by amino acid deletions, insertions, or substitutions when compared to the CDR regions depicted in a particular sequence. In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the framework regions that have been mutated by amino acid deletions, insertions or substitutions when compared to the framework regions in a particular sequence.
在一些实施方式中，编码本发明抗体的多核苷酸分子包括已通过核苷酸缺失、插入或置换突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少约60、70、80、90、95或100％同一性的多核苷酸分子。In some embodiments, the polynucleotide molecules encoding the antibodies of the invention include those that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60, Polynucleotide molecules of 70, 80, 90, 95 or 100% identity.
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。In some embodiments, it may be desirable to generate cysteine-engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues.
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl - 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof.
抗体表达Antibody expression
在又一个方面，本发明提供了一种多核苷酸分子，其编码本文所述的抗体或其抗原结合片段。所述多核苷酸分子可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的多核苷酸分子，或包含编码抗体的轻链和/或重链的氨基酸序列的多核苷酸分子。In yet another aspect, the present invention provides a polynucleotide molecule encoding an antibody or antigen-binding fragment thereof as described herein. The polynucleotide molecule may comprise a polynucleotide molecule encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or a polynucleotide comprising the amino acid sequence encoding the light chain and/or heavy chain of an antibody. Glycolic acid molecule.
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸分子，优选地，所述载体为真核表达载体。在一些实施方式中，如本文所述的多核苷酸分子包含在一个或多个表达载体中。In yet another aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the vector is a eukaryotic expression vector. In some embodiments, the polynucleotide molecules as described herein are contained in one or more expression vectors.
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸分子或如本文所述的表达载体或表达本文所述的抗体或其抗原结合片段。优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。In yet another aspect, the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein or expressing an antibody or antigen-binding fragment thereof as described herein. Preferably, the host cells are eukaryotic cells, more preferably mammalian cells.
在又一个方面，本发明提供了一种用于制备如本文所述的抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞，使其表达所述抗体或其抗原结合片段，并回收所表达的抗体或其抗原结合片段。In yet another aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof as described herein, the method comprising culturing the antibody or antigen-binding fragment thereof as described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof The host cell is used to express the antibody or antigen-binding fragment thereof, and the expressed antibody or antigen-binding fragment thereof is recovered.
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。The invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, Monkey Kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
在一个实施方式中，本发明提供制备本文所述的抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中，来产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。In one embodiment, the present invention provides a method of making an antibody as described herein, wherein the method comprises, when introducing an expression vector into a mammalian host cell, by culturing the host cell for a period of time sufficient to allow the antibody to grow in the host The antibody is produced by expression in the cell, or more preferably by secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。It is likely that antibodies expressed by different cell lines or in transgenic animals have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies. Also, in certain embodiments, afucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic , because their carbohydrate structure is a normal component of native human serum IgG.
药物组合物和药物制剂Pharmaceutical compositions and pharmaceutical preparations
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体或本文所述的宿主细胞，和药学上可接受的载体或赋形剂。应理解，本发明提供的抗体或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。In yet another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, or a host as described herein cells, and a pharmaceutically acceptable carrier or excipient. It will be appreciated that the antibodies or pharmaceutical compositions thereof provided by the present invention may incorporate suitable carriers, excipients and other agents in formulations for co-administration to provide improved transfer, delivery, tolerance, and the like.
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。The term "pharmaceutical composition" refers to a formulation that allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered.
可以通过将具有所需纯度的本发明的抗体与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的抗体的药物制剂，优选地以水溶液或冻干制剂的形式。Antibodies of the invention can be prepared by mixing an antibody of the invention of the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)) containing the compounds described herein. Pharmaceutical formulations of antibodies, preferably in the form of aqueous solutions or lyophilized formulations.
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码本文所述的抗体的多核苷酸分子的组合物。The pharmaceutical compositions or formulations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other . In some embodiments, the other active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a growth inhibitory agent, an antibiotic, or various anti-tumor or anti-cancer agents known, in a suitable amount effective for the intended use exist in combination. In some embodiments, the pharmaceutical compositions of the present invention further comprise compositions of polynucleotide molecules encoding the antibodies described herein.
在又一个方面，本发明提供了一种药物组合，其包含本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一种或多种另外的治疗剂。In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.
在又一个方面，本发明提供了一种试剂盒，其包括本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物。In yet another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or the pharmaceutical compositions described herein.
医药用途及治疗方法Medicinal uses and methods of treatment
本文提供的任何抗体均可用于治疗方法。还应当理解，在讨论“抗体”时，也包括包含抗体的组合物。本发明的抗体可以治疗有效量或预防有效量用于本发明任一实施方式所述的治疗或预防方法中。Any of the antibodies provided herein can be used in therapeutic methods. It should also be understood that when discussing an "antibody", compositions comprising the antibody are also included. The antibodies of the present invention may be used in a therapeutically or prophylactically effective amount in any of the methods of treatment or prophylaxis described in any of the embodiments of the present invention.
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物在制备用于治疗和/或预防SARS-CoV-2或其变体感染的药物中的用途；优选地，所述SARS-CoV-2变体包括阿尔法突变株(Alpha突变株)、贝塔突变株(Beta突变株)、伽马突变株(Gamma突变株)、德尔塔突变株(Delta突变株)、Epsilon突变株、Zeta突变株、Eta突变株、 Theta突变株、Iota突变株、卡帕突变株(Kappa突变株)、缪突变株(Mu突变株)和奥密克戎突变株(omicron突变株)中的至少一种；优选为奥密克戎突变株。In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical combination described herein Use of a compound in the preparation of a medicine for the treatment and/or prevention of infection by SARS-CoV-2 or its variants; preferably, the SARS-CoV-2 variants include alpha mutants (Alpha mutants), beta mutants strain (Beta mutant), Gamma mutant (Gamma mutant), Delta mutant (Delta mutant), Epsilon mutant, Zeta mutant, Eta mutant, Theta mutant, Iota mutant, Kappa mutant At least one of a strain (Kappa mutant strain), a Mu mutant strain (Mu mutant strain), and an Omicron mutant strain (omicron mutant strain); preferably an Omicron mutant strain.
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，其用于治疗和/或预防SARS-CoV-2或其变体感染。In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical combination described herein substances for the treatment and/or prevention of infection with SARS-CoV-2 or variants thereof.
在又一个方面，本发明提供了一种治疗和/或预防SARS-CoV-2或其变体感染的方法，其包括向有需要的受试者施用本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物或药物组合。In yet another aspect, the present invention provides a method of treating and/or preventing infection by SARS-CoV-2 or a variant thereof, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, A polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition or combination described herein.
本发明的给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或病灶内给予等。Modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), by inhalation, aerosol delivery, or intralesional administration, and the like.
本发明还包括向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体、其抗原结合片段或药物组合物。在一些实施方式中，本发明抗体、其抗原结合片段或药物组合物与至少一种另外的治疗剂共施用。The invention also includes co-administration to a subject of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents). In some embodiments, the therapy includes surgery and/or radiation therapy. The antibodies, antigen-binding fragments thereof, or pharmaceutical compositions of the invention can be used alone or in combination with other therapeutic agents in therapy. In some embodiments, the antibody, antigen-binding fragment or pharmaceutical composition thereof of the invention is co-administered with at least one additional therapeutic agent.
用于诊断和检测的试剂、检测方法和试剂盒Reagents, test methods and kits for diagnosis and detection
在又一个方面，本发明提供了一种使用本文所述的抗体或其抗原结合片段检测SARS-CoV-2或其变体在样品中的存在的方法。术语“检测”用于本文中时，包括定量或定性检测。在一些实施方式中，所述样品是生物样品。在某些实施方式中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方式中，生物样品包含细胞或组织。所述方法包括使本文所述的抗体或其抗原结合片段或含有所述抗体或其抗原结合片段的检测组合物与样品接触的步骤，以及检测是否存在所述抗体或其抗原结合片段与SARS-CoV-2或其变体CBD结合产生的结合物或结合信号的步骤。用于检测用途时，本文所述的抗体或其抗原结合片段可被标记，以指示是否形成了所述结合物。In yet another aspect, the present invention provides a method of detecting the presence of SARS-CoV-2 or a variant thereof in a sample using the antibody or antigen-binding fragment thereof described herein. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other fluid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues. The method comprises the steps of contacting the antibody or antigen-binding fragment thereof described herein, or a detection composition comprising the antibody or antigen-binding fragment thereof, with a sample, and detecting the presence or absence of the antibody or antigen-binding fragment thereof and SARS- Steps in which CoV-2 or its variant CBD binds to produce a conjugate or a binding signal. For detection purposes, the antibodies or antigen-binding fragments thereof described herein can be labeled to indicate whether the conjugate is formed.
在一些实施方案中，本发明提供一种检测试剂，其含有本文任一实施方案所述的抗体或其抗原结合片段。检测试剂中可含有合适的载体，如溶剂，如水。在一些实施方案中，用脱脂牛奶稀释C端连接有鼠Fc的重组人ACE蛋白，再用所得稀释液稀释本文任一实施方案所述的抗体或其抗原结合片段，可获得本文的检测试剂。检测试剂中的所述抗体或其抗原结合片段可以任何合适的浓度存在，使用时可将其稀释到所需的浓度。在一些实施方案中，本发明提供一种检测试纸或检测芯片，其包被有本文任一实施方案所述的抗体或其抗原结合片段。可采用周知的方法将本文所述的抗体或其抗原结合片段包被到常用的试 纸或芯片上。例如，在一些实施方案中，经由蛋白A将本文任一实施方案所述的抗体或其抗原结合片段包被到蛋白A芯片上。In some embodiments, the present invention provides a detection reagent comprising the antibody or antigen-binding fragment thereof of any of the embodiments herein. The detection reagent may contain a suitable carrier such as a solvent such as water. In some embodiments, the detection reagents herein can be obtained by diluting the recombinant human ACE protein with murine Fc linked at its C-terminus with skim milk, and then diluting the antibody or antigen-binding fragment thereof described in any of the embodiments herein with the resulting dilution. The antibody or antigen-binding fragment thereof in the detection reagent may be present at any suitable concentration, and may be diluted to the desired concentration for use. In some embodiments, the present invention provides a test strip or a test chip coated with the antibody or antigen-binding fragment thereof described in any of the embodiments herein. The antibodies or antigen-binding fragments thereof described herein can be coated on commonly used test strips or chips using well-known methods. For example, in some embodiments, the antibody or antigen-binding fragment thereof described in any of the embodiments herein is coated onto a Protein A chip via Protein A.
在一些实施方案中，本文提供一种试剂盒，其用于检测SARS-CoV-2或其变体在样品中的存在。该试剂盒可含有本文任一实施方案所述的抗体或其抗原结合片段、检测试剂和/或检测试纸或芯片。In some embodiments, provided herein is a kit for detecting the presence of SARS-CoV-2 or a variant thereof in a sample. The kit may contain the antibody or antigen-binding fragment thereof described in any of the embodiments herein, detection reagents, and/or detection strips or chips.
在另外一些实施方案中，本文提供一种药盒，其可用于治疗治疗和/或预防SARS-CoV-2或其变体感染。该药盒可含有本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体、宿主细胞或药物组合物。In other embodiments, provided herein is a kit for use in the therapeutic treatment and/or prevention of infection by SARS-CoV-2 or a variant thereof. The kit may contain the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector, host cell or pharmaceutical composition described in any of the embodiments herein.
在一些实施方案中，本文还提供以下用途：本文任一实施方案所述的试剂盒在制备诊断SARS-CoV-2或其变体感染的药物中的用途；或本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体或宿主细胞在制备用于检测样品中是否存在SARS-CoV-2或其变体的检测试剂、检测试纸或检测芯片中的应用；或本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体、宿主细胞、检测试剂和/或检测试纸或检测芯片在制备用于检测样品中是否存在SARS-CoV-2或其变体的试剂盒中的应用。Also provided herein, in some embodiments, is the use of the kit of any of the embodiments herein in the manufacture of a medicament for diagnosing infection by SARS-CoV-2 or a variant thereof; or of any of the embodiments herein Use of antibodies or antigen-binding fragments thereof, polynucleotide molecules, expression vectors or host cells in the preparation of detection reagents, test strips or detection chips for the presence of SARS-CoV-2 or variants thereof in a sample; or herein Antibodies or antigen-binding fragments thereof, polynucleotide molecules, expression vectors, host cells, detection reagents, and/or detection strips or detection chips of any of the embodiments are prepared for use in detecting the presence of SARS-CoV-2 or SARS-CoV-2 in a sample. Kit application of its variants.
本发明包括所述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。The present invention includes all combinations of the specific embodiments described. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be hereafter The detailed description will become apparent to those skilled in the art. All publications, patents and patent applications cited herein, including citations, are hereby incorporated by reference in their entirety for all purposes.
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other methods, and equivalent substitutions known to those skilled in the art By way of example, preferred embodiments include, but are not limited to, the embodiments of the present invention.
