Monoclonal antibody against novel coronavirus and application thereof

Abstract

The present document relates to the field of immunology and the field of molecular virology, in particular the field of diagnosis, prevention and treatment of the novel coronavirus. In particular, the present document relates to monoclonal antibodies against the novel coronavirus, as well as compositions (e.g. diagnostic and therapeutic agents) comprising said antibodies. Furthermore, the present document also relates to uses of said antibodies. The antibodies described herein can be used for the diagnosis, prevention and / or treatment of an infection with the novel coronavirus and / or a disease caused by said infection (e.g. COVID-19).

Description

Technical Field

[0001] This article relates to the fields of immunology and molecular virology, particularly to the diagnosis, prevention, and treatment of the novel coronavirus. Specifically, this article relates to antibodies against the novel coronavirus, and compositions comprising said antibodies (e.g., diagnostic and therapeutic agents). Furthermore, this article also relates to the uses of said antibodies. The antibodies described herein can be used for the diagnosis, prevention, and / or treatment of infection with the novel coronavirus and / or the disease caused by said infection (e.g., COVID-19). Background Technology

[0002] The novel coronavirus SARS-CoV-2 is the pathogen that causes COVID-19. It is a single-stranded RNA virus, belonging to the same family Coronaviridae as the severe acute respiratory syndrome coronavirus (SARS-CoV) that caused the 2002-2003 outbreak and the Middle East respiratory syndrome coronavirus (MERS-CoV) that caused the 2012 outbreak. Coronavirus particles are round or oval, and also pleomorphic, with a diameter of 50-200 nm, making them relatively large viruses. Coronaviruses are enveloped viruses, with a lipid envelope surrounding the viral capsid, on which a wide spike protein (S protein, SEQ ID No: 1460) is arranged, resembling a sun halo. Studies have confirmed that the surface of the novel coronavirus SARS-CoV-2 possesses the S protein, which can bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2) molecule through its receptor-binding domain (RBD) during viral infection, thereby initiating the fusion of the viral membrane with the host cell membrane, leading to viral infection of the host cell.

[0003] To date, neutralizing antibodies have proven to be an effective treatment for viral diseases. Generally, when B lymphocytes in a patient's body are stimulated by antigens, they become activated, transforming and differentiating into various cell types and producing antibodies. Existing research reports that antibodies against the novel coronavirus are present in the peripheral blood of recovered COVID-19 patients; these antibodies are produced and secreted by activated B cells. However, various B cells exist in the plasma of recovered patients, and the binding activity and neutralizing titer of antibodies produced by different B cells vary. To date, no studies have reported antibodies against the novel coronavirus with high binding activity and / or moderate neutralizing activity.

[0004] Therefore, there is a need to develop antibodies with high binding activity and / or high neutralizing activity against the novel coronavirus SARS-CoV-2 in order to provide an effective means of diagnosing, preventing and / or treating novel coronavirus infection. Summary of the Invention

[0005] The technical solutions provided in this paper meet the above requirements and offer relevant advantages.

[0006] On the one hand, this article provides an antigen-binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3; wherein the VH CDR3 comprises a sequence selected from SEQ ID NO: 1-360 and 2971-3005 or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1-360 and 2971-3005, and / or wherein the VL CDR3 comprises a sequence selected from SEQ ID NO: 361-720 and 3076-3110 or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 361-720 and 3076-3110.

[0007] In some embodiments, the antigen-binding unit binds to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein with an equilibrium dissociation constant (KD) of less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, or less than 0.01 nM.

[0008] In some embodiments, the antigen-binding unit neutralizes the novel coronavirus (SARS-CoV-2) with an IC50 of less than 20 μg / ml, less than 10 μg / ml, less than 9 μg / ml, less than 8 μg / ml, less than 7 μg / ml, less than 6 μg / ml, less than 5 μg / ml, less than 4 μg / ml, less than 3 μg / ml, less than 2 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.25 μg / ml, less than 0.2 μg / ml, less than 0.1 μg / ml, less than 0.05 μg / ml, or less than 0.001 μg / ml.

[0009] In some embodiments, the VH CDR1 of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1461-1820 and 2901-2935. In some embodiments, the VH CDR1 of the antigen-binding unit comprises a sequence comprising 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1461-1820 and 2901-2935. In some embodiments, the VH CDR1 of the antigen-binding unit comprises the same sequence as the CDR1 contained in SEQ ID NO: 721-1080 and 3111-3145.

[0010] In some embodiments, the VH CDR2 of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1821-2180 and 2936-2970. In some embodiments, the VH CDR2 of the antigen-binding unit comprises a sequence comprising 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1821-2180 and 2936-2970. In some embodiments, the VH CDR2 of the antigen-binding unit comprises the same sequence as the CDR2 contained in SEQ ID NO: 721-1080 and 3111-3145.

[0011] In some embodiments, the VL CDR1 of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 2181-2540 and 3006-3040, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 2181-2540 and 3006-3040. In some embodiments, the VL CDR1 of the antigen-binding unit comprises a sequence comprising 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 2181-2540 and 3006-3040. In some embodiments, the VL CDR1 of the antigen-binding unit comprises the same sequence as the CDR1 contained in SEQ ID NO: 1081-1440 and 3146-3180.

[0012] In some embodiments, the VL CDR2 of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 2541-2900 and 3041-3075. In some embodiments, the VL CDR2 of the antigen-binding unit comprises a sequence comprising 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 2541-2900 and 3041-3075. In some embodiments, the VL CDR2 of the antigen-binding unit comprises the same sequence as the CDR2 contained in SEQ ID NO: 1081-1440 and 3146-3180.

[0013] In some embodiments, the VH of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 721-1080 and 3111-3145. In some embodiments, the VH of the antigen-binding unit comprises a sequence comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 721-1080 and 3111-3145. In some embodiments, the VH of the antigen-binding unit comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 721-1080 and 3111-3145.

[0014] In some embodiments, the VL of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1081-1440 and 3146-3180. In some embodiments, the VL of the antigen-binding unit comprises a sequence comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1081-1440 and 3146-3180. In some embodiments, the VL of the antigen-binding unit comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180.

[0015] On the other hand, this article provides an antigen-binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VLCDR2, and VL CDR3; wherein the VH CDR1 comprises a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935, a sequence containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1461-1820 and 2901-2935, or a sequence identical to the CDR1 contained in SEQ ID NO: 721-1080 and 3111-3145, wherein the VH CDR2 comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970, and a sequence containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1461-1820 and 2901-2935, or a sequence identical to the CDR1 contained in SEQ ID NO: 721-1080 and 3111-3145, and wherein the VH CDR2 comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970, and a sequence identical to the CDR1 contained in SEQ ID NO: 721-1080 and 3111-3145 ... Compared to SEQ ID NOs 1821-2180 and 2936-2970, the VH CDR3 contains a sequence with one or more amino acid additions, deletions, or substitutions, or is identical to the CDR2 contained in SEQ ID NOs 721-1080 and 3111-3145; and / or the VL CDR1 contains a sequence selected from SEQ ID NOs 2181-2540 and 3006-3040, a sequence with one or more amino acid additions, deletions, or substitutions compared to SEQ ID NOs 2181-2540 and 3006-3040, or is identical to the CDR2 contained in SEQ ID NOs 721-1080 and 3111-3145; and / or the VL CDR1 contains a sequence selected from SEQ ID NOs 2181-2540 and 3006-3040, a sequence with one or more amino acid additions, deletions, or substitutions compared to SEQ ID NOs 2181-2540 and 3006-3040; or is identical to the CDR2 contained in SEQ ID NOs 721-1080 and 3111-3145. The VL CDR2 comprises a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075, a sequence containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 2541-2900 and 3041-3075, or a sequence identical to the CDR2 contained in SEQ ID NO: 1081-1440 and 3146-3180. The VL CDR3 comprises a sequence selected from SEQ ID NO: 361-720 and 3076-3110, a sequence containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 361-720 and 3076-3110, or a sequence identical to the CDR3 contained in SEQ ID NO: 1081-1440 and 3146-3180.

[0016] On the other hand, this article provides an antigen-binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VLCDR2, and VL CDR3; wherein VH CDR1 comprises a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935 or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1461-1820 and 2901-2935, wherein VH CDR2 comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970 or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1821-2180 and 2936-2970, and wherein VH CDR3 comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970, wherein VH CDR3 comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970, wherein VH CDR3 comprises a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1821-2180 and 2936-2970, and wherein VH CDR3 comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970, wherein VH CDR3 comprises a sequence comprising one or more amino acid additions, deletions, or substitutions compared to ... Sequences selected from SEQ ID NO: 1-360 and 2971-3005, or sequences containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1-360 and 2971-3005; and / or wherein the VL CDR1 contains a sequence selected from SEQ ID NO: 2181-2540 and 3006-3040, or a sequence containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 2181-2540 and 3006-3040; the VL CDR2 contains a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075, or a sequence containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 2541-2900 and 3041-3075; the VL CDR3 contains a sequence selected from SEQ ID NO: 361-720 and 3076-3110, or a sequence containing one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1-360 and 2971-3005; NO: 361-720 contains a sequence with one or more added, deleted, or substituted amino acids compared to 3076-3110.

[0017] In some embodiments, the VH of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 721-1080 and 3111-3145. In some embodiments, the VH of the antigen-binding unit comprises a sequence comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 721-1080 and 3111-3145. In some embodiments, the VH of the antigen-binding unit comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 721-1080 and 3111-3145.

[0018] In some embodiments, the VL of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180, or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1081-1440 and 3146-3180. In some embodiments, the VL of the antigen-binding unit comprises a sequence comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1081-1440 and 3146-3180. In some embodiments, the VL of the antigen-binding unit comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180.

[0019] In some embodiments, the antigen-binding unit binds to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein with an equilibrium dissociation constant (KD) of less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, or less than 0.01 nM.

[0020] In some embodiments, the antigen-binding unit neutralizes the novel coronavirus (SARS-CoV-2) with an IC50 of less than 20 μg / ml, less than 10 μg / ml, less than 9 μg / ml, less than 8 μg / ml, less than 7 μg / ml, less than 6 μg / ml, less than 5 μg / ml, less than 4 μg / ml, less than 3 μg / ml, less than 2 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.25 μg / ml, less than 0.2 μg / ml, less than 0.1 μg / ml, less than 0.05 μg / ml, or less than 0.001 μg / ml.

