WO2022188829A1 - Anticorps anti-sars-cov-2 et son application - Google Patents

Anticorps anti-sars-cov-2 et son application 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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amino acid
acid sequence
chain variable
variable region
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PCT/CN2022/080100
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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/fr

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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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    • 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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    • 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

L'invention concerne un anticorps anti-SARS-CoV-2 et une application de celui-ci. L'invention concerne un anticorps ou un fragment de liaison à l'antigène de celui-ci qui se lie spécifiquement à un domaine de liaison au récepteur (RBD) du SARS-CoV-2 ou à un variant de celui-ci. L'invention concerne également une molécule d'acide nucléique codant pour l'anticorps ou son fragment de liaison à l'antigène, un vecteur et une cellule hôte pour exprimer l'anticorps ou le fragment de liaison à l'antigène de celui-ci, ainsi qu'un procédé thérapeutique et diagnostique et une application de l'anticorps ou du fragment de liaison à l'antigène de celui-ci.
PCT/CN2022/080100 2021-03-10 2022-03-10 Anticorps anti-sars-cov-2 et son application WO2022188829A1 (fr)

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