本发明采用下述缩略词：The present invention adopts the following abbreviations:
OD代表光密度；OD stands for optical density;
HRP代表辣根过氧化物酶；HRP stands for horseradish peroxidase;
IPTG代表异丙基-β-D-硫代半乳糖苷；IPTG stands for isopropyl-β-D-thiogalactoside;
TMB代表3,3',5,5'-四甲基联苯胺；TMB stands for 3,3',5,5'-tetramethylbenzidine;
PBS代表磷酸缓冲盐溶液；PBS stands for Phosphate Buffered Saline;
PBST代表PBS+0.05％Tween 20。PBST stands for PBS + 0.05% Tween 20.
实施例Example
通过以下实施例对本发明进行说明，但并不旨在对本发明作出任何限制。本文已经详细描述了本发明，其中也公开了其具体实施方式。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。The present invention is illustrated by the following examples, but is not intended to limit the present invention in any way. The present invention has been described in detail herein, wherein specific embodiments thereof are also disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.
实施例1：噬菌体展示技术Example 1: Phage Display Technology
1.1 Fab构建1.1 Fab Construction
以CB6-HC的质粒(君实生物自制)为模板通过PCR获得抗体可变区VH片段，以CB6-LC的质粒(君实生物自制)模板通过PCR获得抗体可变区VL片段，然后分别通过与CH1,Cκ重叠PCR获得VH-CH1(氨基酸序列如SEQ ID NO：11所示)和VL-Cκ(氨基酸序列如SEQ ID NO：10所示)。VH-CH1和VL-Cκ通过重叠PCR组装成Fab片段。将Fab片段分别和pCOS载体(君实生物自制)用SfiI(购自NEB公司)酶切并用试剂盒回收。上述酶切片段和载体用T4连接酶(购自NEB公司)连接，得到含有Fab序列的pCOS-CB6质粒，转化至大肠杆菌Top10，铺板第二天送测序，经序列比对分析得到正确的Fab克隆。The VH fragment of antibody variable region was obtained by PCR with the plasmid of CB6-HC (made by Junshi Biotechnology) as the template, and the VL fragment of antibody variable region was obtained by PCR with the plasmid of CB6-LC (made by Junshi Biotechnology), and then respectively passed through VH-CH1 (amino acid sequence shown in SEQ ID NO: 11) and VL-Cκ (amino acid sequence shown in SEQ ID NO: 10) were obtained by overlapping PCR with CH1 and Cκ. VH-CH1 and VL-CK were assembled into Fab fragments by overlapping PCR. The Fab fragments were digested with pCOS vector (manufactured by Junshi Biotechnology) with SfiI (purchased from NEB Company) and recovered with a kit. The above-mentioned restriction fragment and the vector were connected with T4 ligase (purchased from NEB Company) to obtain the pCOS-CB6 plasmid containing the Fab sequence, which was transformed into E. clone.
1.2 CDR关键位点突变文库的构建及文库的淘选1.2 Construction of a CDR key site mutation library and panning of the library
采用overlap PCR构建CDR关键位点(图1所示)突变文库片段。如图2所示，第一轮PCR以pCOS-CB6为模板用引物RSC-F和H1-R扩增片段H1；用引物H2-F和Dp-EX扩增片段H2；用引物RSC-F和H3-R扩增片段H3；用引物H3-F和Dp-EX扩增片段H4；用引物H2-F和H3-R扩增片段H5；用引物RSC-F和L1-R扩增片段L1；用引物L1-F和L3-R扩增片段L2；用引物L3-F和Dp-EX扩增片段L3。第二轮PCR以片段H1和片段H2为模板用引物RSC-F和Dp-EX overlap PCR得到HCDR1和HCDR2的组合突变。以片段H1和片段H4和H5为模板用引物RSC-F和Dp-EX overlap PCR得到HCDR1、HCDR2和HCDR3的组合突变。以片段H3和片段H4为模板用引物RSC-F和Dp-EX overlap PCR得到HCDR3的突变。以片段L1和片段L2和L3为模板用引物RSC-F和Dp-EX overlap PCR得到LCDR1、LCDR2和LCDR3的组合突变。1％琼脂糖凝胶电泳分离PCR产物，用胶回收试剂盒回收目的片段。组合突变的片段和pCOS载体用SfiI酶切，试剂盒回收。随后酶切片段和载体用T4连接酶连接。连接产物乙醇沉淀回收，电转化法将连接产物转入到TG1菌种得到突变文库。将构建好的重链文库和轻链文库包装成噬菌 体，第一轮淘选分别取100ul噬菌体加入50nM生物素化的SARS-CoV-2 RBD(君实自制)孵育结合，之后加入链霉亲和素偶链的磁珠结合生物素化的RBD，用PBST(中文)洗掉不结合的噬菌体，用PH 2.0的甘氨酸洗脱结合在RBD上的噬菌体，感染TG1，扩增包装出噬菌体用于第二轮的淘选。通过三轮淘选，第三轮的噬菌体感染TG1并铺板成单克隆。突变文库构建引物序列如下所示(N为A、T、G或C)：Use overlap PCR to construct CDR key site (shown in Figure 1) mutation library fragments. As shown in Figure 2, the first round of PCR used pCOS-CB6 as a template to amplify fragment H1 with primers RSC-F and H1-R; amplify fragment H2 with primers H2-F and Dp-EX; use primers RSC-F and H3-R amplified fragment H3; amplified fragment H4 with primers H3-F and Dp-EX; amplified fragment H5 with primers H2-F and H3-R; amplified fragment L1 with primers RSC-F and L1-R; Fragment L2 was amplified with primers L1-F and L3-R; fragment L3 was amplified with primers L3-F and Dp-EX. The second round of PCR took fragment H1 and fragment H2 as templates and used primers RSC-F and Dp-EX overlap PCR to obtain the combined mutation of HCDR1 and HCDR2. Combination mutations of HCDR1, HCDR2 and HCDR3 were obtained by PCR with primers RSC-F and Dp-EX overlap using fragment H1 and fragments H4 and H5 as templates. The mutation of HCDR3 was obtained by PCR with primers RSC-F and Dp-EX overlap using fragment H3 and fragment H4 as templates. Combinatorial mutations of LCDR1, LCDR2 and LCDR3 were obtained by PCR with primers RSC-F and Dp-EX overlap using fragment L1 and fragments L2 and L3 as templates. The PCR products were separated by 1% agarose gel electrophoresis, and the target fragments were recovered with a gel recovery kit. The combined mutated fragment and pCOS vector were digested with SfiI and recovered by the kit. The digested fragment and vector were then ligated with T4 ligase. The ligation product was recovered by ethanol precipitation, and the ligation product was transferred into the TG1 strain by electroporation to obtain a mutant library. The constructed heavy chain library and light chain library were packaged into phages. In the first round of panning, 100ul of phages were added to 50nM biotinylated SARS-CoV-2 RBD (self-made by Junshi) for incubation and binding, and then streptavidin was added. The magnetic beads of the paired chain bind to the biotinylated RBD, wash off the unbound phage with PBST (Chinese), eluate the phage bound to the RBD with glycine of pH 2.0, infect TG1, and amplify and package the phage for the first step. Second round of panning. Through three rounds of panning, the third round of phage was infected with TG1 and plated as single clones. The primer sequences for mutant library construction are shown below (N is A, T, G or C):
CB6H-F:GCT GCC CAA CCA GCC ATG GCC GAGGTGCAGCTGGTGGAG(SEQ ID NO:12)CB6H-F: GCT GCC CAA CCA GCC ATG GCC GAGGTGCAGCTGGTGGAG (SEQ ID NO: 12)
CB6H-R:CGA TGG GCC CTT GGT GGA GGC GCTGCTCACGGTCACCAG(SEQ ID NO:13)CB6H-R: CGA TGG GCC CTT GGT GGA GGC GCTGCTCACGGTCACCAG (SEQ ID NO: 13)
CB6L-F:GGG CCC AGG CGG CCG AGC TC GACATCGTGATGACCCAG(SEQ ID NO:14)CB6L-F: GGG CCC AGG CGG CCG AGC TC GACATCGTGATGACCCAG (SEQ ID NO: 14)
CB6L-R:GAAGACAGATGGTGCAGCCACAGTTCG CTTGATCTCCAGCTTGGT(SEQ ID NO:15)CB6L-R: GAAGACAGATGGTGCAGCCACAGTTCG CTTGATCTCCAGCTTGGT (SEQ ID NO: 15)
CB6H1-R:CAGGCCCTTGCCGGGGGCCTGTCTCACCCAGCTCATGTAGTTSNNSNNCACSNNGAAGCCGCTGGC(SEQ ID NO:16)CB6H1-R: CAGGCCCTTGCCGGGGGCCTGTCTCACCCAGCTCATGTAGTTSNNSNNCACSNNGAAGCCGCTGGC (SEQ ID NO: 16)
CB6H2-F:GTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCTACNNSNNSGGCNNSACCTTCTACGCC(SEQ ID NO:17)CB6H2-F: GTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGTGATCTACNNSNNSGGCNNSACCTTCTACGCC (SEQ ID NO: 17)
CB6H3-R:CACCAGGGTGCCCTGGCCCCAGTAGTCCAGGTASTCSNNSNNSNNSNNSNNCACTYTGGCGCAGTAGTA(SEQ ID NO:18)CB6H3-R: CACCAGGGTGCCCTGGCCCCAGTAGTCCAGGTASTCSNNSNNSNNSNNSNNCACTYTGGCGCAGTAGTA (SEQ ID NO: 18)
CB6H3-F:GACTACTGGGGCCAGGGCACCCTGGTG(SEQ ID NO:19)CB6H3-F: GACTACTGGGGCCAGGGCACCCTGGTG (SEQ ID NO: 19)
CB6L1-R:CAGCTTGGGGGCCTTGCCGGGCTTCTGCTGGTACCAGTTCAGGTASNNSNNGATSNNCTGGCTGGCTCT(SEQ ID NO:20)CB6L1-R: CAGCTTGGGGGCCTTGCCGGGCTTCTGCTGGTACCAGTTCAGGTASNNSNNGATSNNCTGGCTGGCTCT (SEQ ID NO: 20)
CB6L1-F:CAGAAGCCCGGCAAGGCCCCCAAGCTG(SEQ ID NO:21)CB6L1-F: CAGAAGCCCGGCAAGGCCCCCAAGCTG (SEQ ID NO: 21)
CB6L3-R:CTTGATCTCCAGCTTGGTGCCCTGGCCGAAGGTGTACTCGGGSNNSNNGCTSNNGCTCTGCTGGCA(SEQ ID NO:22)CB6L3-R: CTTGATCTCCAGCTTGGTGCCCTGGCCGAAGGTGTACTCGGGSNNSNNGCTSNNGCTCTGCTGGCA (SEQ ID NO: 22)
CB6L3-F:GGCCAGGGCACCAAGCTGGAGATCAAG(SEQ ID NO:23)CB6L3-F: GGCCAGGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 23)
RSC-F:GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TC(SEQ ID NO:24)RSC-F: GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TC (SEQ ID NO:24)
Dp-EX:GAG GAG GAG GAG GAG GAG AGA AGC GTA GTC CGG AAC GTC(SEQ ID NO:25)Dp-EX: GAG GAG GAG GAG GAG GAG AGA AGC GTA GTC CGG AAC GTC (SEQ ID NO:25)
1.3 ELISA筛选1.3 ELISA screening
从平板上挑取单克隆于含有250ul体积的0.05％葡萄糖、2×YT培养基(1L含16g胰蛋 白胨，10g酵母提取物，5g氯化钠)的96孔板中37度培养4小时。待OD 600值≥0.6时，加入1M IPTG至终浓度1mM，摇匀，30度培养过夜。将链霉亲和素稀释至5μg/ml包被，每孔加入50ul并4度过夜。洗板并用2％脱脂牛奶封闭。用PBST洗涤液洗板4次，将生物素化的SARS-CoV-2 RBD(君实自制)抗原稀释至3ug/ml包被，每孔50ul，室温孵育0.5小时，洗板后备用。将过夜培养好的96孔板以4000rpm/分钟离心10分钟，收集上清。将上清40ul和10ul 10％脱脂牛奶加入包被抗原的板中，室温孵育1小时。洗板后再与1:3000稀释的山羊抗人IgG(Fab特异性)过氧化物酶抗体37℃孵育1小时，然后与0.1mg/ml的HRP底物TMB 37℃孵育15分钟显色，检测OD 450信号。共获得43个OD 450读值较高的克隆，并送测序分析。 A single clone was picked from the plate and cultured in a 96-well plate containing 250ul volume of 0.05% glucose, 2×YT medium (1L containing 16g tryptone, 10g yeast extract, 5g sodium chloride) at 37°C for 4 hours. When the OD 600 value is greater than or equal to 0.6, add 1M IPTG to a final concentration of 1mM, shake well, and incubate at 30 degrees overnight. Dilute streptavidin to 5μg/ml coating, add 50ul per well and leave overnight at 4°C. Plates were washed and blocked with 2% skim milk. The plate was washed 4 times with PBST washing solution, and the biotinylated SARS-CoV-2 RBD (self-made by Junshi) antigen was diluted to 3ug/ml for coating, 50ul per well, incubated at room temperature for 0.5 hours, and used after washing. The 96-well plate cultured overnight was centrifuged at 4000 rpm/min for 10 minutes, and the supernatant was collected. 40ul of the supernatant and 10ul of 10% skim milk were added to the antigen-coated plate and incubated for 1 hour at room temperature. After washing the plate, it was incubated with goat anti-human IgG (Fab-specific) peroxidase antibody diluted 1:3000 at 37°C for 1 hour, and then incubated with 0.1 mg/ml HRP substrate TMB at 37°C for 15 minutes for color development and detection. OD 450 signal. A total of 43 clones with higher OD 450 readings were obtained and sent for sequencing analysis.