[0021] On the other hand, this document provides an antigen-binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 721-1080 and 3111-3145, and / or wherein the VL comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180.

[0022] In some embodiments, the antigen-binding unit binds to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein with an equilibrium dissociation constant (KD) of less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, or less than 0.01 nM.

[0023] In some embodiments, the antigen-binding unit neutralizes the novel coronavirus (SARS-CoV-2) with an IC50 of less than 20 μg / ml, less than 10 μg / ml, less than 9 μg / ml, less than 8 μg / ml, less than 7 μg / ml, less than 6 μg / ml, less than 5 μg / ml, less than 4 μg / ml, less than 3 μg / ml, less than 2 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.25 μg / ml, less than 0.2 μg / ml, less than 0.1 μg / ml, less than 0.05 μg / ml, or less than 0.001 μg / ml.

[0024] In some embodiments, the antigen-binding unit further comprises a heavy chain constant region (CH). In some embodiments, the CH of the antigen-binding unit comprises a sequence of SEQ ID NO: 1457 or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1457. In some embodiments, the CH of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 1457. In some embodiments, the CH of the antigen-binding unit comprises a sequence comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid addition, deletion, or substitution compared to SEQ ID NO: 1457. In some embodiments, the CH of the antigen-binding unit comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 1457.

[0025] In some embodiments, the antigen-binding unit further comprises a light chain constant region (CL). In some embodiments, the CL of the antigen-binding unit comprises the sequence of SEQ ID NO: 1458 or a sequence comprising one or more amino acid additions, deletions, or substitutions compared to SEQ ID NO: 1458. In some embodiments, the CL of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 1458. In some embodiments, the CL of the antigen-binding unit comprises a sequence comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid addition, deletion, or substitution compared to SEQ ID NO: 1458. In some embodiments, the CL of the antigen-binding unit comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with a sequence selected from SEQ ID NO: 1458.

[0026] In another respect, this article provides isolated nucleic acid molecules that encode antigen-binding units as defined above.

[0027] In another aspect, this document provides a vector comprising isolated nucleic acid molecules as defined above. The vector described herein can be a cloning vector or an expression vector. In some embodiments, the vector described herein is, for example, a plasmid, a granule, or a bacteriophage, etc.

[0028] In another aspect, host cells containing the isolated nucleic acid molecules or vectors described herein are also provided. Such host cells include, but are not limited to, prokaryotic cells such as *E. coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The cells described herein can also be cell lines, such as HEK293 cells.

[0029] In another aspect, a method for preparing the antigen-binding unit described herein is also provided, comprising culturing the host cells described herein under suitable conditions and recovering the antigen-binding unit described herein from the cell culture.

[0030] In another aspect, this article provides a composition comprising an antigen-binding unit, an isolated nucleic acid molecule, a vector, or a host cell as described above.

[0031] In another aspect, this document provides a kit comprising the antigen-binding unit described herein. In some embodiments, the antigen-binding unit described herein further includes a detectable label. In some embodiments, the kit further includes a second antibody that specifically recognizes the antigen-binding unit described herein. Preferably, the second antibody further includes a detectable label. Such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (e.g., horseradish peroxidase).

[0032] In another aspect, this document provides a method for detecting the presence or level of the novel coronavirus or its S protein or the RBD of the S protein in a sample, comprising using an antigen-binding unit as described herein. In some embodiments, the antigen-binding unit described herein further includes a detectable label. In another preferred embodiment, the method further includes detecting the antigen-binding unit described herein using a second antibody carrying the detectable label. The method can be used for diagnostic purposes (e.g., the sample is from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not from a patient).

[0033] In another aspect, this document provides a method for diagnosing whether a subject is infected with the novel coronavirus, comprising: detecting the presence of the novel coronavirus or its S protein or the RBD of the S protein in a sample from the subject using the antigen-binding unit described herein. In some embodiments, the antigen-binding unit described herein further includes a detectable marker. In another preferred embodiment, the method further includes detecting the antigen-binding unit described herein using a second antibody carrying the detectable marker.

[0034] In another aspect, the use of the antigen-binding unit described herein in the preparation of a kit for detecting the presence or level of the novel coronavirus or its S protein or the RBD of the S protein in a sample, or for diagnosing whether a subject is infected with the novel coronavirus.

[0035] In another aspect, this article provides a pharmaceutical composition comprising the antigen-binding unit described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0036] In another aspect, this document provides a method for neutralizing the virulence of the novel coronavirus in a sample, comprising contacting a sample containing the novel coronavirus with the antigen-binding unit described herein. Such methods can be used for therapeutic purposes or non-therapeutic purposes (e.g., the sample is a cell sample, not a patient or a sample from a patient).

[0037] In another aspect, the use of the antigen-binding unit described herein for preparing a medicament for neutralizing the virulence of a novel coronavirus in a sample is provided. In yet another aspect, the antigen-binding unit as described above is provided for neutralizing the virulence of a novel coronavirus in a sample.

[0038] In another aspect, the use of the antigen-binding unit described herein in the preparation of a pharmaceutical composition for the prevention or treatment of a subject with novel coronavirus infection or a disease associated with novel coronavirus infection (e.g., COVID-19). In another aspect, the antigen-binding unit as described above is provided herein for the prevention or treatment of a subject with novel coronavirus infection or a disease associated with novel coronavirus infection (e.g., COVID-19).

[0039] In another aspect, this document provides a method for preventing or treating a subject with novel coronavirus infection or a disease related to novel coronavirus infection (e.g., COVID-19), comprising administering to a subject in need a preventive or therapeutically effective amount of the antigen-binding unit described herein, or the pharmaceutical composition described herein.

[0040] In some implementations, the subject is a mammal, such as a human.

[0041] The antigen-binding unit or the pharmaceutical composition described herein may be administered to a subject via any appropriate route of administration. Such routes of administration include, but are not limited to, oral, oral, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

[0042] The pharmaceuticals and pharmaceutical compositions provided herein can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., antiviral drugs such as favipiravir, remdesivir, and interferon). In some embodiments, the pharmaceutical compositions also contain pharmaceutically acceptable carriers and / or excipients.

[0043] In another aspect, this document provides conjugates comprising an antigen-binding unit as described above, wherein the antigen-binding unit is conjugated to a chemically functional moiety. In some embodiments, the chemically functional moiety is selected from radioactive isotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compound substrate cofactors, and inhibitors. Attached Figure Description

[0044] Figure 1A-1C The SDS-PAGE results of antigen-binding units ABU-174, ABU-175, and ABU190 are shown as an example.

[0045] Figure 2A-2E The results of the assay for the affinity of antigen-binding units ABU-174(A), ABU-175(B), ABU190(C), ABU297(D), and ABU367(E) for the S protein are shown exemplarily.

[0046] Figure 3A-3C The results of the assay of neutralizing and inhibitory activity of antigen-binding units ABU-174(A), ABU-175(B), and ABU190(C) against SARS-CoV-2 pseudovirus are shown exemplarily.

[0047] Figure 4 The results of the CPE assay for the neutralizing and inhibitory activity of the ABU-175 antibody against SARS-CoV-2 are shown as an example.

[0048] Figure 5 The PRNT assay results of the neutralizing and inhibitory activity of antigen-binding units ABU-174, ABU-175, and ABU190 against SARS-CoV-2 virus are shown as an example. Detailed Implementation Plan

[0049] While preferred embodiments described herein have been shown and described, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and alternatives will now occur to those skilled in the art without departing from the description herein. It should be understood that various alternatives to the embodiments described herein may be employed in carrying out the practice described herein. The scope of the description is intended to be defined by the following claims, and therefore covers the methods and structures within the scope of these claims and their equivalents.

[0050] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of this range (accurate to one-tenth of the lower limit unit, unless the context explicitly specifies otherwise) and any other indicated or intermediate values ​​within the range are included in this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also included in this invention, except for any specifically excluded limits within the range. When the range contains one or two limits, the range excluding any one or two included limits is also included in this invention.

[0051] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to an amino acid polymer of any length. The polymer may be linear, cyclic, or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also includes modified amino acid polymers, such modifications are achieved through processes such as sulfation, glycosylation, esterification, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic treatment, phosphorylation, isopentenylation, racemization, selenization, transfer RNA-mediated addition of amino acids to proteins (e.g., arginination), pervasive proteination, or any other manipulation, such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptides. A polypeptide or amino acid sequence “derived” from a specified protein refers to the origin of the polypeptide. Preferably, the polypeptide has an amino acid sequence substantially identical to, or a portion thereof, the amino acid sequence of the polypeptide encoded in the sequence, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or is immunologically identifiable by the polypeptide encoded in the sequence. The term also includes polypeptides expressed from a specified nucleic acid sequence. As used herein, the term “domain” refers to a portion of a protein that is physically or functionally distinct from other portions of that protein or peptide. Physically defined domains include highly hydrophobic or hydrophilic amino acid sequences, such as those that are membrane-bound or cytoplasm-bound. Domains can also be defined by, for example, internal homology caused by gene replication. Functionally defined domains have different biological functions. For example, an antigen-binding domain refers to the portion of an antigen-binding unit or antibody that binds to an antigen. Functionally defined domains do not need to be encoded by a continuous amino acid sequence, and functionally defined domains may contain one or more physically defined domains.

[0052] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptides. Standard single-letter or three-letter codes are used to designate amino acids. In this document, amino acids are generally represented by single-letter and three-letter abbreviations well known in the art. For example, alanine may be represented by A or Ala.

[0053] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The assignment of amino acids to the various regions or domains follows the definitions in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883, or IMGT (ImMunoGenTics) (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)). Unless otherwise stated, the CDRs in the VH and VL of the antibodies in this application are based on the definitions of the IMGT coding system. In the Kabat numbering system, the CDR amino acid residues in VH are numbered 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3); the CDR amino acid residues in VL are numbered 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3).Under the Chothia system, the CDR amino acid residues in VH are numbered 26-32 (CDR1), 52-56 (CDR2), and 95-102 (CDR3); and the amino acid residues in VL are numbered 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). Under the IMGT numbering system, the CDR amino acid residues in VH are approximately numbered 26-33 (CDR1), 51-56 (CDR2), and 93-102 (CDR3); and the CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-51 (CDR2), and 89-97 (CDR3) (as disclosed at https: / / www.novoprolabs.com / tools / cdr).

[0054] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0055] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabolas, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv) in which the VL and VH domains enable them to pair as linkers to form monovalent molecules for a single polypeptide chain (see, for example, Bird et al., Science 242: 423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA). 85: 5879 5883 (1988)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used as described herein are from Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J.Mol.Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol. Description.

[0056] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, in which the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444 6448 (1993), and Poljak RJ et al., Structure 2: 1121 1123 (1994)).

[0057] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody fragments described herein) can be obtained from a given antibody (e.g., the antibody fragments described herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0058] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0059] In this document, unless the context clearly indicates otherwise, the term "antigen-binding unit" includes antibodies and their antigen-binding fragments as defined above.

[0060] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see Journal of Virological Methods, 2009, 158(1-2): 171-179).

[0061] As used in this article, "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence (e.g., the ability to infect cells) of a target virus.

[0062] As used herein, in the case of peptides, a “sequence” is the order of amino acids in the peptide from the amino terminus to the carboxyl terminus, wherein adjacent residues in the sequence are continuous in the primary structure of the peptide. A sequence can also be a linear sequence of a peptide known to contain additional residues in one or both directions.

[0063] As used herein, “identity,” “homology,” or “sequence identity” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using programs such as Emboss Needle or BestFit to determine sequence identity, similarity, or homology between two different amino acid sequences, default settings can be used, or an appropriate scoring matrix, such as blosum45 or blosum80, can be selected to optimize the identity, similarity, or homology score. Preferably, homologous polynucleotides are those that hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to these sequences. When performing optimal alignment of sequences of comparable length, homologous polypeptides preferably have at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity.

[0064] For the purposes of this document, “sequence identity percentage (%)” is defined as the percentage of amino acid residues in the query sequence that are identical to amino acid residues in the second, reference polypeptide sequence or portions thereof, after aligning the sequences and introducing gaps where necessary to obtain the maximum sequence identity percentage, and without considering any conserved substitutions as part of sequence identity. Alignment aimed at determining the amino acid sequence identity percentage can be performed in various ways within the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment over the full length of the sequences being compared. The identity percentage can be measured over the entire length of the defined polypeptide sequence, or over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, such as a fragment of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, or at least 200 consecutive residues. These lengths are merely exemplary, and it should be understood that any segment length supported by the sequences shown in the tables, figures, or sequence listings herein can be used to describe the length on which a percentage of identity can be measured.

[0065] The antigen-binding unit described herein may have one or more modifications relative to a reference sequence. These modifications may be deletions, insertions, or additions, or substitutions or replacements of amino acid residues. "Deletion" refers to an amino acid sequence change resulting from the absence of one or more amino acid residues. "Insertion" or "addition" refers to an amino acid sequence change resulting from the addition of one or more amino acid residues compared to the reference sequence. "Substitution" or "replacement" refers to the substitution of one or more amino acids by a different amino acid. In this document, mutations in the antigen-binding unit relative to the reference sequence can be determined by comparing the antigen-binding unit to the reference sequence. Optimal alignment of the sequences used for comparison can be performed according to any method known in the art.

[0066] As used herein, the term "antigen" refers to a substance that is recognized and specifically bound by an antigen-binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides, and lipids; portions thereof, and combinations thereof. Non-limiting exemplary antigens include proteins from coronaviruses such as SARS-CoV-2, and other homologs thereof.

[0067] As used herein, the term "isolated" means separated from cellular and other components, which are normally associated with polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof in nature. Those skilled in the art will recognize that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not require "isolation" to distinguish them from their naturally occurring counterparts. Furthermore, "concentrated," "isolated," or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are distinguishable from their naturally occurring counterparts because the molecular concentration or number per unit volume is greater than ("concentrated") or less than that of their naturally occurring counterparts ("isolated"). Enrichment can be measured based on absolute amounts, such as the weight of a solution per unit volume, or it can be measured relative to a second, potentially interfering substance present in the source mixture.

[0068] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably. They refer to polymeric forms of nucleotides (whether deoxyribonucleotides or ribonucleotides) or their analogues of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci identified from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, oligonucleotides, or synthetic DNA. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure can be conferred before or after polymer assembly, if present. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components.

[0069] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of various combinations of cloning, restriction enzyme digestion and / or ligation steps, as well as other procedures that produce constructs different from those found in nature.

[0070] The terms “gene” or “gene segment” are used interchangeably in this document. They refer to a polynucleotide containing at least one open reading frame (OPF) that encodes a specific protein after transcription and translation. A gene or gene segment may be genomic, cDNA, or synthetic, provided that the polynucleotide contains at least one OPF that may cover an entire coding region or a segment thereof.

[0071] The terms "operably linked" or "effectively linked" refer to juxtaposition, where the relationship between the components described in this way allows them to function in their intended manner. For example, if a promoter sequence promotes transcription of a coding sequence, then the promoter sequence is operably linked to that coding sequence.

[0072] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA, and / or the transcribed mRNA (also called “transcription”) is subsequently translated into peptides, polypeptides, or proteins. Transcriptions and encoded polypeptides are collectively referred to as gene products. If the polynucleotides are derived from genomic DNA, expression can include the splicing of mRNA in eukaryotic cells.

[0073] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0074] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0075] As used herein, the term "biological sample" encompasses a variety of sample types obtained from a living organism and that can be used in diagnostic or monitoring tests. This term includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells derived therefrom and their progeny. This term includes samples that have been processed in any way after acquisition, such as by treatment with reagents, dissolution, or enrichment for certain components. This term includes clinical samples and also includes cells in cell cultures, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0076] As used herein, the terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject, particularly a human, to whom diagnosis, treatment, or therapy is desired.

[0077] As used herein, the terms “treatment,” “treatment,” etc., are used generically to refer to achieving the desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete stabilization or cure of the disease and / or adverse reactions attributable to the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals such as mice, rats, rabbits, pigs, primates, including humans and other apes, particularly humans, and the term includes: (a) preventing the occurrence of a disease or symptoms in subjects who may be susceptible to the disease or symptoms but have not yet been diagnosed; (b) suppressing disease symptoms; (c) halting the development of the disease; (d) alleviating disease symptoms; (e) causing the remission of the disease or symptoms; or any combination thereof. As used herein, the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binds to the antigen.

[0078] As used herein, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. In this paper, KD is defined as the ratio of two kinetic rate constants Ka / Kd, where "Ka" refers to the rate constant for antibody binding to the antigen, and "Kd" refers to the rate constant for antibody dissociation from the antibody / antigen complex. The smaller the equilibrium dissociation constant KD, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies exhibit a dissociation rate less than approximately 10-1. -5 The dissociation equilibrium constant (KD) of M binds to the antigen. The specific binding properties between the two molecules can be determined using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.

[0079] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing viral infection of cells and / or the maturation and / or release of viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection. In some embodiments, said neutralizing activity is achieved through an IC50 of the antibody or antibody fragment inhibiting the virus. 50 The term "half-maximum inhibitory concentration" (IC50) indicates the level of inhibition. 50 IC50 is a measure of the efficacy of drugs, such as antibodies, in inhibiting biological or biochemical functions (e.g., viral activity). In this article, IC50 is used to measure... 50 The neutralization inhibition rate of the antigen-binding fragment against virus-infected cells (e.g., pseudoviruses or eukaryotes) was calculated using the Reed-Muench method. This paper presents an antigen-binding unit capable of specifically recognizing and targeting the S protein of the novel coronavirus, particularly the receptor-binding domain (RBD) of the S protein, and exhibiting highly efficient viral neutralization capabilities. Therefore, the antigen-binding unit described herein is particularly suitable for the diagnosis, prevention, and treatment of novel coronavirus infection or diseases related to novel coronavirus infection (e.g., COVID-19).

[0080] Antigen binding unit

[0081] On the one hand, the antigen-binding unit described herein includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region includes VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region includes VL CDR1, VL CDR2 and VL CDR3.

[0082] The VH of the antigen-binding unit described herein may contain a sequence selected from SEQ ID NO.: 721-1080 and 3111-3145, a sequence that contains one or more amino acid additions, deletions, or substitutions compared to the sequence selected from SEQ ID NO.: 721-1080 and 3111-3145, or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 721-1080 and 3111-3145. When the VH of the antigen-binding unit described herein differs from the reference polypeptide sequence in terms of amino acid additions, deletions, or substitutions, the VH of the antigen-binding unit described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions, deletions, or substitutions compared to the reference polypeptide sequence. When the VH of the antigen-binding unit described herein differs from the reference polypeptide sequence in terms of amino acid additions, deletions, or substitutions, the VH of the antigen-binding unit described herein may contain more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions, deletions, or substitutions compared to the reference polypeptide sequence. When the VH of the antigen-binding unit described herein has amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH of the antigen-binding unit described herein may have fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions, deletions, or substitutions compared to the reference polypeptide.

[0083] The VH CDR1 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935, containing one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO.: 1461-1820 and 2901-2935, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO.: 1461-1820 and 2901-2935. When the VH CDR1 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VH CDR1 of the antigen-binding unit described herein may contain one, two, three, four, or five additions, deletions, or substitutions compared to the reference peptide. When the VH CDR1 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH CDR1 of the antigen-binding unit described herein may contain more than one, two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence. Conversely, when the VH CDR1 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH CDR1 of the antigen-binding unit described herein may contain fewer than two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence.

[0084] The VH CDR2 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970, containing one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO.: 1821-2180 and 2936-2970, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO.: 1821-2180 and 2936-2970. When the VH CDR2 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH CDR2 of the antigen-binding unit described herein may contain one, two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide. When the VH CDR2 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH CDR2 of the antigen-binding unit described herein may contain more than one, two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence. Conversely, when the VH CDR2 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH CDR2 of the antigen-binding unit described herein may contain fewer than two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence.