1.4克隆与SARS-CoV-2 RBD的结合1.4 Binding of clones to SARS-CoV-2 RBD
从43个克隆保存的菌液中挑取2ul至含有250ul体积的0.05％葡萄糖、2YT培养基的96孔板中37度培养4小时。待OD 600值≥6时，加入1M IPTG至终浓度1mM，30度培养过夜。4000rpm/分钟离心10分钟后弃上清。加入100ul预冷的1×TES缓冲液重悬，然后加入150ul预冷的0.2×TES缓冲液，冰上静置0.5小时，4000rpm/分钟离心10分钟，收集上清，获得周质腔提取液。 Pick 2 ul from the bacterial solution stored in 43 clones to a 96-well plate containing 250 ul volume of 0.05% glucose and 2YT medium and culture at 37 degrees for 4 hours. When the OD 600 value is ≥ 6, add 1M IPTG to the final concentration of 1mM, and incubate at 30 degrees overnight. The supernatant was discarded after centrifugation at 4000 rpm/min for 10 minutes. Add 100ul of pre-cooled 1×TES buffer to resuspend, then add 150ul of pre-cooled 0.2×TES buffer, let stand on ice for 0.5 hours, centrifuge at 4000rpm/min for 10 minutes, collect supernatant, and obtain periplasmic cavity extract.
将链霉亲和素稀释至5μg/ml包被，每孔加入50ul并4度过夜。洗板并用2％脱脂牛奶封闭。用PBST洗涤液洗板4次，将生物素化的SARS-CoV-2 RBD(君实自制)抗原稀释至3ug/ml包被，每孔50ul，室温孵育0.5小时，洗板后备用。将初步定量的周质腔提取液按3倍梯度稀释加入包被抗原的板中，室温孵育1小时。洗板后再与1:3000稀释的山羊抗人IgG(Fab特异性)过氧化物酶抗体37℃孵育1小时，然后与0.1mg/ml的HRP底物TMB 37℃孵育15分钟显色，检测OD 450信号，使用软件GraphPad Prism四参数拟合EC 50。 Dilute streptavidin to 5μg/ml coating, add 50ul per well and leave overnight at 4°C. Plates were washed and blocked with 2% skim milk. The plate was washed 4 times with PBST washing solution, and the biotinylated SARS-CoV-2 RBD (self-made by Junshi) antigen was diluted to 3ug/ml for coating, 50ul per well, incubated at room temperature for 0.5 hours, and used after washing. Preliminarily quantified periplasmic cavity extract was added to the antigen-coated plate by 3-fold serial dilution, and incubated at room temperature for 1 hour. After washing the plate, it was incubated with goat anti-human IgG (Fab-specific) peroxidase antibody diluted 1:3000 at 37°C for 1 hour, and then incubated with 0.1 mg/ml HRP substrate TMB at 37°C for 15 minutes for color development and detection. OD450 signal, EC50 fitted with four parameters using software GraphPad Prism.
如表1所示，43个克隆中大部分克隆EC 50都有5到10倍的提升，根据EC 50的排序，挑选亲和力较高的8个重链和11个轻链序列进行排列组合，并构建成IgG形式的分子用于进一步验证。表1中，CB6-HC的氨基酸序列如SEQ ID NO：9所示，重链CB6-1-HC到CB6-16-HC的VH序列分别如SEQ ID NO：58-73所示、其余部分为SEQ ID NO：9中的相应部分；CB6-LC的氨基酸序列如SEQ ID NO：10所示，轻链CB6-17-LC到CB6-43-LC的VL部分分别如SEQ ID NO：74-100所示、其余部分为SEQ ID NO：10中的相应部分。 As shown in Table 1, most of the 43 clones had a 5- to 10-fold increase in EC 50. According to the ranking of EC 50 , 8 heavy chain and 11 light chain sequences with higher affinity were selected for permutation and combination, and Molecules constructed in IgG format were used for further validation. In Table 1, the amino acid sequence of CB6-HC is shown in SEQ ID NO: 9, the VH sequences of heavy chain CB6-1-HC to CB6-16-HC are respectively shown in SEQ ID NO: 58-73, and the rest are The corresponding part in SEQ ID NO: 9; the amino acid sequence of CB6-LC is shown in SEQ ID NO: 10, and the VL parts of light chain CB6-17-LC to CB6-43-LC are shown in SEQ ID NO: 74-100, respectively The remainder shown is the corresponding portion in SEQ ID NO:10.
表1：43个克隆及EC 50值 Table 1: 43 clones and EC50 values
Figure PCTCN2022080100-appb-000056
Figure PCTCN2022080100-appb-000056
Figure PCTCN2022080100-appb-000057
Figure PCTCN2022080100-appb-000057
Figure PCTCN2022080100-appb-000058
Figure PCTCN2022080100-appb-000058
实施例2：候选抗体的构建与筛选Example 2: Construction and Screening of Candidate Antibodies
2.1候选抗体的构建2.1 Construction of candidate antibodies
从实施例1的表1中挑选亲和力较高的8个重链和11个轻链序列进行排列组合，并构建成IgG形式的分子用于进一步测试亲和力，抗体编号如表2所示。8 heavy chain and 11 light chain sequences with higher affinity were selected from Table 1 of Example 1 for permutation and combination, and were constructed into molecules in the form of IgG for further affinity testing. The antibody numbers are shown in Table 2.
表2：CB6突变轻重链库组合Table 2: CB6 mutant light and heavy chain library combinations
组合combination CB6-28-LCCB6-28-LC CB6-29-LCCB6-29-LC CB6-31-LCCB6-31-LC CB6-27-LCCB6-27-LC CB6-38-LCCB6-38-LC CB6-32-LCCB6-32-LC CB6-37-LCCB6-37-LC CB6-41-LCCB6-41-LC CB6-33-LCCB6-33-LC CB6-26-LCCB6-26-LC CB6-36-LCCB6-36-LC CB6-LCCB6-LC
CB6-16-HCCB6-16-HC JS016-1JS016-1 JS016-10JS016-10 JS016-19JS016-19 JS016-28JS016-28 JS016-37JS016-37 JS016-46JS016-46 JS016-55JS016-55 JS016-64JS016-64 JS016-73JS016-73 JS016-82JS016-82 JS016-91JS016-91 JS016-100JS016-100
CB6-4-HCCB6-4-HC JS016-2JS016-2 JS016-11JS016-11 JS016-20JS016-20 JS016-29JS016-29 JS016-38JS016-38 JS016-47JS016-47 JS016-56JS016-56 JS016-65JS016-65 JS016-74JS016-74 JS016-83JS016-83 JS016-92JS016-92 JS016-101JS016-101
CB6-7-HCCB6-7-HC JS016-3JS016-3 JS016-12JS016-12 JS016-21JS016-21 JS016-30JS016-30 JS016-39JS016-39 JS016-48JS016-48 JS016-57JS016-57 JS016-66JS016-66 JS016-75JS016-75 JS016-84JS016-84 JS016-93JS016-93 JS016-102JS016-102
CB6-2-HCCB6-2-HC JS016-4JS016-4 JS016-13JS016-13 JS016-22JS016-22 JS016-31JS016-31 JS016-40JS016-40 JS016-49JS016-49 JS016-58JS016-58 JS016-67JS016-67 JS016-76JS016-76 JS016-85JS016-85 JS016-94JS016-94 JS016-103JS016-103
CB6-6-HCCB6-6-HC JS016-5JS016-5 JS016-14JS016-14 JS016-23JS016-23 JS016-32JS016-32 JS016-41JS016-41 JS016-50JS016-50 JS016-59JS016-59 JS016-68JS016-68 JS016-77JS016-77 JS016-86JS016-86 JS016-95JS016-95 JS016-104JS016-104
CB6-3-HCCB6-3-HC JS016-6JS016-6 JS016-15JS016-15 JS016-24JS016-24 JS016-33JS016-33 JS016-42JS016-42 JS016-51JS016-51 JS016-60JS016-60 JS016-69JS016-69 JS016-78JS016-78 JS016-87JS016-87 JS016-96JS016-96 JS016-105JS016-105
CB6-11-HCCB6-11-HC JS016-7JS016-7 JS016-16JS016-16 JS016-25JS016-25 JS016-34JS016-34 JS016-43JS016-43 JS016-52JS016-52 JS016-61JS016-61 JS016-70JS016-70 JS016-79JS016-79 JS016-88JS016-88 JS016-97JS016-97 JS016-106JS016-106
CB6-15-HCCB6-15-HC JS016-8JS016-8 JS016-17JS016-17 JS016-26JS016-26 JS016-35JS016-35 JS016-44JS016-44 JS016-53JS016-53 JS016-62JS016-62 JS016-71JS016-71 JS016-80JS016-80 JS016-89JS016-89 JS016-98JS016-98 JS016-107JS016-107
CB6-HCCB6-HC JS016-9JS016-9 JS016-18JS016-18 JS016-27JS016-27 JS016-36JS016-36 JS016-45JS016-45 JS016-54JS016-54 JS016-63JS016-63 JS016-72JS016-72 JS016-81JS016-81 JS016-90JS016-90 JS016-99JS016-99 JS016JS016
2.2候选抗体的筛选2.2 Screening of candidate antibodies
将重组SARS-CoV-2 RBD-His(君实生物自制)稀释至3.0μg/ml包被，37℃孵育90分钟，洗板并用2％脱脂牛奶封闭。加入不同浓度的CB6(JS016)和表2所示组合库抗体(从2μg/ml到2ng/ml，10倍梯度稀释，共4个浓度梯度)，37℃孵育1小时并洗板。再与1:5000稀释的山羊抗人IgG(Fc特异性)过氧化物酶抗体37℃孵育1小时，然后与0.1mg/ml的HRP底物TMB 37℃孵育15分钟显色，检测结合信号。将JS016-1到JS016-107号抗体浓度在20ng/ml时的OD 450读数与CB6抗体浓度在20ng/ml时的OD 450读数的比值作为相对活性，比值越高，表明结合活性越强。将JS016-1到JS016-107号抗体蛋白表达水平(单位mg/L)与CB6蛋白表达水平的比值作为相对表达水平，比值越高，表明表达越好。如表3所示，CB6突变轻重链库组合产生的抗体相对活性绝大多数都优于对照抗体CB6，提示亲和力获得提高。选择相对结合活性和相对表达水平均较高的JS016-38、39、40、73、83、84、88、106和107号分子进一步开展验证。 Recombinant SARS-CoV-2 RBD-His (self-made by Junshi Biotechnology) was diluted to 3.0 μg/ml for coating, incubated at 37°C for 90 minutes, washed and blocked with 2% skim milk. Different concentrations of CB6 (JS016) and combinatorial library antibodies shown in Table 2 (from 2 μg/ml to 2 ng/ml, 10-fold serial dilution, a total of 4 concentration gradients) were added, incubated at 37°C for 1 hour and the plate was washed. It was then incubated with 1:5000 diluted goat anti-human IgG (Fc specific) peroxidase antibody at 37°C for 1 hour, and then incubated with 0.1 mg/ml HRP substrate TMB at 37°C for 15 minutes for color development, and the binding signal was detected. The ratio of the OD 450 reading of JS016-1 to JS016-107 at 20 ng/ml and the OD 450 reading of CB6 antibody at 20 ng/ml was taken as the relative activity. The higher the ratio, the stronger the binding activity. The ratio of JS016-1 to JS016-107 antibody protein expression level (unit mg/L) and CB6 protein expression level was taken as the relative expression level. The higher the ratio, the better the expression. As shown in Table 3, the relative activity of the antibodies produced by the combination of the CB6 mutant light and heavy chain libraries was overwhelmingly better than that of the control antibody CB6, indicating that the affinity was improved. JS016-38, 39, 40, 73, 83, 84, 88, 106 and 107 molecules with higher relative binding activity and relative expression level were selected for further verification.