[0085] The VH CDR3 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 1-360 and 2971-3005, containing one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO.: 1-360 and 2971-3005, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO.: 1-360 and 2971-3005. When the VH CDR3 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VH CDR3 of the antigen-binding unit described herein may contain one, two, three, four, or five additions, deletions, or substitutions compared to the reference peptide. When the VH CDR3 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH CDR3 of the antigen-binding unit described herein may contain more than one, two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence. When the VH CDR2 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VH CDR3 of the antigen-binding unit described herein may contain fewer than two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence.

[0086] The VL of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 1081-1440 and 3146-3180, a sequence comprising one or more amino acid additions, deletions, or substitutions compared to the sequence selected from SEQ ID NO.: 1081-1440 and 3146-3180, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 1081-1440 and 3146-3180. When the VL of the antigen-binding unit described herein differs from the reference polypeptide sequence in terms of amino acid additions, deletions, or substitutions, the VL of the antigen-binding unit described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions, deletions, or substitutions compared to the reference polypeptide sequence. When the VL of the antigen-binding unit described herein differs from the reference polypeptide sequence in terms of amino acid additions, deletions, or substitutions, the VL of the antigen-binding unit described herein may contain more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions, deletions, or substitutions compared to the reference polypeptide sequence. When the VL of the antigen-binding unit described herein differs from the reference polypeptide sequence in that there are additions, deletions, or substitutions of amino acids, the VL of the antigen-binding unit described herein may have fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additions, deletions, or substitutions compared to the reference polypeptide sequence.

[0087] The VL CDR1 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 2181-2540 and 3006-3040, containing one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO.: 2181-2540 and 3006-3040, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO.: 2181-2540 and 3006-3040. When the VL CDR1 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VL CDR1 of the antigen-binding unit described herein may contain one, two, three, four, or five additions, deletions, or substitutions compared to the reference peptide. When the VL CDR1 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VL CDR1 of the antigen-binding unit described herein may contain more than one, two, three, four, or five additions, deletions, or substitutions compared to the reference peptide. Conversely, when the VL CDR1 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VL CDR1 of the antigen-binding unit described herein may contain fewer than two, three, four, or five additions, deletions, or substitutions compared to the reference peptide.

[0088] The VL CDR2 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 2541-2900 and 3041-3075, containing one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO.: 2541-2900 and 3041-3075, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO.: 2541-2900 and 3041-3075. When the VL CDR2 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VL CDR2 of the antigen-binding unit described herein may contain one, two, three, four, or five additions, deletions, or substitutions compared to the reference peptide. When the VL CDR2 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VL CDR2 of the antigen-binding unit described herein may contain more than one, two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence. Conversely, when the VL CDR2 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference polypeptide sequence, the VL CDR2 of the antigen-binding unit described herein may contain fewer than two, three, four, or five additions, deletions, or substitutions compared to the reference polypeptide sequence.

[0089] The VL CDR3 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 361-720 and 3076-3110, containing one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO.: 361-720 and 3076-3110, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO.: 361-720 and 3076-3110. When the VL CDR3 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VL CDR3 of the antigen-binding unit described herein may contain one, two, three, four, or five additions, deletions, or substitutions compared to the reference peptide. When the VL CDR3 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VL CDR3 of the antigen-binding unit described herein may contain more than one, two, three, four, or five additions, deletions, or substitutions compared to the reference peptide. Conversely, when the VL CDR3 of the antigen-binding unit described herein contains amino acid additions, deletions, or substitutions compared to the reference peptide sequence, the VL CDR3 of the antigen-binding unit described herein may contain fewer than two, three, four, or five additions, deletions, or substitutions compared to the reference peptide.

[0090] The VH CDR1 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935, which, compared to the sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935, contains one or more amino acid additions, deletions, or substitutions, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935; and the VL CDR1 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 2181-2540 and 3006-3040, which, compared to the sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935, contains one or more amino acid additions, deletions, or substitutions, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935. The sequences NO: 2181-2540 and 3006-3040 contain one or more amino acid additions, deletions, or substitutions, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO: 2181-2540 and 3006-3040.

[0091] The VH CDR2 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970, which, compared to the sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970, contains one or more amino acid additions, deletions, or substitutions, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970; and the VL CDR2 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 2541-2900 and 3041-3075, which, compared to the sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970, contains one or more amino acid additions, deletions, or substitutions, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970. The sequences NO: 2541-2900 and 3041-3075 contain one or more amino acid additions, deletions, or substitutions, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO: 2541-2900 and 3041-3075.

[0092] The VH CDR3 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 1-360 and 2971-3005, which, compared to the sequence selected from SEQ ID NO.: 1-360 and 2971-3005, contains one or more amino acid additions, deletions, or substitutions, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 1-360 and 2971-3005; and the VL CDR3 of the antigen-binding unit described herein may comprise a sequence selected from SEQ ID NO.: 361-720 and 3076-3110, which, compared to the sequence selected from SEQ ID NO.: 361-720 and 3076-3110, contains one or more amino acid additions, deletions, or substitutions, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequence selected from SEQ ID NO.: 1-360 and 2971-3005. The sequences NO: 361-720 and 3076-3110 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity.

[0093] The VH of the antigen-binding unit described herein may comprise VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 is selected from sequences of SEQ ID NO.: 1461-1820 and 2901-2935, and comprises one or more amino acid additions, deletions, or substitutions compared to sequences of SEQ ID NO.: 1461-1820 and 2901-2935, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with sequences of SEQ ID NO.: 1461-1820 and 2901-2935; wherein VH CDR2 is selected from sequences of SEQ ID NO.: 1821-2180 and 2936-2970, and comprises one or more amino acid additions, deletions, or substitutions compared to sequences of SEQ ID NO.: 1461-1820 and 2901-2935, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with sequences of SEQ ID NO.: 1461-1820 and 2901-2935; wherein VH CDR1 is selected from sequences of SEQ ID NO.: 1821-2180 and 2936-2970, and comprises one or more amino acid additions, deletions, or substitutions compared to sequences of SEQ ID NO.: 1461-1820 and 2901-2935, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 The sequences NO: 1821-2180 and 2936-2970 contain one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO: 1821-2180 and 2936-2970, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO: 1821-2180 and 2936-2970; and the VH CDR3 is selected from the sequences selected from SEQ ID NO: 1-360 and 2971-3005, and contains one or more amino acid additions, deletions, or substitutions compared to the sequences selected from SEQ ID NO: 1821-2180 and 2936-2970, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO: 1821-2180 and 2976-2970; and the VH CDR3 is selected from the sequences selected from SEQ ID NO: 1-360 and 2971-3005, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with the sequences selected from SEQ ID NO: 1821-2180 and 2976-2970. Sequences NO: 1-360 and 2971-3005 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity.

[0094] The VL of the antigen-binding unit described herein may comprise VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 is selected from sequences of SEQ ID NO.: 2181-2540 and 3006-3040, and comprises one or more amino acid additions, deletions, or substitutions compared to sequences of SEQ ID NO.: 2181-2540 and 3006-3040, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with sequences of SEQ ID NO.: 2181-2540 and 3006-3040; wherein VL CDR2 is selected from sequences of SEQ ID NO.: 2541-2900 and 3041-3075, and comprises one or more amino acid additions, deletions, or substitutions compared to sequences of SEQ ID NO.: 2181-2540 and 3006-3040, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with sequences of SEQ ID NO.: 2181-2540 and 3006-3040; wherein VL CDR2 is selected from sequences of SEQ ID NO.: 2181-2540 and 3041-3075, and comprises one or more amino acid additions, deletions, or substitutions compared to sequences of SEQ ID NO.: 2181-2540 and 3006-3040, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 Compared to sequences NO: 2541-2900 and 3041-3075, the sequences contain one or more amino acid additions, deletions, or substitutions, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with sequences selected from SEQ ID NO: 2541-2900 and 3041-3075; and wherein the VL CDR3 is selected from sequences of SEQ ID NO: 361-720 and 3076-3110, and compared to sequences selected from SEQ ID NO: 361-720 and 3076-3110, the sequences contain one or more amino acid additions, deletions, or substitutions, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity with sequences selected from SEQ ID NO: 2541-2900 and 3041-3075. The sequences NO: 361-720 and 3076-3110 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity.

[0095] The VH of the antigen-binding unit described herein may contain sequences selected from combinations of the following CDR1, CDR2, and CDR3:

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] The VH CDR1 of the antigen-binding unit described herein may contain the same sequence as CDR1 contained in SEQ ID NO: 721-1080 and 3111-3145; the VH CDR2 of the antigen-binding unit described herein may contain the same sequence as CDR2 contained in SEQ ID NO: 721-1080 and 3111-3145; the VH CDR3 of the antigen-binding unit described herein may contain the same sequence as CDR3 contained in SEQ ID NO: 721-1080 and 3111-3145; the VL CDR1 of the antigen-binding unit may contain the same sequence as CDR1 contained in SEQ ID NO: 1081-1440 and 3146-3180; the VL CDR2 of the antigen-binding unit may contain the same sequence as CDR2 contained in SEQ ID NO: 1081-1440 and 3146-3180; and / or the VL of the antigen-binding unit CDR3 may contain the same sequence as CDR3 contained in SEQ ID NO: 1081-1440 and 3146-3180.

[0127] In one embodiment, the antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0128] a. CDR1 amino acid sequence of the light chain variable region: SEQ ID NO: 2354, SEQ ID NO: 2355, SEQ ID NO: 2370, SEQ ID NO: 2477, and SEQ ID NO: 3012;

[0129] b. CDR2 amino acid sequence of the light chain variable region: SEQ ID NO: 2714, SEQ ID NO: 2715, SEQ ID NO: 2730, SEQ ID NO: 2837, and SEQ ID NO: 3047;

[0130] c. CDR3 amino acid sequence of the light chain variable region: SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 550, SEQ ID NO: 657, and SEQ ID NO: 3082;

[0131] d. CDR1 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1634, SEQ ID NO: 1635, SEQ ID NO: 1650, SEQ ID NO: 1757, and SEQ ID NO: 2907;

[0132] e. CDR2 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1994, SEQ ID NO: 1995, SEQ ID NO: 2010, SEQ ID NO: 2117, and SEQ ID NO: 2942; and

[0133] f. CDR3 amino acid sequence of heavy chain variable region: SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 190, SEQ ID NO: 297 and SEQ ID NO: 2977.

[0134] In one embodiment, the antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0135] a. CDR1 amino acid sequence of the light chain variable region: SEQ ID NO: 2354;

[0136] b. CDR2 amino acid sequence of the light chain variable region: SEQ ID NO: 2714;

[0137] c. CDR3 amino acid sequence of the light chain variable region: SEQ ID NO: 534;

[0138] d. CDR1 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1634;

[0139] e. CDR2 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1994; and

[0140] f. CDR3 amino acid sequence of the heavy chain variable region: SEQ ID NO: 174.

[0141] In one embodiment, the antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0142] a. CDR1 amino acid sequence of the light chain variable region: SEQ ID NO: 2355;

[0143] b. CDR2 amino acid sequence of the light chain variable region: SEQ ID NO: 2715;

[0144] c. CDR3 amino acid sequence of the light chain variable region: SEQ ID NO: 535;

[0145] d. CDR1 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1635;

[0146] e. CDR2 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1995; and

[0147] f. CDR3 amino acid sequence of the heavy chain variable region: SEQ ID NO: 175.

[0148] In one embodiment, the antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0149] a. CDR1 amino acid sequence of the light chain variable region: SEQ ID NO: 2370;

[0150] b. CDR2 amino acid sequence of the light chain variable region: SEQ ID NO: 2730;

[0151] c. CDR3 amino acid sequence of the light chain variable region: SEQ ID NO: 550;

[0152] d. CDR1 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1650;

[0153] e. CDR2 amino acid sequence of the heavy chain variable region: SEQ ID NO: 2010; and

[0154] f. CDR3 amino acid sequence of the heavy chain variable region: SEQ ID NO: 190.

[0155] In one embodiment, the antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0156] a. CDR1 amino acid sequence of the light chain variable region: SEQ ID NO: 2477;

[0157] b. CDR2 amino acid sequence of the light chain variable region: SEQ ID NO: 2837;

[0158] c. CDR3 amino acid sequence of the light chain variable region: SEQ ID NO: 657;

[0159] d. CDR1 amino acid sequence of the heavy chain variable region: SEQ ID NO: 1757;

[0160] e. CDR2 amino acid sequence of the heavy chain variable region: SEQ ID NO: 2117; and

[0161] f. CDR3 amino acid sequence of the heavy chain variable region: SEQ ID NO: 297.

[0162] In one embodiment, the antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0163] a. CDR1 amino acid sequence of the light chain variable region: SEQ ID NO: 3012;

[0164] b. CDR2 amino acid sequence of the light chain variable region: SEQ ID NO: 3047;

[0165] c. CDR3 amino acid sequence of the light chain variable region: SEQ ID NO: 3082;

[0166] d. CDR1 amino acid sequence of the heavy chain variable region: SEQ ID NO: 2907;

[0167] e. CDR2 amino acid sequence of the heavy chain variable region: SEQ ID NO: 2942; and

[0168] f. CDR3 amino acid sequence of the heavy chain variable region: SEQ ID NO: 2977.

[0169] In one embodiment, the antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0170] a. Amino acid sequence of the light chain variable region: SEQ ID NO: 1377 and SEQ ID NO: 3152; and

[0171] b. Amino acid sequence of the heavy chain variable region: SEQ ID NO: 1017 and SEQ ID NO: 3117.

[0172] In one embodiment, the antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0173] a. Amino acid sequence of the light chain variable region: SEQ ID NO: 1254; and

[0174] b. Amino acid sequence of the heavy chain variable region: SEQ ID NO: 894.

[0175] In one embodiment, the antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0176] a. Amino acid sequence of the light chain variable region: SEQ ID NO: 1255; and

[0177] b. Amino acid sequence of the heavy chain variable region: SEQ ID NO: 895.

[0178] In one embodiment, the antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0179] a. Amino acid sequence of the light chain variable region: SEQ ID NO: 1270; and

[0180] b. Amino acid sequence of the heavy chain variable region: SEQ ID NO: 910.

[0181] In one embodiment, the antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0182] a. Amino acid sequence of the light chain variable region: SEQ ID NO: 1377; and

[0183] b. Amino acid sequence of the heavy chain variable region: SEQ ID NO: 1017.

[0184] In one embodiment, the antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

[0185] a. Amino acid sequence of the light chain variable region: SEQ ID NO: 3152; and

[0186] b. Amino acid sequence of the heavy chain variable region: SEQ ID NO: 3117.

[0187] The antigen-binding unit described herein can bind to the SARS-CoV-2 S protein. The antigen-binding unit described herein can bind to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein. The binding of the antigen-binding unit to the RBD can be characterized or represented by any method known in the art. For example, binding can be characterized by binding affinity, which can be the strength of the interaction between the antigen-binding unit and the antigen. Binding affinity can be determined by any method known in the art, such as in vitro binding assays. The binding affinity of the antigen-binding unit described herein can be expressed as KD, which is defined as the ratio of two kinetic rate constants Ka / Kd, where "Ka" refers to the rate constant of antibody binding to the antigen, and "Kd" refers to the rate constant of antibody dissociation from the antibody / antigen complex. The antigen-binding unit disclosed herein specifically binds to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein with a KD in the range of about 10 μM to about 1 fM. For example, the antigen-binding unit can specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein at KD values ​​of less than about 10 μM, 1 μM, 0.1 μM, 50 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 50 pM, 10 pM, 1 pM, 0.1 pM, 10 fM, 1 fM, 0.1 fM, or less than 0.1 fM. The antigen-binding unit disclosed herein can bind to the receptor-binding domain (RBD) of the novel coronavirus (SARS-CoV-2) S protein with an equilibrium dissociation constant (KD) of less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, or less than 0.01 nM.

[0188] The antigen-binding unit described herein exhibits neutralizing activity against the novel coronavirus (SARS-CoV-2). This neutralizing activity can be analyzed using pseudoviruses. Pseudoviruses possess cell infection characteristics similar to herpesviruses, mimicking the early stages of true viral infection in cells, and can be safely and rapidly detected and analyzed. The neutralizing activity of the antigen-binding unit described herein against SARS-CoV-2 can be detected using methods known in the art, such as a microwell cell neutralization assay, as described in Temperton NJ et al., EmergInfect Dis, 2005, 11(3), 411-416.

[0189] The neutralizing activity of the antigen-binding unit described in this article against the novel coronavirus (SARS-CoV-2) can be detected using experimental cells such as Huh-7 cells and the pseudovirus SARS-CoV-2. The antigen-binding unit described in this article can be used at concentrations, for example, less than 100 μg / ml, less than 50 μg / ml, less than 20 μg / ml, less than 10 μg / ml, less than 9 μg / ml, less than 8 μg / ml, less than 7 μg / ml, less than 6 μg / ml, less than 5 μg / ml, less than 4 μg / ml, less than 3 μg / ml, less than 2 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.25 μg / ml, less than 0.2 μg / ml, and less than 0.1 μg / ml. The IC50 of less than 0.05 μg / ml, less than 1 ng / ml, less than 0.5 ng / ml, less than 0.25 ng / ml, less than 0.2 ng / ml, less than 0.1 ng / ml, less than 50 pg / ml, less than 25 pg / ml, less than 20 pg / ml, less than 10 pg / ml, less than 5 pg / ml, less than 2.5 pg / ml, less than 2 pg / ml, or less than 1 pg / ml can neutralize the novel coronavirus (SARS-CoV-2) pseudovirus.

[0190] The neutralizing activity of the antigen-binding unit described herein against the novel coronavirus (SARS-CoV-2) can be detected using the Plaque Reduction Neutralization Test (PRNT) with SARS-CoV-2 evoked virus. The IC50 of the antigen-binding unit described herein against SARS-CoV-2 evoked virus is calculated by the reduction of plaques after incubation. The antigen-binding unit described herein can be used at concentrations, for example, less than 100 μg / ml, less than 50 μg / ml, less than 20 μg / ml, less than 10 μg / ml, less than 9 μg / ml, less than 8 μg / ml, less than 7 μg / ml, less than 6 μg / ml, less than 5 μg / ml, less than 4 μg / ml, less than 3 μg / ml, less than 2 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.25 μg / ml, less than 0.2 μg / ml, and less than 0.1 μg / ml. The IC50 values ​​of less than 0.05 μg / ml, less than 1 ng / ml, less than 0.5 ng / ml, less than 0.25 ng / ml, less than 0.2 ng / ml, less than 0.1 ng / ml, less than 50 pg / ml, less than 25 pg / ml, less than 20 pg / ml, less than 10 pg / ml, less than 5 pg / ml, less than 2.5 pg / ml, less than 2 pg / ml, or less than 1 pg / ml can neutralize the novel coronavirus (SARS-CoV-2) virus.

[0191] Preparation of antigen-binding units

[0192] This document provides a method for generating any antigen-binding unit disclosed herein, wherein the method includes culturing host cells expressing the antigen-binding unit under conditions suitable for expressing the antigen-binding unit, and isolating the antigen-binding unit expressed by the host cells.

[0193] The expressed antigen-binding units can be isolated using a variety of protein purification techniques known in the art. Typically, antigen-binding units are isolated from the culture medium as secreted peptides, although they can also be recovered from host cell lysates or bacterial periplasm when produced directly without a signal peptide. If the antigen-binding units are membrane-bound, they can be dissolved using appropriate detergent solutions commonly used by those skilled in the art. The recovered antigen-binding units can be further purified by salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., running on a cation or anion exchange column at neutral pH and eluting with a gradient of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and labeled affinity column chromatography, or by affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulins.

[0194] In the methods and compositions described herein, derived immunoglobulins with the following components may be used: chemical linkers, detectable components such as fluorescent dyes, enzymes, substrates, chemiluminescent components, specific binding components such as streptavidin, avidin, or biotin, or drug conjugates.

[0195] This document also provides antigen-binding units conjugated to a chemically functional moiety. Typically, this moiety is a label capable of generating a detectable signal. These conjugated antigen-binding units can be used, for example, in detection systems such as imaging of viral infection severity, foci of infection, etc. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compound substrate cofactors, and inhibitors. Examples of patents teaching the use of such labels are found in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. The moiety can be covalently linked to, recombinantly linked to, or conjugated to the antigen-binding unit via a second agent such as a second antibody, protein A, or a biotin-avidin complex.