表3：CB6突变轻重链库组合产生抗体的相对活性Table 3: Relative activity of antibodies produced by combinations of CB6 mutant light and heavy chain repertoires
编号Numbering 相对活性Relative activity 编号Numbering 相对活性Relative activity 编号Numbering 相对活性Relative activity
JS016-1JS016-1 1.691.69 JS016-37JS016-37 0.010.01 JS016-73JS016-73 2.272.27
JS016-2JS016-2 1.941.94 JS016-38JS016-38 2.312.31 JS016-74JS016-74 2.342.34
JS016-3JS016-3 1.861.86 JS016-39JS016-39 1.791.79 JS016-75JS016-75 2.102.10
JS016-4JS016-4 1.731.73 JS016-40JS016-40 1.661.66 JS016-76JS016-76 2.062.06
JS016-5JS016-5 1.961.96 JS016-41JS016-41 1.821.82 JS016-77JS016-77 2.232.23
JS016-6JS016-6 1.981.98 JS016-42JS016-42 1.751.75 JS016-78JS016-78 2.192.19
JS016-7JS016-7 1.661.66 JS016-43JS016-43 1.981.98 JS016-79JS016-79 2.272.27
JS016-8JS016-8 1.661.66 JS016-44JS016-44 1.881.88 JS016-80JS016-80 2.222.22
JS016-9JS016-9 1.591.59 JS016-45JS016-45 1.891.89 JS016-81JS016-81 2.122.12
JS016-10JS016-10 1.841.84 JS016-46JS016-46 1.721.72 JS016-82JS016-82 2.192.19
JS016-11JS016-11 2.102.10 JS016-47JS016-47 1.921.92 JS016-83JS016-83 2.512.51
JS016-12JS016-12 2.052.05 JS016-48JS016-48 1.851.85 JS016-84JS016-84 2.262.26
JS016-13JS016-13 1.661.66 JS016-49JS016-49 1.811.81 JS016-85JS016-85 2.192.19
JS016-14JS016-14 1.831.83 JS016-50JS016-50 1.991.99 JS016-86JS016-86 2.312.31
JS016-15JS016-15 1.671.67 JS016-51JS016-51 1.941.94 JS016-87JS016-87 2.202.20
JS016-16JS016-16 1.901.90 JS016-52JS016-52 1.801.80 JS016-88JS016-88 2.282.28
JS016-17JS016-17 1.761.76 JS016-53JS016-53 1.751.75 JS016-89JS016-89 2.132.13
JS016-18JS016-18 1.711.71 JS016-54JS016-54 2.062.06 JS016-90JS016-90 2.242.24
JS016-19JS016-19 1.691.69 JS016-55JS016-55 2.012.01 JS016-91JS016-91 2.222.22
JS016-20JS016-20 1.561.56 JS016-56JS016-56 1.621.62 JS016-92JS016-92 2.312.31
JS016-21JS016-21 1.591.59 JS016-57JS016-57 1.761.76 JS016-93JS016-93 2.252.25
JS016-22JS016-22 1.541.54 JS016-58JS016-58 1.801.80 JS016-94JS016-94 2.232.23
JS016-23JS016-23 0.220.22 JS016-59JS016-59 1.781.78 JS016-95JS016-95 2.272.27
JS016-24JS016-24 1.481.48 JS016-60JS016-60 1.521.52 JS016-96JS016-96 2.242.24
JS016-25JS016-25 1.511.51 JS016-61JS016-61 1.801.80 JS016-97JS016-97 2.292.29
JS016-26JS016-26 1.261.26 JS016-62JS016-62 1.371.37 JS016-98JS016-98 2.132.13
JS016-27JS016-27 1.471.47 JS016-63JS016-63 1.531.53 JS016-99JS016-99 2.142.14
JS016-28JS016-28 1.621.62 JS016-64JS016-64 1.551.55 JS016-100JS016-100 1.801.80
JS016-29JS016-29 1.761.76 JS016-65JS016-65 1.891.89 JS016-101JS016-101 2.212.21
JS016-30JS016-30 1.541.54 JS016-66JS016-66 1.741.74 JS016-102JS016-102 1.871.87
JS016-31JS016-31 1.521.52 JS016-67JS016-67 1.911.91 JS016-103JS016-103 1.891.89
JS016-32JS016-32 1.601.60 JS016-68JS016-68 1.981.98 JS016-104JS016-104 1.871.87
JS016-33JS016-33 1.631.63 JS016-69JS016-69 1.901.90 JS016-105JS016-105 1.971.97
JS016-34JS016-34 1.591.59 JS016-70JS016-70 2.082.08 JS016-106JS016-106 2.032.03
JS016-35JS016-35 1.631.63 JS016-71JS016-71 1.961.96 JS016-107JS016-107 2.032.03
JS016-36JS016-36 0.880.88 JS016-72JS016-72 1.621.62 JS016JS016 1.001.00
实施例3：本发明抗体体外结合活性Example 3: In vitro binding activity of the antibodies of the present invention
将重组SARS-CoV-2-RBD-His(君实生物自制)稀释至3.0μg/ml包被，37℃孵育90分钟，洗板并用2％脱脂牛奶封闭。加入不同浓度的JS016抗体(阳性对照CB6(JS016)从40μg/ml到0.009537ng/ml，候选抗体从8μg/mL到0.001907ng/mL，4倍梯度稀释)，37℃孵育1小时并洗板。再与1:5000稀释的山羊抗人IgG(Fc特异性)过氧化物酶抗体37℃孵育1小时，然后与0.1mg/ml的HRP底物TMB 37℃孵育15分钟显色，检测抗体的结合信号。使用软件GraphPad Prism四参数拟合EC 50。 Recombinant SARS-CoV-2-RBD-His (self-made by Junshi Biotechnology) was diluted to 3.0 μg/ml for coating, incubated at 37°C for 90 minutes, washed and blocked with 2% skim milk. Add different concentrations of JS016 antibody (positive control CB6 (JS016) from 40 μg/ml to 0.009537ng/ml, candidate antibody from 8 μg/mL to 0.001907ng/mL, 4-fold serial dilution), incubate at 37°C for 1 hour and wash the plate. Incubate with 1:5000 diluted goat anti-human IgG (Fc-specific) peroxidase antibody at 37°C for 1 hour, and then with 0.1 mg/ml HRP substrate TMB at 37°C for 15 minutes for color development to detect the binding of the antibody Signal. The EC50 was fitted with four parameters using the software GraphPad Prism.
结果如表4和图3A-3D所示，通过结合ELISA测定，本发明抗体与重组SARS-CoV-2-RBD的结合能力均强于CB6抗体，结合能力提高约3-6倍，其中JS016-38及JS016-40两个抗体结合能力最强。The results are shown in Table 4 and Figures 3A-3D. The binding ability of the antibody of the present invention to recombinant SARS-CoV-2-RBD was stronger than that of the CB6 antibody, and the binding ability was increased by about 3-6 times. Among them, JS016- 38 and JS016-40 have the strongest binding ability.
实施例4：本发明抗体体外阻断活性Example 4: In vitro blocking activity of the antibodies of the present invention
将重组SARS-CoV-2-RBD-His稀释至5.0μg/ml包板，37℃温育90分钟。洗板并用2％脱脂牛奶封闭。用2％脱脂牛奶将重组人ACE2，C端鼠Fc标签稀释至5.0μg/ml，再用其稀释JS016抗体(阳性对照CB6(JS016)从400μg/ml到2.26ng/ml，候选抗体从80μg/mL到0.452ng/ml，3倍梯度稀释)。将混合物加入板中37℃孵育1小时并洗板。通过与1:5000稀释的过氧化物酶标记羊抗鼠Fc片段二抗37℃孵育1小时，再加入0.1mg/ml的TMB并37℃孵育20分钟。使用软件GraphPad Prism四参数拟合IC 50。 Recombinant SARS-CoV-2-RBD-His was diluted to 5.0 μg/ml for plating and incubated at 37°C for 90 minutes. Plates were washed and blocked with 2% skim milk. Recombinant human ACE2, C-terminal mouse Fc tag was diluted to 5.0 μg/ml with 2% skim milk, and then used to dilute JS016 antibody (positive control CB6 (JS016) from 400 μg/ml to 2.26 ng/ml, candidate antibody from 80 μg/ml mL to 0.452ng/ml, 3-fold serial dilution). The mixture was added to the plate and incubated at 37°C for 1 hour and the plate was washed. By incubating with 1:5000 diluted peroxidase-conjugated goat anti-mouse Fc fragment secondary antibody for 1 hour at 37°C, then adding 0.1 mg/ml of TMB and incubating at 37°C for 20 minutes. IC50s were fitted with four parameters using the software GraphPad Prism .
结果如表5和图4A-4D所示，通过阻断ELISA测定，本发明抗体均能更有效的阻断SARS-CoV-2病毒S蛋白的RBD与其受体ACE2结合，阻断能力高约2.5-4.5倍，其中JS016-38/40/83/106阻断能力略高于其余抗体。The results are shown in Table 5 and Figures 4A-4D. As determined by blocking ELISA, the antibodies of the present invention can more effectively block the binding of the RBD of the SARS-CoV-2 virus S protein to its receptor ACE2, and the blocking ability is about 2.5% higher. -4.5 times, in which the blocking ability of JS016-38/40/83/106 is slightly higher than that of other antibodies.
表4：ELISA结合与阻断活性Table 4: ELISA binding and blocking activities
Figure PCTCN2022080100-appb-000059
Figure PCTCN2022080100-appb-000059
实施例5：octet Red 96e测定本发明抗体对SARS-CoV-2 RBD的亲和力Example 5: Determination of the affinity of the antibody of the present invention to SARS-CoV-2 RBD with octet Red 96e
使用protein A捕获法测定JS016-38和抗原RBD-his(SARS-CoV-2 RBD)结合的动力学参数。将浓度为5μg/ml的JS016-38结合在Protein A探针上(Cat No:18-5010；lot：2001131)，将抗原RBD-his用1X Fortebio工作液(1X PBS+0.05％吐温20)从120nM往下2倍稀释设5个浓度梯度与抗体结合，于1X Fortebio工作液中解离。Kinetic parameters of binding of JS016-38 to the antigen RBD-his (SARS-CoV-2 RBD) were determined using a protein A capture method. JS016-38 at a concentration of 5μg/ml was combined with the Protein A probe (Cat No: 18-5010; lot: 2001131), and the antigen RBD-his was used in 1X Fortebio working solution (1X PBS+0.05% Tween 20) 2-fold dilution from 120nM down to 5 concentration gradients for antibody binding and dissociation in 1X Fortebio working solution.
使用protein A捕获法测定JS016-40和抗原RBD-his结合的动力学参数。将浓度为5μg/ml的JS016-40结合在Protein A探针上(Cat No:18-5010；lot：2001131)，将抗原RBD-his 用1X Fortebio工作液(1X PBS+0.05％P20)从120nM往下2倍稀释设5个浓度梯度与抗体结合，于1X Fortebio工作液中解离。Kinetic parameters of binding of JS016-40 to antigen RBD-his were determined using protein A capture method. JS016-40 at a concentration of 5 μg/ml was bound to the Protein A probe (Cat No: 18-5010; lot: 2001131), and the antigen RBD-his was treated with 1X Fortebio working solution (1X PBS+0.05% P20) from 120nM 2-fold dilution to set 5 concentration gradients to bind to the antibody and dissociate in 1X Fortebio working solution.
使用protein A捕获法测定CB6和抗原RBD-his结合的动力学参数。将浓度为5μg/ml的CB6结合在Protein A探针上(Cat No:18-5010；lot：2001131)，将抗原RBD-his用1X Fortebio工作液(1X PBS+0.05％P20)从120nM往下2倍稀释设5个浓度梯度与抗体结合，于1X Fortebio工作液中解离。Kinetic parameters of binding of CB6 to antigen RBD-his were determined using protein A capture method. CB6 at a concentration of 5 μg/ml was bound to the Protein A probe (Cat No: 18-5010; lot: 2001131), and the antigen RBD-his was used 1X Fortebio working solution (1X PBS+0.05% P20) from 120nM down 2-fold dilutions set 5 concentration gradients to bind to the antibody and dissociate in 1X Fortebio working solution.
抗体JS016-38、抗体JS016-40和抗体CB6与RBD-his结合的动力学参数见表5。The kinetic parameters of antibody JS016-38, antibody JS016-40 and antibody CB6 binding to RBD-his are shown in Table 5.
JS016-38和JS016-40的结合速率与CB6相当，解离速率比CB6慢约100倍，亲和力常数比CB6提高约100倍。JS016-38和JS016-40的结合能力提高主要是解离变慢贡献的。The on-rate of JS016-38 and JS016-40 is comparable to that of CB6, the dissociation rate is about 100 times slower than that of CB6, and the affinity constant is about 100 times higher than that of CB6. The improved binding ability of JS016-38 and JS016-40 was mainly contributed by the slower dissociation.
表5：本发明抗体的动力学参数Table 5: Kinetic parameters of antibodies of the invention
抗体Antibody kon(1/Ms)kon(1/Ms) kdis(1/s)kdis(1/s) KD(M)KD(M)
CB6CB6 1.89E+051.89E+05 4.97E-034.97E-03 2.63E-082.63E-08
JS016-38JS016-38 1.35E+051.35E+05 <1.00E-05<1.00E-05 <7.41E-11<7.41E-11
JS016-40JS016-40 1.71E+051.71E+05 2.03E-052.03E-05 1.19E-101.19E-10
注：KD为亲和力常数；kon为抗原抗体结合速率；kdis为抗原抗体解离速率；KD＝kdis/kon。Note: KD is the affinity constant; kon is the antigen-antibody binding rate; kdis is the antigen-antibody dissociation rate; KD=kdis/kon.
实施例6：本发明抗体活病毒中和活性Example 6: Live virus neutralization activity of the antibody of the present invention
准备1个96孔平底细胞培养板，在第一列8个孔中加入90μL抗体稀释液(DMEM完全培养基，Gibco)，其余孔中加入50μL抗体稀释液。取40μg/mL的JS016-38抗体向第一列的8个孔中各加入10μL，使抗体终浓度为4μg/mL。用8道移液器将第一列中液体混匀后，吸取50μL加入到第二列中混匀，再吸取50μL加入到下一列中混匀，依次操作，进行2倍的系列稀释，共稀释10次，总计11个不同浓度抗体，最后一列不加抗体的对照。JS016-40和CB6抗体(JS016)稀释方法相同。CB6抗体最高浓度5μg/mL，共稀释10次，总计11个不同浓度抗体。Prepare a 96-well flat-bottom cell culture plate, add 90 μL of antibody dilution (DMEM complete medium, Gibco) to 8 wells in the first column, and add 50 μL of antibody dilution to the remaining wells. Take 40 μg/mL of JS016-38 antibody and add 10 μL to each of the 8 wells in the first column to make the final antibody concentration 4 μg/mL. After mixing the liquid in the first column with an 8-channel pipette, pipette 50 μL into the second column and mix, then pipet 50 μL and add it to the next column to mix well, and operate in sequence to perform a 2-fold serial dilution to achieve a total dilution. 10 times, a total of 11 different concentrations of antibody, the last column without antibody control. JS016-40 and CB6 antibody (JS016) were diluted in the same way. The highest concentration of CB6 antibody was 5 μg/mL, and a total of 10 dilutions were made, a total of 11 antibodies of different concentrations.
取出培养箱中事先准备好的Vero E6细胞(汇合率达80％～90％)，以T75培养瓶为例，吸去瓶中的培养基，加入5mL PBS缓冲液清洗细胞，倾去PBS后，加入3mL 0.25％胰酶-EDTA，使其浸没细胞消化1分钟，倾去胰酶，置于细胞培养箱中消化5分钟，轻轻拍打培养瓶侧壁使细胞脱落，加入10mL培养基中和胰酶，吹打几次后转移至离心管中，210g离心5分钟，倾去上清，用10mL DMEM完全培养基(Gibco)重悬细胞，细胞计数，用DMEM完全培养基将细胞稀释至1.5×10 5个/mL。 Take out the Vero E6 cells prepared in the incubator (the confluence rate is 80% to 90%), take the T75 culture flask as an example, aspirate the medium in the flask, add 5 mL of PBS buffer to wash the cells, and pour off the PBS. Add 3 mL of 0.25% trypsin-EDTA to submerge the cells for 1 minute of digestion, pour off the trypsin, and place them in a cell incubator to digest for 5 minutes. Enzyme, pipetting several times and then transferring to a centrifuge tube, centrifuging at 210g for 5 minutes, decanting the supernatant, resuspending the cells with 10 mL DMEM complete medium (Gibco), counting the cells, and diluting the cells with DMEM complete medium to 1.5×10 5 /mL.
在P3实验室生物安全柜中，用抗体稀释液将SARS-CoV-2稀释至2×10 3TCID 50/ml(依据提供的病毒原液滴度计算稀释倍数进行稀释)；在有抗体的96孔板中，每孔加入50μL病毒，使每孔含病毒的量为100TCID 50/孔；每个抗体浓度做8个复孔，设置不加抗体孔，不加病毒孔(额外的96板)作为对照。将上述96孔板置于细胞培养箱中(37℃，5％CO 2)孵育1小时。 In the P3 laboratory biological safety cabinet, dilute SARS-CoV-2 with antibody diluent to 2×10 3 TCID 50 /ml (calculate the dilution factor according to the provided virus titer); in 96 wells with antibodies In the plate, add 50 μL of virus to each well, so that the amount of virus in each well is 100TCID 50 /well; make 8 duplicate wells for each antibody concentration, set up no antibody well, no virus well (an additional 96 plates) as a control . The above 96-well plate was placed in a cell culture incubator (37°C, 5% CO 2 ) and incubated for 1 hour.
孵育1小时后，向96孔板中每孔加100μl之前稀释好的Vero E6细胞，使每孔细胞为1.5×10 4个。将96孔板前后左右轻轻晃动，使细胞在孔中分散均匀，将96孔板放入细胞培养箱中，37℃，5％CO 2培养72小时。72小时后从细胞培养箱中取出96孔板，置于光学显微镜下观察并统计细胞病变效应(CPE)的情况。中和抑制率＝100-病变孔数/8。根据中和抑制率结果，利用生物统计学软件Graphpad拟合计算ND 50。 After 1 hour of incubation, 100 μl of the previously diluted Vero E6 cells were added to each well of a 96-well plate, so that the number of cells per well was 1.5×10 4 . Gently shake the 96-well plate back and forth to make the cells evenly dispersed in the wells, and place the 96-well plate in a cell culture incubator for 72 hours at 37°C, 5% CO 2 . After 72 hours, the 96-well plate was taken out from the cell culture incubator and placed under a light microscope to observe and count the cytopathic effect (CPE). Neutralization inhibition rate=100-number of lesions/8. According to the results of neutralization inhibition rate, ND50 was calculated by fitting with biostatistics software Graphpad.
由图5可以看出两株单克隆抗体均能够抑制SARS-CoV-2对Vero E6细胞的侵染，具有浓度梯度依赖效应，JS016-38和JS016-40的ND 50分别为0.47±0.16μg/mL和0.09±0.02μg/mL。经过亲和力成熟突变后，JS016-40的中和活性与CB6相比提高了4倍，而JS016-38的中和活性与CB6相当。 It can be seen from Figure 5 that both monoclonal antibodies can inhibit the infection of Vero E6 cells by SARS-CoV-2 with a concentration gradient dependent effect. The ND 50 of JS016-38 and JS016-40 are 0.47±0.16μg/ mL and 0.09 ± 0.02 μg/mL. After affinity maturation mutation, the neutralizing activity of JS016-40 was increased 4-fold compared with CB6, while the neutralizing activity of JS016-38 was comparable to CB6.
实施例7：本发明抗体与SARS-CoV-2-RBD突变体结合活性Example 7: Binding activity of the antibody of the present invention to SARS-CoV-2-RBD mutant
使用3.0μg/ml的重组SARS-CoV-2-RBD及其突变体(君实自制)蛋白包板，37℃温育90分钟；用2％脱脂牛奶封闭；使用梯度稀释的候选抗体(从40μg/ml至0.009537ng/ml，按4倍梯度稀释)与其结合，37℃温育1小时。用1:5000稀释的山羊抗人IgG(Fc特异性)过氧化物酶抗体进行检测，37℃温育1小时，最后用0.1mg/ml TMB 37℃温育显色15分钟后终止，使用软件GraphPad Prism四参数拟合EC 50。 Plates were coated with 3.0 μg/ml recombinant SARS-CoV-2-RBD and its mutants (self-made by Junshi), and incubated at 37°C for 90 minutes; blocked with 2% skim milk; candidate antibodies (from 40 μg /ml to 0.009537ng/ml, 4-fold serial dilution) combined with it and incubated at 37°C for 1 hour. Detected with 1:5000 diluted goat anti-human IgG (Fc-specific) peroxidase antibody, incubated at 37 °C for 1 hour, and finally incubated with 0.1 mg/ml TMB at 37 °C for 15 minutes and terminated after color development, using the software GraphPad Prism Four parameter fit EC50 .
其中，重组SARS-CoV-2-RBD突变体分别为：K417N、E484K和N501Y突变。Among them, the recombinant SARS-CoV-2-RBD mutants were: K417N, E484K and N501Y mutations.
如表6所示，与最初报道的SARS-CoV-2-RBD野生型(抗原名称RBD-his)相比，抗体CB6与突变型RBD-K417N-his不结合，与RBD-N501Y-his及RBD-E484K-his的结合很弱(图6A)。本发明抗体JS016-38及JS016-40与RBD-his、突变型RBD-N501Y-his、RBD-K417N-his及RBD-E484K-his均具有较强的结合能力(图6B和6C)，且EC 50显著优于对照抗体CB6。 As shown in Table 6, compared with the wild type of SARS-CoV-2-RBD (antigen name RBD-his) originally reported, antibody CB6 does not bind to mutant RBD-K417N-his, and binds to RBD-N501Y-his and RBD-N501Y-his The binding of -E484K-his was weak (Fig. 6A). The antibodies JS016-38 and JS016-40 of the present invention all have strong binding ability to RBD-his, mutant RBD-N501Y-his, RBD-K417N-his and RBD-E484K-his (Fig. 6B and 6C), and EC 50 was significantly better than the control antibody CB6.
表6：本发明抗体与SARS-CoV-2RBD及其突变体蛋白结合Table 6: Binding of the antibodies of the present invention to SARS-CoV-2 RBD and its mutant proteins
Figure PCTCN2022080100-appb-000060
Figure PCTCN2022080100-appb-000060
Figure PCTCN2022080100-appb-000061
Figure PCTCN2022080100-appb-000061
注：”NA”表示不结合。Note: "NA" means no binding.
实施例8：本发明抗体阻断SARS-CoV-2突变体与ACE2结合Example 8: Antibodies of the present invention block the binding of SARS-CoV-2 mutants to ACE2
使用5.0μg/ml的重组SARS-CoV-2-RBD及其突变体蛋白包板，37℃温育90分钟，用2％脱脂牛奶封闭。用2％脱脂牛奶将重组人ACE2-mFc稀释至5.0μg/ml，再用其稀释JS016抗体(从400μg/ml到2.26ng/ml，按3倍梯度稀释)，将混合物加到封闭后的板上37℃温育，孵育1小时。用1:5000稀释的过氧化物酶标记羊抗鼠Fc片段二抗进行检测，37℃温育，孵育1小时。最后用0.1mg/ml TMB显色15分钟后终止，使用软件GraphPad Prism四参数拟合IC 50。 Plates were coated with 5.0 μg/ml of recombinant SARS-CoV-2-RBD and its mutant proteins, incubated at 37°C for 90 minutes, and blocked with 2% skim milk. Recombinant human ACE2-mFc was diluted to 5.0 μg/ml with 2% skim milk, and then JS016 antibody was diluted with it (from 400 μg/ml to 2.26 ng/ml, in a 3-fold gradient), and the mixture was added to the blocked plate Incubate at 37°C for 1 hour. Detection was performed with peroxidase-conjugated goat anti-mouse Fc fragment secondary antibody diluted 1:5000, and incubated at 37°C for 1 hour. Finally, color development was terminated after 15 minutes with 0.1 mg/ml TMB, and the IC 50 was fitted with four parameters using the software GraphPad Prism.
其中，重组SARS-CoV-2-RBD突变体分别为：K417N、E484K和N501Y突变。Among them, the recombinant SARS-CoV-2-RBD mutants were: K417N, E484K and N501Y mutations.
如表7所示，与最初报道的SARS-CoV-2-RBD野生型(抗原名称RBD-his)相比，CB6抗体需要很高浓度才能阻断RBD-N501Y-his及RBD-E484K-his与ACE2的结合，不能阻断RBD-K417N-his与ACE2的结合(图7A)。本发明抗体JS016-38及JS016-40均能分别阻断RBD-his、RBD-N501Y-his、RBD-K417N-his及RBD-E484K-his突变体与ACE2的结合，且IC 50显著优于对照抗体CB6(图7B，7C)。 As shown in Table 7, the CB6 antibody required very high concentrations to block the interaction of RBD-N501Y-his and RBD-E484K-his with Binding of ACE2 did not block the binding of RBD-K417N-his to ACE2 (Fig. 7A). The antibodies JS016-38 and JS016-40 of the present invention can block the binding of RBD-his, RBD-N501Y-his, RBD-K417N-his and RBD-E484K-his mutants to ACE2 respectively, and the IC 50 is significantly better than the control Antibody CB6 (Figure 7B, 7C).
表7：本发明抗体阻断SARS-CoV-2RBD及其突变体与ACE2结合Table 7: Antibodies of the present invention block the binding of SARS-CoV-2 RBD and its mutants to ACE2
Figure PCTCN2022080100-appb-000062
Figure PCTCN2022080100-appb-000062
Figure PCTCN2022080100-appb-000063
Figure PCTCN2022080100-appb-000063
注：”NA”表示不结合。Note: "NA" means no binding.