[0196] Other functional components include signal peptides, agents that enhance immune reactivity, agents that promote coupling with solid supports, vaccine carriers, biological response modifiers, paramagnetic labels, and drugs. Signal peptides are short amino acid sequences that guide newly synthesized proteins across the cell membrane (typically the endoplasmic reticulum in eukaryotic cells) and the inner or outer membrane of bacteria. Signal peptides can be located at the N-terminal or C-terminal portion of a polypeptide and can be enzymatically removed from the cell between polypeptide biosynthesis and secretion. These peptides can be introduced into antigen-binding units to allow the secretion of synthetic molecules.

[0197] Agents that enhance immune reactivity include, but are not limited to, bacterial superantigens. Agents that promote coupling with solid supports include, but are not limited to, biotin or avidin. Immunogen carriers include, but are not limited to, any physiologically acceptable buffer solution. Biological response modifiers include cytokines, particularly tumor necrosis factor (TNF), interleukin-2, interleukin-4, granulocyte-macrophage colony-stimulating factor, and gamma interferon.

[0198] The chemically functional moiety can be prepared recombinantly, for example by generating a fusion gene encoding an antigen-binding unit and the functional moiety. Alternatively, the antigen-binding unit can be chemically bonded to the moiety using any of a variety of well-known chemical procedures. For example, when the moiety is a protein, the linking can be done using a heterobifunctional cross-linking agent, such as SPDP, carbodiimide glutaraldehyde, etc. The moiety can be covalently linked or conjugated using a second agent, such as a second antibody, protein A, or a biotin-avidin complex. Paramagnetic moieties and their conjugation with antibodies are well known in the art. See, for example, Miltenyi et al. (1990) Cytometry 11:231-238.

[0199] Nucleic acid

[0200] In one respect, this article provides isolated polynucleotides encoding the antigen-binding units described herein. Nucleotide sequences corresponding to the various regions of the L or H chains of existing antibodies can be readily obtained and sequenced using conventional techniques, including but not limited to hybridization, PCR, and DNA sequencing. Hybridoma cells that produce monoclonal antibodies are a preferred source of antibody nucleotide sequences. Large quantities of hybridoma cells producing a range of monoclonal antibodies are available from public or private repositories. The largest repository is the American Type Culture Collection, which provides a variety of well-characterized hybridoma cell lines. Alternatively, antibody nucleotides can be obtained from immunized or non-immunized rodents or humans, as well as from organs such as spleen and peripheral blood lymphocytes. Specific techniques applicable to the extraction and synthesis of antibody nucleotides are described in Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3833-3837; Larrick et al. (1989) Biochem. Biophys. Res. Commun. 160: 1250-1255; Sastry et al. (1989) Proc. Natl. Acad. Sci., USA 86: 5728-5732; and US Patent No. 5,969,108.

[0201] The antibody nucleotide sequence can also be modified, for example, by replacing the homologous non-human sequence with the coding sequence in the constant regions of the human heavy and light chains. In this way, chimeric antibodies are prepared that retain the binding specificity of the original antibody.

[0202] Additionally, polynucleotides encoding heavy and / or light chains of antigen-binding units can be codon-optimized to achieve optimized expression of the subject antigen-binding unit in desired host cells. For example, in one codon optimization method, natural codons are replaced with the most common codons from a reference genome, wherein the codon translation rate for each amino acid is designed to be high. Further exemplary methods for generating codon-optimized polynucleotides for expressing desired proteins are described in Kanaya et al., Gene, 238:143-155 (1999), Wang et al., Mol. Biol. Evol., 18(5):792-800 (2001), U.S. Patent No. 5,795,737, U.S. Publication No. 2008 / 0076161 and WO 2008 / 000632, and these methods can be applied to the heavy and / or light chains of antigen-binding units.

[0203] The polynucleotides described herein include polynucleotides encoding functional equivalents of exemplary polypeptides and fragments thereof.

[0204] Due to the degeneracy of the genetic code, the nucleotides of the L and H sequences, as well as the heterodimerization sequences suitable for constructing the polynucleotides and vectors described herein, can exhibit considerable variation. These variations are encompassed throughout this document.

[0205] Treatment

[0206] This article provides a method for preventing or treating novel coronavirus (SARS-CoV-2) infection in a subject using the antigen-binding unit described herein, including administering the antigen-binding unit described herein to the subject.

[0207] This article provides a method for treating diseases, conditions, or ailments in mammals using the antigen-binding unit described herein in combination with a second agent. The second agent may be administered together with, before, or after the antibody. The second agent may be an antiviral agent. Antiviral agents include, but are not limited to, telaprevir, boceprevir, semiprevir, sofosbuvir, daclastavir, asunaprevir, lamivudine, adefovir, entecavir, tenofovir, telbivudine, interferon-alpha, and PEGylated interferon-alpha. The second agent may be selected from hydroxychloroquine, chloroquine, favipiravir, gimsilumab, AdCOVID (University of Alabamaat Birmingham), AT-100 (Airway Therapeutics), TZLS-501 (Tiziana Life Sciences), OYA1 (OyaGen), BPI-002 (BeyondSpring), INO-4800 (Inovio Pharmaceutical), NP-120 (ifenprodil), remdesivir (GS-5734), Actemra (Roche), gallydivir (BCX4430), SNG001 (Synairgen Research), or a combination thereof.

[0208] The second agent may be an agent used to relieve symptoms of inflammatory conditions concurrent in the subject. The anti-inflammatory agent includes nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. NSAIDs include, but are not limited to, salicylates such as acetylsalicylic acid; diflunisal, salicylic acid, and disalicylate; propionic acid derivatives such as ibuprofen; naproxen; dextro ibuprofen, dextro ketoprofen, flurbiprofen, oxaprazin, fenofoprofen, loxoprofen, and ketoprofen; acetic acid derivatives such as indomethacin, diclofenac, tolmetine, aceclofenac, sulindac, nabumetone, etodoxacin, and ketoroxyprofen; and enolic acid derivatives such as piroxicam. Lornoxicam, meloxicam, isoxicam, tenoxicam, phenylbutazone, and droxoxicam; anthranilic acid derivatives, such as mefenamic acid, flufenamic acid, meclofenamic acid, and tofenamic acid; selective COX-2 inhibitors, such as celecoxib, romecoxib, rofecoxib, etorcoxib, vardicoxib, fenicoxib, and parecoxib; sulfanilicin, such as nimesulide; and other nonsteroidal anti-inflammatory drugs (NSAIDs) such as clonisin and ricofibrone. Corticosteroids include, but are not limited to, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone.

[0209] The second agent may be an immunosuppressant. Immunosuppressants that can be used in combination with antigen-binding units include, but are not limited to, hydroxychloroquine, sulfasalazine, leflunomide, etanercept, infliximab, adalimumab, D-penicillamine, oral gold compounds, injectable gold compounds (intramuscular injection), minocycline, sodium gold thiomalate, auronoxine, D-penicillamine, chlorbenzabride, butyrazine, actalide, cyclophosphamide, azathioprine, methotrexate, imidazoribine, cyclosporine, and tacrolimus.

[0210] The specific dosage will vary depending on the specific antigen-binding unit selected, the dosing regimen to be followed, whether it is administered in combination with other drugs, the time of administration, the tissue to which it is administered, and the physical delivery system that carries the specific antigen-binding unit. In some implementations, during the treatment cycle, subjects are administered an average of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 mg of antigen-binding units per week. For example, subjects may be administered antigen-binding units in the range of approximately 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg weekly. In some embodiments, subjects may be administered antigen-binding units in the range of approximately 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg weekly.

[0211] During the treatment cycle, the antigen-binding unit may be administered to the subject at an average daily dose greater than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg. For example, during the treatment cycle, the antigen-binding unit may be administered to the subject at an average daily dose of approximately 6 to 10 mg, approximately 6.5 to 9.5 mg, approximately 6.5 to 8.5 mg, approximately 6.5 to 8 mg, or approximately 7 to 9 mg.

[0212] The dosage of the antigen-binding unit can be about, at least about, or at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 30 0, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000 mg or mg / kg, or any range derived therefrom. The expected dose in mg / kg refers to the amount of antigen-binding units in mg per kilogram of the subject's total body weight. When multiple doses are administered to a patient, the doses may vary in amount or they may be the same.

[0213] Pharmaceutical Composition

[0214] This article provides pharmaceutical compositions comprising a subject antibody or a functional fragment thereof and a pharmaceutically acceptable carrier, excipient, or stabilizer, including but not limited to inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers, and adjuvants. (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)).

[0215] The pharmaceutical composition may be a unit dosage form suitable for a precise single-dose administration. The pharmaceutical composition may further comprise an antigen-binding unit as the active ingredient and may include conventional drug carriers or excipients. Furthermore, it may include other drugs or agents, carriers, adjuvants, etc. Exemplary parenteral formulations include solutions or suspensions of active peptides and / or PEG-modified peptides in sterile aqueous solutions, such as propylene glycol aqueous solutions or dextran solutions. If desired, such dosage forms may be appropriately buffered with salts such as histidine and / or phosphate.

[0216] The composition may further comprise one or more pharmaceutically acceptable additives and excipients. These additives and excipients include, but are not limited to, anti-sticking agents, defoamers, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tension modifiers, flavoring agents, coloring agents, flavor enhancers, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[0217] Reagent test kit

[0218] The kits described herein contain the antigen-binding units described herein or their conjugates as described herein. Use of the antigen-binding units described herein in the preparation of kits for detecting the presence or level of the novel coronavirus or its S protein or the RBD of the S protein in a sample, or for diagnosing whether a subject is infected with the novel coronavirus, is also provided.

[0219] In some implementations, the sample includes, but is not limited to, excrement, oral or nasal secretions, bronchoalveolar lavage fluid, etc., from a subject (e.g., a mammal, preferably a human).

[0220] General methods for using antibodies or their antigen-binding fragments to detect the presence or level of a target virus or antigen (e.g., novel coronavirus or its S protein or RBD) in a sample are well known to those skilled in the art. In some embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar detection methods.

[0221] Example

[0222] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0223] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this article are generally based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Laboratory Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Where specific conditions are not specified in the examples, conventional conditions or manufacturer-recommended conditions are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are illustrative of the invention and are not intended to limit the scope of the invention.