实施例9：Biacore TM 8K测定抗体对抗原的亲和力 Example 9: Biacore ™ 8K Determination of Antibody Affinity for Antigen
使用proteinA捕获法分别测定抗体CB6、JS016-40、JS016-41-YTE和JS016-77-YTE和抗原RBD-his(君实自制)或RBD-Omicron-his(购自义翘神州，货号40592-V08H121)结合的动力学参数。本文所述的JS016-41-YTE、JS016-77-YTE分别由JS016-41、JS016-77替换重链恒定区得到，抗体JS016-41-YTE的重链氨基酸序列如SEQ ID NO:148所示，轻链氨基酸序列如SEQ ID NO:149所示；JS016-77-YTE的重链氨基酸序列如SEQ ID NO:148所示，轻链氨基酸序列如SEQ ID NO:150所示。The antibody CB6, JS016-40, JS016-41-YTE and JS016-77-YTE and the antigen RBD-his (made by Junshi) or RBD-Omicron-his (purchased from Yiqiao Shenzhou, item number 40592- Kinetic parameters of V08H121) binding. The JS016-41-YTE and JS016-77-YTE described herein are obtained by replacing the heavy chain constant region with JS016-41 and JS016-77, respectively. The heavy chain amino acid sequence of the antibody JS016-41-YTE is shown in SEQ ID NO: 148 , the light chain amino acid sequence is shown in SEQ ID NO: 149; the heavy chain amino acid sequence of JS016-77-YTE is shown in SEQ ID NO: 148, and the light chain amino acid sequence is shown in SEQ ID NO: 150.
具体而言，将浓度为1μg/ml的抗体结合在Protein A芯片上(label No:29139131-AA；lot：10261132)，将抗原用1X HBS-EP工作液(购自Life science，BR-1006-69)从72nM或36nM往下2倍稀释设6个浓度梯度与抗体结合，于HBS-EP工作液中解离。Specifically, the antibody at a concentration of 1 μg/ml was bound to a Protein A chip (label No: 29139131-AA; lot: 10261132), and the antigen was treated with 1X HBS-EP working solution (purchased from Life science, BR-1006- 69) 2-fold dilution from 72nM or 36nM down to set 6 concentration gradients to bind to the antibody, and dissociate in HBS-EP working solution.
各抗体和RBD-his、RBD-Omicron-his结合的动力学参数见表8，动力学特征参数检测结果分别如图8A、8B、8C和8D所示。结果显示，CB6对Omicron无结合，JS016-40、JS016-41-YTE和JS016-77-YTE对Omicron有较好的结合；JS016-41-YTE和JS016-77-YTE与RBD-his的结合速率与CB6相当，解离速率比CB6慢约100倍，与RBD-his的亲和力常数比CB6提高约100倍。The kinetic parameters of the binding of each antibody to RBD-his and RBD-Omicron-his are shown in Table 8, and the detection results of kinetic characteristic parameters are shown in Figures 8A, 8B, 8C and 8D, respectively. The results showed that CB6 did not bind to Omicron, while JS016-40, JS016-41-YTE and JS016-77-YTE had better binding to Omicron; the binding rates of JS016-41-YTE and JS016-77-YTE to RBD-his Comparable to CB6, the dissociation rate is about 100 times slower than that of CB6, and the affinity constant with RBD-his is about 100 times higher than that of CB6.
表8：抗体和RBD-his、RBD-Omicron-his结合的动力学参数Table 8: Kinetic parameters of antibody binding to RBD-his, RBD-Omicron-his
Figure PCTCN2022080100-appb-000064
Figure PCTCN2022080100-appb-000064
Figure PCTCN2022080100-appb-000065
Figure PCTCN2022080100-appb-000065
实施例10：抗体与SARS-CoV-2的omicron突变株S蛋白体外结合活性(ELISA)Example 10: Binding activity of antibody to S protein of omicron mutant strain of SARS-CoV-2 in vitro (ELISA)
将重组SARS-CoV-2的omicron突变株S蛋白(购自Acro，货号：SPN-C52Hz)稀释至3.0μg/mL，置于包被板中，37℃孵育90分钟，洗板并用含有2％脱脂牛奶的PBS溶液封闭。加入不同浓度的抗体(从10μg/mL到0.61ng/mL，4倍梯度稀释，共8个浓度)，37℃孵育1小时并洗板。再与1:5000(v/v)稀释的山羊抗人IgG(Fc特异性)过氧化物酶抗体(购自Sigma，货号A0170，作为检测抗体)37℃孵育1小时，然后与0.1mg/mL的HRP底物TMB 37℃孵育15分钟显色，最后用2M HCl溶液终止反应，在450nm/620nm下读板，检测得到结合信号。使用软件GraphPad Prism进行四参数对数回归(4PL)模型拟合，得到EC50。The recombinant SARS-CoV-2 omicron mutant S protein (purchased from Acro, product number: SPN-C52Hz) was diluted to 3.0 μg/mL, placed in a coated plate, incubated at 37°C for 90 minutes, washed and washed with 2% Skim milk in PBS for blocking. Antibodies at different concentrations (from 10 μg/mL to 0.61 ng/mL, 4-fold serial dilution, 8 concentrations in total) were added, incubated at 37°C for 1 hour and the plate was washed. It was then incubated with 1:5000 (v/v) diluted goat anti-human IgG (Fc-specific) peroxidase antibody (purchased from Sigma, Cat. No. A0170, as detection antibody) at 37°C for 1 hour, and then mixed with 0.1 mg/mL The HRP substrate TMB was incubated at 37°C for 15 minutes for color development. Finally, the reaction was terminated with 2M HCl solution, and the plate was read at 450nm/620nm, and the binding signal was detected. Four-parameter logistic regression (4PL) model fitting was performed using the software GraphPad Prism to obtain EC50.
测试结果如表9和图9所示。CB6对omicron突变株S蛋白无结合活性，JS016-40、JS016-41-YTE、JS016-77-YTE均优于CB6。The test results are shown in Table 9 and Figure 9. CB6 has no binding activity to S protein of omicron mutant strain, and JS016-40, JS016-41-YTE and JS016-77-YTE are better than CB6.
表9：抗体与SARS-CoV-2的omicron突变株S蛋白体外结合活性Table 9: In vitro binding activity of antibody to S protein of omicron mutant strain of SARS-CoV-2
Figure PCTCN2022080100-appb-000066
Figure PCTCN2022080100-appb-000066
实施例11：假病毒中和活性Example 11: Pseudovirus neutralizing activity
利用荧光素酶报告基因系统，检测待测抗体对SARS-CoV-2 omicron突变株(购自Vazyme，货号：DD1568-03)假病毒感染293-ACE2细胞(诺唯赞，产品编号：DD1401-01)的阻断作用。Using the luciferase reporter gene system, the detection of the antibody to be tested against SARS-CoV-2 omicron mutant strain (purchased from Vazyme, product number: DD1568-03) pseudovirus infected 293-ACE2 cells (Novezan, product number: DD1401-01) ) blocking effect.
将SARS-CoV-2 Omicron突变株突变株(1μL病毒/孔)假病毒分别与待测抗体(从100μg/mL到0.1pg/mL，10倍梯度稀释)在37℃预孵育1h。然后用实验缓冲液(DMEM培养基(1X)+10v/v％FBS)重悬293-ACE2细胞，并按每孔20000个细胞加入到假病毒与抗体混合液中，放于37℃培养箱孵育24h。孵育结束后，向每孔加入50μL荧光素酶报告基因检测试剂(Bright-Lite Luciferase Assay System，购自Vazyme，货号：DD1204)，并用酶标仪检测荧光信号，之后用GraphPad Prism软件进行四参数对数回归(4PL)模型拟合曲线，得出IC50值。The SARS-CoV-2 Omicron mutant mutant (1 μL virus/well) pseudovirus was pre-incubated with the test antibody (from 100 μg/mL to 0.1 pg/mL, 10-fold serial dilution) at 37 °C for 1 h. Then resuspend 293-ACE2 cells with experimental buffer (DMEM medium (1X) + 10v/v% FBS), add 20,000 cells per well to the mixture of pseudovirus and antibody, and incubate at 37°C 24h. After the incubation, 50 μL of luciferase reporter gene detection reagent (Bright-Lite Luciferase Assay System, purchased from Vazyme, product number: DD1204) was added to each well, and the fluorescence signal was detected with a microplate reader. Plot regression (4PL) model was used to fit the curve to obtain the IC50 value.
测试结果如表10和图10A、10B、10C所示。CB6对omicron突变株无中和活性，JS016-40、JS016-41-YTE、JS016-77-YTE的IC50分别为24.57ng/ml、17.62ng/ml、776.4ng/ml，均优于CB6。The test results are shown in Table 10 and Figures 10A, 10B, and 10C. CB6 has no neutralizing activity against omicron mutants. The IC50 of JS016-40, JS016-41-YTE and JS016-77-YTE are 24.57ng/ml, 17.62ng/ml and 776.4ng/ml, respectively, which are all better than CB6.
表10：抗体的假病毒中和活性Table 10: Pseudovirus neutralizing activity of antibodies
样品名称sample name IC50(ng/ml)IC50(ng/ml)
CB6CB6 无活性inactive
JS016-40JS016-40 24.5724.57
JS016-41-YTEJS016-41-YTE 17.6217.62
JS016-71-YTEJS016-71-YTE 776.4776.4

Claims (20)

  1. 一种抗体或其抗原结合片段，其特异性结合SARS-CoV-2或其变体的受体结合结构域(RBD)，其中所述抗体或其抗原结合片段包含如下所示的重链可变区的HCDR1、HCDR2和HCDR3，和/或如下所示的轻链可变区的LCDR1、LCDR2和LCDR3：An antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain (RBD) of SARS-CoV-2 or a variant thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable as shown below HCDR1, HCDR2 and HCDR3 of the light chain variable region, and/or LCDR1, LCDR2 and LCDR3 of the light chain variable region as follows:
    HCDR1：GFX 1VX 2X 3NY，其中，X 1选自L、T、E、R、Q、V、W、I或S，优选地，X 1选自L、T、E、R、Q、V或I；X 2选自Q、G、D、R、P、M、K、V、A、N或Y，优选地，X 2选自Q、G、D、R、P、N或Y；X 3选自R、W、Y、A、F、V或H，优选地，X3选自W、R、A、F或V； HCDR1: GFX 1 VX 2 X 3 NY, wherein X 1 is selected from L, T, E, R, Q, V, W, I or S, preferably, X 1 is selected from L, T, E, R, Q , V or I; X 2 is selected from Q, G, D, R, P, M, K, V, A, N or Y, preferably, X 2 is selected from Q, G, D, R, P, N or Y; X is selected from R , W, Y, A, F, V or H, preferably, X is selected from W, R, A, F or V;
    HCDR2：IYPGGX 4T，其中，X 4为T或S； HCDR2: IYPGGX 4 T, where X 4 is T or S;
    HCDR3：ARVLPMYGDYLDY；HCDR3:ARVLPMYGDYLDY;
    LCDR1：QX 5IX 6X 7Y，其中，X 5选自V、D、Q、A、W、R、N、S、D、M、K或P，优选地，X 5选自Q、A、R、N、S、D或M；X 6选自N、H、L、G、P、S、M、E、V、R、D、A或I，优选地，X 6选自E、L、V、R、D、E或A；X 7选自H、V、F、P、N、S、R、Q、G、Y或T，优选地，X 7选自Q、P、S、G、P、R或Y； LCDR1: QX 5 IX 6 X 7 Y, wherein X 5 is selected from V, D, Q, A, W, R, N, S, D, M, K or P, preferably, X 5 is selected from Q, A , R, N, S, D or M; X 6 is selected from N, H, L, G, P, S, M, E, V, R, D, A or I, preferably, X 6 is selected from E, L, V, R, D, E or A ; X is selected from H, V, F, P, N, S, R, Q, G, Y or T, preferably, X is selected from Q, P, S , G, P, R or Y;
    LCDR2：AAS；LCDR2: AAS;
    LCDR3：QQSX 8SX 9X 10PEYT，其中，X 8选自G、Y、T、S、K、A、N、E或P，优选地，X 8选自A、N、S或P；X 9选自P、S、I、N、A、W或F，优选地，X 9选自S、P或A；X 10选自T、V、L、I、R、K、S、M或F，优选地，X 10选自S、R、T、K、V、L或F。 LCDR3: QQSX 8 SX 9 X 10 PEYT, wherein X 8 is selected from G, Y, T, S, K, A, N, E or P, preferably, X 8 is selected from A, N, S or P; X 9 is selected from P, S, I, N, A, W or F, preferably, X 9 is selected from S, P or A; X 10 is selected from T, V, L, I, R, K, S, M or F, preferably, X 10 is selected from S, R, T, K, V, L or F.