[0224] Example 1: Isolation of memory B cells

[0225] Blood samples were collected from individuals infected with SARS-CoV-2 who had recovered and been discharged from the hospital (provided by Beijing You'an Hospital), and processed in a P2+ biosafety laboratory using STEMCELL SepMate. TMPBMCs were extracted using a Stemcell Technologies (catalog number: 86415) device. Subsequently, memory B cells were enriched in the extracted PBMCs using the STEMCELL EasySep Human Memory B Cell Isolation Kit (Stemcell Technologies, catalog number: 17864) according to the manufacturer's instructions.

[0226] Example 2: Obtaining and Identifying Antigen-Binding Unit Sequences

[0227] According to the manufacturer's instructions, single-cell transcriptome VDJ sequencing was performed on the enriched memory B cells using Chromium Single Cell V(D)J Reagent Kits (purchased from 10X genomics, catalog number: 100006). Analysis of the sequencing results yielded 360 antigen-binding units, named ABU 1-395. The sequence information of the obtained antigen-binding units is shown in Table 1 below.

[0228] Table 1. Exemplary antigen-binding units obtained in this paper.

[0229] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-1 721 1081

[0230] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-2 722 1082 ABU-3 723 1083 ABU-4 724 1084 ABU-5 725 1085 ABU-6 726 1086 ABU-7 727 1087 ABU-8 728 1088 ABU-9 729 1089 ABU-10 730 1090 ABU-11 731 1091 ABU-12 732 1092 ABU-13 733 1093 ABU-14 734 1094 ABU-15 735 1095 ABU-16 736 1096 ABU-17 737 1097 ABU-18 738 1098 ABU-19 739 1099 ABU-20 740 1100 ABU-21 741 1101 ABU-22 742 1102 ABU-23 743 1103 ABU-24 744 1104 ABU-25 745 1105 ABU-26 746 1106 ABU-27 747 1107 ABU-28 748 1108 ABU-29 749 1109 ABU-30 750 1110 ABU-31 751 1111 ABU-32 752 1112 ABU-33 753 1113 ABU-34 754 1114 ABU-35 755 1115 ABU-36 756 1116 ABU-37 757 1117 ABU-38 758 1118 ABU-39 759 1119 ABU-40 760 1120 ABU-41 761 1121 ABU-42 762 1122 ABU-43 763 1123 ABU-44 764 1124 ABU-45 765 1125 ABU-46 766 1126

[0231] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-47 767 1127 ABU-48 768 1128 ABU-49 769 1129 ABU-50 770 1130 ABU-51 771 1131 ABU-52 772 1132 ABU-53 773 1133 ABU-54 774 1134 ABU-55 775 1135 ABU-56 776 1136 ABU-57 777 1137 ABU-58 778 1138 ABU-59 779 1139 ABU-60 780 1140 ABU-61 781 1141 ABU-62 782 1142 ABU-63 783 1143 ABU-64 784 1144 ABU-65 785 1145 ABU-66 786 1146 ABU-67 787 1147 ABU-68 788 1148 ABU-69 789 1149 ABU-70 790 1150 ABU-71 791 1151 ABU-72 792 1152 ABU-73 793 1153 ABU-74 794 1154 ABU-75 795 1155 ABU-76 796 1156 ABU-77 797 1157 ABU-78 798 1158 ABU-79 799 1159 ABU-80 800 1160 ABU-81 801 1161 ABU-82 802 1162 ABU-83 803 1163 ABU-84 804 1164 ABU-85 805 1165 ABU-86 806 1166 ABU-87 807 1167 ABU-88 808 1168 ABU-89 809 1169 ABU-90 810 1170 ABU-91 811 1171

[0232] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-92 812 1172 ABU-93 813 1173 ABU-94 814 1174 ABU-95 815 1175 ABU-96 816 1176 ABU-97 817 1177 ABU-98 818 1178 ABU-99 819 1179 ABU-100 820 1180 ABU-101 821 1181 ABU-102 822 1182 ABU-103 823 1183 ABU-104 824 1184 ABU-105 825 1185 ABU-106 826 1186 ABU-107 827 1187 ABU-108 828 1188 ABU-109 829 1189 ABU-110 830 1190 ABU-111 831 1191 ABU-112 832 1192 ABU-113 833 1193 ABU-114 834 1194 ABU-115 835 1195 ABU-116 836 1196 ABU-117 837 1197 ABU-118 838 1198 ABU-119 839 1199 ABU-120 840 1200 ABU-121 841 1201 ABU-122 842 1202 ABU-123 843 1203 ABU-124 844 1204 ABU-125 845 1205 ABU-126 846 1206 ABU-127 847 1207 ABU-128 848 1208 ABU-129 849 1209 ABU-130 850 1210 ABU-131 851 1211 ABU-132 852 1212 ABU-133 853 1213 ABU-134 854 1214 ABU-135 855 1215 ABU-136 856 1216

[0233] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-137 857 1217 ABU-138 858 1218 ABU-139 859 1219 ABU-140 860 1220 ABU-141 861 1221 ABU-142 862 1222 ABU-143 863 1223 ABU-144 864 1224 ABU-145 865 1225 ABU-146 866 1226 ABU-147 867 1227 ABU-148 868 1228 ABU-149 869 1229 ABU-150 870 1230 ABU-151 871 1231 ABU-152 872 1232 ABU-153 873 1233 ABU-154 874 1234 ABU-155 875 1235 ABU-156 876 1236 ABU-157 877 1237 ABU-158 878 1238 ABU-159 879 1239 ABU-160 880 1240 ABU-161 881 1241 ABU-162 882 1242 ABU-163 883 1243 ABU-164 884 1244 ABU-165 885 1245 ABU-166 886 1246 ABU-167 887 1247 ABU-168 888 1248 ABU-169 889 1249 ABU-170 890 1250 ABU-171 891 1251 ABU-172 892 1252 ABU-173 893 1253 ABU-174 894 1254 ABU-175 895 1255 ABU-176 896 1256 ABU-177 897 1257 ABU-178 898 1258 ABU-179 899 1259 ABU-180 900 1260 ABU-181 901 1261

[0234] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-182 902 1262 ABU-183 903 1263 ABU-184 904 1264 ABU-185 905 1265 ABU-186 906 1266 ABU-187 907 1267 ABU-188 908 1268 ABU-189 909 1269 ABU-190 910 1270 ABU-191 911 1271 ABU-192 912 1272 ABU-193 913 1273 ABU-194 914 1274 ABU-195 915 1275 ABU-196 916 1276 ABU-197 917 1277 ABU-198 918 1278 ABU-199 919 1279 ABU-200 920 1280 ABU-201 921 1281 ABU-202 922 1282 ABU-203 923 1283 ABU-204 924 1284 ABU-205 925 1285 ABU-206 926 1286 ABU-207 927 1287 ABU-208 928 1288 ABU-209 929 1289 ABU-210 930 1290 ABU-211 931 1291 ABU-212 932 1292 ABU-213 933 1293 ABU-214 934 1294 ABU-215 935 1295 ABU-216 936 1296 ABU-217 937 1297 ABU-218 938 1298 ABU-219 939 1299 ABU-220 940 1300 ABU-221 941 1301 ABU-222 942 1302 ABU-223 943 1303 ABU-224 944 1304 ABU-225 945 1305 ABU-226 946 1306

[0235] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-227 947 1307 ABU-228 948 1308 ABU-229 949 1309 ABU-230 950 1310 ABU-231 951 1311 ABU-232 952 1312 ABU-233 953 1313 ABU-234 954 1314 ABU-235 955 1315 ABU-236 956 1316 ABU-237 957 1317 ABU-238 958 1318 ABU-239 959 1319 ABU-240 960 1320 ABU-241 961 1321 ABU-242 962 1322 ABU-243 963 1323 ABU-244 964 1324 ABU-245 965 1325 ABU-246 966 1326 ABU-247 967 1327 ABU-248 968 1328 ABU-249 969 1329 ABU-250 970 1330 ABU-251 971 1331 ABU-252 972 1332 ABU-253 973 1333 ABU-254 974 1334 ABU-255 975 1335 ABU-256 976 1336 ABU-257 977 1337 ABU-258 978 1338 ABU-259 979 1339 ABU-260 980 1340 ABU-261 981 1341 ABU-262 982 1342 ABU-263 983 1343 ABU-264 984 1344 ABU-265 985 1345 ABU-266 986 1346 ABU-267 987 1347 ABU-268 988 1348 ABU-269 989 1349 ABU-270 990 1350 ABU-271 991 1351

[0236] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-272 992 1352 ABU-273 993 1353 ABU-274 994 1354 ABU-275 995 1355 ABU-276 996 1356 ABU-277 997 1357 ABU-278 998 1358 ABU-279 999 1359 ABU-280 1000 1360 ABU-281 1001 1361 ABU-282 1002 1362 ABU-283 1003 1363 ABU-284 1004 1364 ABU-285 1005 1365 ABU-286 1006 1366 ABU-287 1007 1367 ABU-288 1008 1368 ABU-289 1009 1369 ABU-290 1010 1370 ABU-291 1011 1371 ABU-292 1012 1372 ABU-293 1013 1373 ABU-294 1014 1374 ABU-295 1015 1375 ABU-296 1016 1376 ABU-297 1017 1377 ABU-298 1018 1378 ABU-299 1019 1379 ABU-300 1020 1380 ABU-301 1021 1381 ABU-302 1022 1382 ABU-303 1023 1383 ABU-304 1024 1384 ABU-305 1025 1385 ABU-306 1026 1386 ABU-307 1027 1387 ABU-308 1028 1388 ABU-309 1029 1389 ABU-310 1030 1390 ABU-311 1031 1391 ABU-312 1032 1392 ABU-313 1033 1393 ABU-314 1034 1394 ABU-315 1035 1395 ABU-316 1036 1396