  2. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含：The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:
    (1)SEQ ID NO:26、28、29、31、32、33、34、35、104、105、106、107、108、109、110和111中任一所示的HCDR1，优选SEQ ID NO:26、28、29、31、32、33、34和35中任一所示的HCDR1；和SEQ ID NO:27和30中任一所示的HCDR2；和(1) HCDR1 shown in any one of SEQ ID NOs: 26, 28, 29, 31, 32, 33, 34, 35, 104, 105, 106, 107, 108, 109, 110 and 111, preferably SEQ ID NO : HCDR1 set forth in any one of 26, 28, 29, 31, 32, 33, 34 and 35; and HCDR2 set forth in any one of SEQ ID NOs: 27 and 30; and
    SEQ ID NO:36、38、40、42、44、46、48、50、52、54、56、112、114、116、118、120、122、124、126、128、130、132、134、136、138、140和142中任一所示的LCDR1，优选SEQ ID NO:36、38、40、42、44、46、48、50、52、54和56中任一所示的LCDR1；和SEQ ID NO:37、39、41、43、45、47、49、51、53、55、57、113、115、117、119、121、123、125、127、129、131、133、135、137、139、141和143中任一所示的LCDR3，优选SEQ ID NO:37、39、41、43、45、47、49、51、 53、55和57中任一所示的LCDR3；或SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, LCDR1 set forth in any one of 136, 138, 140 and 142, preferably LCDRl set forth in any one of SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56; and SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, The LCDR3 shown in any one of 137, 139, 141 and 143, preferably the LCDR3 shown in any one of SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57; or
    (2)氨基酸序列如SEQ ID NO:144、145和3所示的HCDR1、HCDR2与HCDR3和氨基酸序列如SEQ ID NO:4、5和6所示的LCDR1、LCDR2和LCDR3，优选氨基酸序列如(1)所示的HCDR1、HCDR2、氨基酸序列如SEQ ID NO:3所示的HCDR3和氨基酸序列如SEQ ID NO:4、5和6所示的LCDR1、LCDR2和LCDR3；或(2) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 144, 145 and 3 and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4, 5 and 6, preferably amino acid sequences such as ( or
    (3)氨基酸序列如SEQ ID NO:1、2和3所示的HCDR1、HCDR2与HCDR3和氨基酸序列如SEQ ID NO:146、5和147所示的LCDR1、LCDR2和LCDR3，优选氨基酸序列如SEQ ID NO:1、2和3所示的HCDR1、HCDR2与HCDR3和氨基酸序列如式(1)所示的LCDR1和LCDR3以及氨基酸序列如SEQ ID NO:5所示的LCDR2。(3) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1, 2 and 3 and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 146, 5 and 147, preferably the amino acid sequences are as shown in SEQ ID NO: 146, 5 and 147 HCDR1, HCDR2 and HCDR3 shown in ID NO: 1, 2 and 3, LCDR1 and LCDR3 whose amino acid sequence is shown in formula (1), and LCDR2 whose amino acid sequence is shown in SEQ ID NO: 5.
  3. 如权利要求1或2所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区和/或轻链可变区，其中：The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, wherein:
    所述重链可变区含有选自以下组的HCDR1、HCDR2和HCDR3：SEQ ID NO:104、27和3；SEQ ID NO:26、27和3；SEQ ID NO:28、27和3；SEQ ID NO:29、30和3；SEQ ID NO:105、27和3；SEQ ID NO:31、30和3；SEQ ID NO:32、30和3；SEQ ID NO:106、30和3；SEQ ID NO:107、30和3；SEQ ID NO:108、27和3；SEQ ID NO:33、30和3；SEQ ID NO:109、27和3；SEQ ID NO:110、30和3；SEQ ID NO:111、30和3；SEQ ID NO:34、30和3；和SEQ ID NO:35、30和3；优选地，所述抗体或其抗原结合片段的轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:4、5和6所示；The heavy chain variable region contains HCDR1, HCDR2 and HCDR3 selected from the group consisting of: SEQ ID NO: 104, 27 and 3; SEQ ID NO: 26, 27 and 3; SEQ ID NO: 28, 27 and 3; SEQ ID NO: 28, 27 and 3; ID NO: 29, 30 and 3; SEQ ID NO: 105, 27 and 3; SEQ ID NO: 31, 30 and 3; SEQ ID NO: 32, 30 and 3; SEQ ID NO: 106, 30 and 3; ID NO: 107, 30 and 3; SEQ ID NO: 108, 27 and 3; SEQ ID NO: 33, 30 and 3; SEQ ID NO: 109, 27 and 3; SEQ ID NO: 110, 30 and 3; ID NO: 111, 30 and 3; SEQ ID NO: 34, 30 and 3; and SEQ ID NO: 35, 30 and 3; preferably, LCDR1, The amino acid sequences of LCDR2 and LCDR3 are shown in SEQ ID NOs: 4, 5 and 6, respectively;
    所述轻链可变区含有选自以下组的LCDR1、LCDR2和LCDR3：SEQ ID NO:112、5、113；SEQ ID NO:114、5、115；SEQ ID NO:116、5、117；SEQ ID NO:118、5、119；SEQ ID NO:120、5、121；SEQ ID NO:122、5、123；SEQ ID NO:124、5、125；SEQ ID NO:126、5、127；SEQ ID NO:128、5、129；SEQ ID NO:130、5、131；SEQ ID NO:132、5、133；SEQ ID NO:134、5、135；SEQ ID NO:136、5、137；SEQ ID NO:138、5、139；SEQ ID NO:140、5、141；SEQ ID NO:142、5、143；SEQ ID NO:36、5、37；SEQ ID NO:38、5、39；SEQ ID NO:40、5、41；SEQ ID NO:42、5、43；SEQ ID NO:44、5、45；SEQ ID NO:46、5、47；SEQ ID NO:48、5、49；SEQ ID NO:50、5、51；SEQ ID NO:52、5、53；SEQ ID NO:54、5、55；和SEQ ID NO:56、5、57；优选地，所述重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:1、2和3所示。The light chain variable region contains LCDR1, LCDR2 and LCDR3 selected from the group consisting of: SEQ ID NO: 112, 5, 113; SEQ ID NO: 114, 5, 115; SEQ ID NO: 116, 5, 117; SEQ ID NO: 116, 5, 117; ID NO: 118, 5, 119; SEQ ID NO: 120, 5, 121; SEQ ID NO: 122, 5, 123; SEQ ID NO: 124, 5, 125; SEQ ID NO: 126, 5, 127; ID NO: 128, 5, 129; SEQ ID NO: 130, 5, 131; SEQ ID NO: 132, 5, 133; SEQ ID NO: 134, 5, 135; SEQ ID NO: 136, 5, 137; ID NO: 138, 5, 139; SEQ ID NO: 140, 5, 141; SEQ ID NO: 142, 5, 143; SEQ ID NO: 36, 5, 37; SEQ ID NO: 38, 5, 39; ID NO: 40, 5, 41; SEQ ID NO: 42, 5, 43; SEQ ID NO: 44, 5, 45; SEQ ID NO: 46, 5, 47; SEQ ID NO: 48, 5, 49; ID NO: 50, 5, 51; SEQ ID NO: 52, 5, 53; SEQ ID NO: 54, 5, 55; and SEQ ID NO: 56, 5, 57; preferably, the heavy chain variable region The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1, 2 and 3, respectively.
  4. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段 包含重链可变区和/或轻链可变区，其中：The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, wherein:
    所述重链可变区包含：The heavy chain variable region comprises:
    (Ⅰ)氨基酸序列分别如SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或(I) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or
    (Ⅱ)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或(II) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 28, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; or
    (Ⅲ)氨基酸序列分别如SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或(III) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or
    (Ⅳ)氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或(IV) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or
    (Ⅴ)氨基酸序列分别如SEQ ID NO32、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或(v) HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO32, SEQ ID NO:30 and SEQ ID NO:3, respectively; or
    (Ⅵ)氨基酸序列分别如SEQ ID NO:33、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或(VI) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or
    (Ⅶ)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或(VII) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 34, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; or
    (Ⅷ)氨基酸序列分别如SEQ ID NO:35、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；(VIII) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 35, SEQ ID NO: 30 and SEQ ID NO: 3, respectively;
    所述轻链可变区包含：The light chain variable region comprises:
    (Ⅰ)氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；或(I) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
    (Ⅱ)氨基酸序列分别如SEQ ID NO:38、SEQ ID NO:5和SEQ ID NO:39所示的LCDR1、LCDR2和LCDR3；或(II) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 38, SEQ ID NO: 5 and SEQ ID NO: 39, respectively; or
    (Ⅲ)氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:5和SEQ ID NO:41所示的LCDR1、LCDR2和LCDR3；或(III) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:40, SEQ ID NO:5 and SEQ ID NO:41, respectively; or
    (Ⅳ)氨基酸序列分别如SEQ ID NO:42、SEQ ID NO:5和SEQ ID NO:43所示的LCDR1、LCDR2和LCDR3；或(IV) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:42, SEQ ID NO:5 and SEQ ID NO:43, respectively; or
    (Ⅴ)氨基酸序列分别如SEQ ID NO:44、SEQ ID NO:5和SEQ ID NO:45所示的LCDR1、LCDR2和LCDR3；或(v) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:44, SEQ ID NO:5 and SEQ ID NO:45, respectively; or
    (Ⅵ)氨基酸序列分别如SEQ ID NO:46、SEQ ID NO:5和SEQ ID NO:47所示的LCDR1、LCDR2和LCDR3；或(VI) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:46, SEQ ID NO:5 and SEQ ID NO:47, respectively; or
    (Ⅶ)氨基酸序列分别如SEQ ID NO:48、SEQ ID NO:5和SEQ ID NO:49所示的LCDR1、LCDR2和LCDR3；或(VII) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 48, SEQ ID NO: 5 and SEQ ID NO: 49, respectively; or
    (Ⅷ)氨基酸序列分别如SEQ ID NO:50、SEQ ID NO:5和SEQ ID NO:51所示的LCDR1、LCDR2和LCDR3；或(VIII) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:50, SEQ ID NO:5 and SEQ ID NO:51, respectively; or
    (Ⅸ)氨基酸序列分别如SEQ ID NO:52、SEQ ID NO:5和SEQ ID NO:53所示的LCDR1、LCDR2和LCDR3；或(IX) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:52, SEQ ID NO:5 and SEQ ID NO:53, respectively; or
    (Ⅹ)氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(X) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
    (Ⅺ)氨基酸序列分别如SEQ ID NO:56、SEQ ID NO:5和SEQ ID NO:57所示的LCDR1、LCDR2和LCDR3。(XI) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:56, SEQ ID NO:5 and SEQ ID NO:57, respectively.
  5. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含：The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:
    (Ⅰ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(I) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
    (Ⅱ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(II) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 32, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
    (Ⅲ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(III) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
    (Ⅳ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:35、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:48、SEQ ID NO:5和SEQ ID NO:49所示的LCDR1、LCDR2和LCDR3；或(IV) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 35, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:48, SEQ ID NO:5 and SEQ ID NO:49, respectively; or
    (Ⅴ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3； 或(v) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences are respectively LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:36, SEQ ID NO:5 and SEQ ID NO:37; or
    (Ⅵ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；或(VI) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:32, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
    (Ⅶ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:33、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:5和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；或(VII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 5 and SEQ ID NO: 37, respectively; or
    (Ⅷ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:33、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或(VIII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or
    (Ⅸ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或(IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:34, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or
    (Ⅹ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:54、SEQ ID NO:5和SEQ ID NO:55所示的LCDR1、LCDR2和LCDR3；或(X) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54, SEQ ID NO:5 and SEQ ID NO:55, respectively; or
    (Ⅺ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:30和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:48、SEQ ID NO:5和SEQ ID NO:49所示的LCDR1、LCDR2和LCDR3。(XI) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:31, SEQ ID NO:30 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:48, SEQ ID NO:5 and SEQ ID NO:49, respectively.
  6. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区和轻链可变区：The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region:
    (Ⅰ)所述重链可变区包含如SEQ ID NO:59、60、61、63、64、68、72或73中任一项所示的氨基酸序列，或与SEQ ID NO:59、60、61、63、64、68、72或73中任一项所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83、84、85、86、88、89、90、93、94、95或98中任一项所示的氨基酸序列，或与SEQ ID NO:83、84、85、86、88、89、90、 93、94、95或98中任一项所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(I) The heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 or 73, or the amino acid sequence shown in any one of SEQ ID NOs: 59, 60 , 61, 63, 64, 68, 72 or 73 amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity; and the light chain can be The variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, 85, 86, 88, 89, 90, 93, 94, 95, or 98, or the same as SEQ ID NOs: 83, 84, 85 An amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any one of , 86, 88, 89, 90, 93, 94, 95 or 98; or
    (Ⅱ)所述重链可变区包含如SEQ ID NO:61所示的氨基酸序列，或与SEQ ID NO:61所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(II) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or has at least 95%, 96%, 97%, 98% or 99% with the amino acid sequence shown in SEQ ID NO: 61 an amino acid sequence of % sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 95, or has at least 95%, 96%, 97%, or at least 95%, 96%, 97 amino acid sequences of %, 98% or 99% sequence identity; or
    (Ⅲ)所述重链可变区包含如SEQ ID NO:64所示的氨基酸序列，或包含与SEQ ID NO:64所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或包含与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(III) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:64, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:64 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98%, or 99% amino acid sequence identity; or
    (Ⅳ)所述重链可变区包含如SEQ ID NO:59所示的氨基酸序列，或包含与SEQ ID NO:59所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或包含与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(IV) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:59, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:59 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
    (Ⅴ)所述重链可变区包含如SEQ ID NO:73所示的氨基酸序列，或包含与SEQ ID NO:73所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:90所示的氨基酸序列，或包含与SEQ ID NO:90所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(V) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:73 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 90 , 97%, 98% or 99% amino acid sequence identity; or
    (Ⅵ)所述重链可变区包含如SEQ ID NO:61所示的氨基酸序列，或包含与SEQ ID NO:61所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列，或包含与SEQ ID NO:83所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(VI) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:61 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
    (Ⅶ)所述重链可变区包含如SEQ ID NO:64所示的氨基酸序列，或包含与SEQ ID NO:64所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列，或包含与SEQ ID NO:83所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基 酸序列；或(VII) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:64, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:64 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
    (Ⅷ)所述重链可变区包含如SEQ ID NO:68所示的氨基酸序列，或包含与SEQ ID NO:68所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列，或包含与SEQ ID NO:83所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(VIII) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 68 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 83 , 97%, 98% or 99% amino acid sequence identity; or
    (Ⅸ)所述重链可变区包含如SEQ ID NO:68所示的氨基酸序列，或包含与SEQ ID NO:68所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列，或包含与SEQ ID NO:8所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(IX) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO: 68 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 8 , 97%, 98%, or 99% amino acid sequence identity; or
    (Ⅹ)所述重链可变区包含如SEQ ID NO:72所示的氨基酸序列，或包含与SEQ ID NO:72所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列，或包含与SEQ ID NO:8所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(X) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:72, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:72 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 8 , 97%, 98% or 99% amino acid sequence identity; or
    (Ⅺ)所述重链可变区包含如SEQ ID NO:63所示的氨基酸序列，或包含与SEQ ID NO:63所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列，或包含与SEQ ID NO:95所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(XI) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:63 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:95, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO:95 , 97%, 98% or 99% amino acid sequence identity; or
    (XII)所述重链可变区包含如SEQ ID NO:63所示的氨基酸序列，或包含与SEQ ID NO:63所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:90所示的氨基酸序列，或包含与SEQ ID NO:90所示的氨基酸序列具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(XII) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63, or comprises at least 95%, 96%, 97%, 98% or the amino acid sequence shown in SEQ ID NO:63 An amino acid sequence of 99% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90, or comprises at least 95%, 96% with the amino acid sequence shown in SEQ ID NO: 90 , 97%, 98%, or 99% amino acid sequence identity; or
    (XIII)所述重链可变区包含如SEQ ID NO:61、64或59所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:95所示的氨基酸序列；或所述重链可变区包含如SEQ ID NO:73所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:90所示的氨基酸序列；或所述重链可变区包含如SEQ ID NO:61、64或68所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:83所示的氨基酸序列；或所述重链可变区包含如SEQ  ID NO:68或72所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列；或(XIII) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, 64 or 59, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 95; or the The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:90; or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:90; The amino acid sequence set forth in SEQ ID NO: 61, 64 or 68, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: The amino acid sequence shown in 68 or 72, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8; or
    (XIV)所述重链可变区选自SEQ ID NO:58-73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区，优选选自SEQ ID NO:59、60、61、63、64、68、72和73中任一所示的重链可变区或与所述任一重链可变区具有至少95％、96％、97％、98％或99％序列同一性的重链可变区，和所述轻链可变区如SEQ ID NO:8所示；或(XIV) The heavy chain variable region is selected from the heavy chain variable region shown in any one of SEQ ID NOs: 58-73 or has at least 95%, 96%, 97% with any of the heavy chain variable regions , 98% or 99% sequence identity heavy chain variable regions, preferably selected from the heavy chain variable regions shown in any one of SEQ ID NOs: 59, 60, 61, 63, 64, 68, 72 and 73 or A heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said heavy chain variable regions, and said light chain variable region as set forth in SEQ ID NO:8 show; or
    (XV)所述轻链可变区选自SEQ ID NO:74-100中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变区，优选选自SEQ ID NO:83、84、85、86、88、89、90、93、94、95和98中任一所示的轻链可变区或与所述任一轻链可变区具有至少95％、96％、97％、98％或99％序列同一性的轻链可变区，和所述重链可变区如SEQ ID NO:7所示。(XV) The light chain variable region is selected from the light chain variable regions shown in any of SEQ ID NOs: 74-100 or has at least 95%, 96%, 97%, A light chain variable region of %, 98% or 99% sequence identity, preferably selected from any one of SEQ ID NOs: 83, 84, 85, 86, 88, 89, 90, 93, 94, 95 and 98 a light chain variable region or a light chain variable region having at least 95%, 96%, 97%, 98% or 99% sequence identity to any of said light chain variable regions, and said heavy chain variable region The region is shown in SEQ ID NO:7.
  7. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体包含重链和轻链：The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a heavy chain and a light chain:
    (Ⅰ)所述重链包含如SEQ ID NO:101所示的氨基酸序列，或与SEQ ID NO:101所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:102所示的氨基酸序列，或与SEQ ID NO:102所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(I) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 101, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 101 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 90%, 92 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
    (Ⅱ)所述重链包含如SEQ ID NO:103所示的氨基酸序列，或与SEQ ID NO:103所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:102所示的氨基酸序列，或与SEQ ID NO:102所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(II) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 103, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 103 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 90%, 92 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
    (III)所述重链包含如SEQ ID NO:101或103所示的氨基酸序列，和所述轻链包含如SEQ ID NO:102所示的氨基酸序列；或(III) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 101 or 103, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102; or
    (Ⅳ)所述重链包含如SEQ ID NO:148所示的氨基酸序列，或与SEQ ID NO:148所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:149所示的氨基酸序列，或与SEQ ID NO:149所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或(IV) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 148, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 148 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 149, or has at least 90%, 92% with the amino acid sequence shown in SEQ ID NO: 149 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences; or
    (Ⅴ)所述重链包含如SEQ ID NO:148所示的氨基酸序列，或与SEQ ID NO:148所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:150所示的氨基酸序列，或与SEQ ID NO:150所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。(V) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 148, or has at least 90%, 92%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO: 148 %, 98% or 99% amino acid sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 150, or has at least 90%, 92% with the amino acid sequence shown in SEQ ID NO: 150 Amino acid sequences of %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  8. 如权利要求1-7中任一项所述的抗体或其抗原结合片段，其中所述抗体为人源化抗体或全人抗体；所述抗原结合片段选自Fab、Fab'、Fab'-SH、Fv、scFv、F(ab')2、sdAb或双抗体。The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody is a humanized antibody or a fully human antibody; the antigen-binding fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, sdAb or diabody.
  9. 如权利要求1-8中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是任何IgG亚型，如IgG1、IgG2、IgG3或IgG4。The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof is of any IgG subtype, such as IgGl, IgG2, IgG3, or IgG4.
  10. 多核苷酸分子，其编码权利要求1-9中任一项所述的抗体或其抗原结合片段。A polynucleotide molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9.
  11. 表达载体，其包含权利要求10所述的多核苷酸分子，优选地，所述载体为真核表达载体。An expression vector comprising the polynucleotide molecule of claim 10, preferably, the vector is a eukaryotic expression vector.
  12. 宿主细胞，其中，所述宿主细胞：A host cell, wherein the host cell:
    (1)表达权利要求1-9中任一项所述的抗体或其抗原结合片段；或(1) expressing the antibody or antigen-binding fragment thereof of any one of claims 1-9; or
    (2)包含权利要求10所述的多核苷酸分子或权利要求11所述的表达载体；(2) comprising the polynucleotide molecule of claim 10 or the expression vector of claim 11;
    优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。Preferably, the host cells are eukaryotic cells, more preferably mammalian cells.
  13. 制备如权利要求1-9中任一项所述的抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养权利要求12所述的宿主细胞，以使其表达所述抗体或其抗原结合片段，并回收所表达的抗体或其抗原结合片段。A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1-9, the method comprising culturing the host of claim 12 under conditions suitable for expression of the antibody or antigen-binding fragment thereof cells to express the antibody or antigen-binding fragment thereof, and recover the expressed antibody or antigen-binding fragment thereof.
  14. 药物组合物，其含有权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求10所述的多核苷酸分子、权利要求11所述的表达载体和/或权利要求12所述的宿主细胞，和药学上可接受的载体或赋形剂。A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9, the polynucleotide molecule of claim 10, the expression vector of claim 11 and/or claim 12 The host cell, and a pharmaceutically acceptable carrier or excipient.
  15. 如权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求10所述的多核苷酸分子、权利要求11所述的表达载体、权利要求12所述的宿主细胞和/或权利要求14所述的药物组合物在制备治疗和/或预防SARS-CoV-2或其变体感染的药物中的用途；优选地，所述SARS-CoV-2变体包括阿尔法突变株、贝塔突变株、伽马突变株、德尔塔突变株、Epsilon突变株、Zeta突变株、Eta突变株、Theta突变株、Iota突变株、卡帕突变株、缪突变株和奥密克戎突变株中的至少一种；优选为奥密克戎突变株。The antibody or antigen-binding fragment thereof of any one of claims 1-9, the polynucleotide molecule of claim 10, the expression vector of claim 11, the host cell of claim 12, and/or Or the use of the pharmaceutical composition of claim 14 in the preparation of a medicine for the treatment and/or prevention of infection by SARS-CoV-2 or its variant; preferably, the SARS-CoV-2 variant comprises alpha mutants, Beta mutants, Gamma mutants, Delta mutants, Epsilon mutants, Zeta mutants, Eta mutants, Theta mutants, Iota mutants, Kappa mutants, Mutant mutants, and Omicron mutants At least one of ; preferably Omicron mutant.
  16. 一种检测试剂，其含有权利要求1-9中任一项所述的抗体或其抗原结合片段。A detection reagent comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9.
  17. 一种检测试纸或检测芯片，其包被有权利要求1-9中任一项所述的抗体或其抗原结合片段。A detection test paper or detection chip, which is coated with the antibody or its antigen-binding fragment according to any one of claims 1-9.
  18. 试剂盒，其含有权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求10所述的多核苷酸分子、权利要求11所述的表达载体、权利要求12所述的宿主细胞、权利要求14所述的药物组合物、权利要求16所述的检测试剂和/或权利要求17所述的检测试纸或芯片。A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9, the polynucleotide molecule of claim 10, the expression vector of claim 11, the The host cell, the pharmaceutical composition according to claim 14, the detection reagent according to claim 16 and/or the detection test strip or chip according to claim 17.
  19. 如权利要求14所述的药物组合物在制备诊断SARS-CoV-2或其变体感染的药物或药盒中的用途，或权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求10所述的多核苷酸分子、权利要求11所述的表达载体或权利要求12所述的宿主细胞在制备用于检测样品中是否存在SARS-CoV-2或其变体的检测试剂、检测试纸或检测芯片中的应用，或权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求10所述的多核苷酸分子、权利要求11所述的表达载体、权利要求12所述的宿主细胞、权利要求16所述的检测试剂和/或权利要求17所述的检测试纸或检测芯片在制备用于检测样品中是否存在SARS-CoV-2或其变体的试剂盒中的应用。Use of the pharmaceutical composition according to claim 14 in the preparation of a medicament or kit for diagnosing SARS-CoV-2 infection or a variant thereof, or the antibody or antigen binding thereof according to any one of claims 1-9 A fragment, the polynucleotide molecule of claim 10, the expression vector of claim 11, or the host cell of claim 12 prepared for detection of the presence or absence of SARS-CoV-2 or a variant thereof in a sample Reagents, detection strips or detection chips, or the antibody or antigen-binding fragment thereof according to any one of claims 1-9, the polynucleotide molecule according to claim 10, and the expression vector according to claim 11 , the host cell according to claim 12, the detection reagent according to claim 16 and/or the detection test strip or detection chip according to claim 17 are prepared for detecting the presence of SARS-CoV-2 or its variants in a sample application of the kit.
  20. 一种检测SARS-CoV-2或其变体在样品中的存在的方法，其包含使权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求16所述的检测试剂或权利要求17所述的检测试纸或检测芯片与所述样品接触，并检测是否存在所述抗体或其抗原结合片段与SARS-CoV-2或其变体CBD结合产生的结合物或结合信号。A method for detecting the presence of SARS-CoV-2 or a variant thereof in a sample, comprising making the antibody or antigen-binding fragment thereof of any one of claims 1-9, the detection reagent of claim 16 Or the detection test strip or detection chip of claim 17 is contacted with the sample, and it is detected whether there is a conjugate or a binding signal produced by the binding of the antibody or its antigen-binding fragment to SARS-CoV-2 or its variant CBD.
PCT/CN2022/080100 2021-03-10 2022-03-10 Sars-cov-2 antibody and application thereof WO2022188829A1 (en)

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