[0237] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-317 1037 1397 ABU-318 1038 1398 ABU-319 1039 1399 ABU-320 1040 1400 ABU-321 1041 1401 ABU-322 1042 1402 ABU-323 1043 1403 ABU-324 1044 1404 ABU-325 1045 1405 ABU-326 1046 1406 ABU-327 1047 1407 ABU-328 1048 1408 ABU-329 1049 1409 ABU-330 1050 1410 ABU-331 1051 1411 ABU-332 1052 1412 ABU-333 1053 1413 ABU-334 1054 1414 ABU-335 1055 1415 ABU-336 1056 1416 ABU-337 1057 1417 ABU-338 1058 1418 ABU-339 1059 1419 ABU-340 1060 1420 ABU-341 1061 1421 ABU-342 1062 1422 ABU-343 1063 1423 ABU-344 1064 1424 ABU-345 1065 1425 ABU-346 1066 1426 ABU-347 1067 1427 ABU-348 1068 1428 ABU-349 1069 1429 ABU-350 1070 1430 ABU-351 1071 1431 ABU-352 1072 1432 ABU-353 1073 1433 ABU-354 1074 1434 ABU-355 1075 1435 ABU-356 1076 1436 ABU-357 1077 1437 ABU-358 1078 1438 ABU-359 1079 1439 ABU-360 1080 1440 ABU-361 3111 3146

[0238] ASH No. VH SEQ ID No. VL SEQ ID NO. ABU-362 3112 3147 ABU-363 3113 3148 ABU-364 3114 3149 ABU-365 3115 3150 ABU-366 3116 3151 ABU-367 3117 3152 ABU-368 3118 3153 ABU-369 3119 3154 ABU-370 3120 3155 ABU-371 3121 3156 ABU-372 3122 3157 ABU-373 3123 3158 ABU-374 3124 3159 ABU-375 3125 3160 ABU-376 3126 3161 ABU-377 3127 3162 ABU-378 3128 3163 ABU-379 3129 3164 ABU-380 3130 3165 ABU-381 3131 3166 ABU-382 3132 3167 ABU-383 3133 3168 ABU-384 3134 3169 ABU-385 3135 3170 ABU-386 3136 3171 ABU-387 3137 3172 ABU-388 3138 3173 ABU-389 3139 3174 ABU-390 3140 3175 ABU-391 3141 3176 ABU-392 3142 3177 ABU-393 3143 3178 ABU-394 3144 3179 ABU-395 3145 3180

[0239] Example 3: Preparation and purification of the antigen-binding unit in this article

[0240] Based on the sequence information of the antigen-binding units obtained in Example 2, Beijing Yiqiao Shenzhou Co., Ltd. was commissioned to express and purify the obtained antigen-binding units, and their antigen reactivity was tested.

[0241] In short, nucleic acid molecules encoding the antibody heavy and light chains were synthesized in vitro and then cloned into expression vectors, respectively, to obtain recombinant expression vectors encoding the antibody heavy and light chains. These recombinant expression vectors were then co-transfected into HEK293 cells. Four to six hours after transfection, the cell culture medium was replaced with serum-free medium, and the cells were cultured at 37°C for another six days. After culture, the antibody proteins expressed by the cells were purified from the culture using an affinity purification column. Subsequently, the purified target proteins were detected by reducing and non-reducing SDS-PAGE. For example, the electrophoresis results of ABU-174, ABU-175, and ABU190 after preparation are shown below. Figure 1A-1C As shown in the figure. The results showed that the purities of the purified ABU-174, ABU-175, and ABU190 were 95.9%, 96.4%, and 98.2%, respectively.

[0242] Subsequently, the antigen reactivity of the purified test antibody was detected by ELISA using recombinant expressed S protein RBD as the coating antigen and horseradish peroxidase (HRP)-labeled Goat anti-human IgG Fc as the secondary antibody. In short, 96-well plates were coated with recombinant expressed S protein RBD (its amino acid sequence is shown in SEQ ID NO: 1459, concentration 0.01 μg / ml or 1 μg / ml), followed by blocking with blocking buffer. Then, the test monoclonal antibodies (control antibody, ABU-174, ABU-175, and ABU190; concentration 0.1 μg / ml) were added and incubated. After washing with ELISA wash buffer, horseradish peroxidase (HRP)-labeled Goat anti-human IgG Fc was added as the secondary antibody (diluted 1:500), and incubation continued. The plates were then washed with PBST and chromogenic reagent was added for color development. The absorbance at OD 450 nm was then read using a microplate reader. The results are shown in Table 2. As can be seen from Table 2, ABU-174, ABU-175, and ABU190 can all specifically recognize and bind to the S protein RBD.

[0243] Table 2: Reactivity of ABU-174, ABU-175, and ABU190 antigen-binding units with the S protein RBD as detected by ELISA (OD450 readings)

[0244]

[0245] Example 4: Evaluation of the binding ability of the antigen-binding unit in this paper to the S protein.

[0246] This embodiment uses surface plasmon resonance (SPR) technology to detect the affinity between the antibody and the RBD region of the Spike protein. Measurements were performed using a Biacore T200. The biotin-labeled SARS-COV-2 RBD domain was first coupled to an SA chip (GE), with a signal resonance unit (RU) increase of 100 units. The running buffer was PBS at pH 7.4 with 0.005% P2O, ensuring the buffer in the analyte (e.g., antibody) was consistent with the running buffer. The purified antibody was serially diluted 3-fold to achieve concentrations between 50 and 0.78125 nM. Measurement results were analyzed using Biacore Evaluation software. A 1:1 model was used to fit all curves to obtain the rate constant Ka for antibody-antigen binding and the rate constant Kd for antibody dissociation from the antibody / antigen complex. The dissociation equilibrium constant KD was calculated, where KD = Kd / Ka. The results are shown in Table 3 below.

[0247] Table 3 lists the binding affinity of the exemplary antigen-binding units in this paper to the RBD region of the Spike protein, where the KD value of each antigen-binding unit is less than 20 nM.

[0248] Table 3. KD values ​​of the binding affinity between the exemplary antigen-binding unit and the RBD region of the Spike protein.

[0249]

[0250]

[0251]

[0252] Figures 2A-2E Further exemplary examples demonstrate the binding affinity of ABU-174, ABU-175, ABU190, ABU297, and ABU367 to the RBD region of the Spike protein. Figures 2A-2E It can be seen that the KD value of ABU-174 is 0.29 nM, the KD value of ABU-175 is 0.039 nM, the KD value of ABU190 is 2.8 nM, the KD value of ABU297 is 0.824 nM, and the KD value of ABU is 0.18 nM. Figures 2A-2E This indicates that ABU-174, ABU-175, ABU190, ABU297, and ABU367 all have good affinity for the novel coronavirus S protein.

[0253] Example 5: Evaluation of the ability of the antigen-binding unit in this paper to neutralize SARS-CoV-2 pseudoviruses

[0254] In this embodiment, referring to the description in Temperton NJ et al., Emerg Infect Dis, 2005, 11(3), 411-416, the neutralizing activity of the antigen-binding unit described herein against SARS-CoV-2 pseudovirus was detected using a microwell cell neutralization assay. The SARS-CoV-2 pseudovirus used in this embodiment was provided by the China National Institutes for Food and Drug Control. It has cell infection characteristics similar to those of the real virus, can simulate the early process of real virus infection of cells, and carries the reporter gene luciferase, allowing for rapid and convenient detection and analysis. The manipulation of the pseudovirus is highly safe; the neutralization experiment can be completed in a P2-level laboratory to detect the neutralization titer of the antibody. The specific steps of the experimental method are as follows.

[0255] 1. Balancing reagent

[0256] Remove the reagents (0.25% trypsin-EDTA, DMEM complete medium) stored at 2-8℃ and allow them to equilibrate at room temperature for at least 30 minutes.

[0257] 2. Experimental Operation

[0258] (1) Take a 96-well plate and arrange the samples as shown in Table 4; wells A2-H2 are set as cell control wells (CC), containing only experimental cells; wells A3-H3 are set as virus control wells (VV), containing experimental cells and pseudovirus; wells A4-A11, B4-B11, C4-C11, D4-D11, E4-E11, F4-F11, G4-G11, and H4-H11 are set as experimental wells, containing experimental cells, pseudovirus, and different concentrations of the antibody to be tested; the remaining wells are set as blanks. The experimental cells and pseudoviruses used in this embodiment are Huh-7 cells and SARS-CoV-2 virus, respectively (both provided by the China National Institutes for Food and Drug Control).

[0259] Table 4.96 Sample Arrangement in the Plate

[0260] 1 2 3 4 5-10 11 12 A - CC VV Dilution 1 Dilution 1 Dilution 1 - B - CC VV Dilution 2 Dilution 2 Dilution 2 - C - CC VV Dilution 3 Dilution 3 Dilution 3 - D - CC VV Dilution 4 Dilution 4 Dilution 4 - E - CC VV Dilution 5 Dilution 5 Dilution 5 - F - CC VV Dilution 6 Dilution 6 Dilution 6 - G - CC VV Dilution 7 Dilution 7 Dilution 7 - H - CC VV Dilution 8 Dilution 8 Dilution 8 -

[0261] (2) Add 100 μl / well of DMEM complete medium (containing 1% antibiotic, 25 mM HEPES, 10% FBS) to the cell control wells; add 100 μl / well of DMEM complete medium to the virus control wells; and add 50 μl / well of the specified concentration of the test antibody diluted in DMEM complete medium to the experimental wells. The antibody concentrations of dilutions 1-8 used in Table 4 are 1 / 30 μg / μl, 1 / 90 μg / μl, 1 / 270 μg / μl, 1 / 810 μg / μl, 1 / 2430 μg / μl, 1 / 7290 μg / μl, 1 / 21870 μg / μl, and 1 / 65610 μg / μl, respectively.

[0262] (3) Dilute the SARS-CoV-2 pseudovirus to approximately 1.3 × 10⁻⁶ using DMEM complete medium. 4 / ml(TCID50); then add 50μl / well of SARS-CoV-2 pseudovirus to the virus control well and experimental well.

[0263] (4) Place the 96-well plate in a cell culture incubator (37°C, 5% CO2) and incubate for 1 hour.

[0264] (5) Dilute the pre-cultured Huh-7 cells to 2×10⁻⁶ using DMEM complete medium. 5 Cells / ml. After the incubation in the previous step, add 100 μl of cells per well to the cell control well, virus control well, and experimental well.

[0265] (6) Place the 96-well plate in a cell culture incubator (37°C, 5% CO2) and incubate for 20-28 hours.

[0266] (7) Remove the 96-well plate from the cell culture incubator, aspirate 150 μl of supernatant from each well, and then add 100 μl of luciferase detection reagent. React at room temperature in the dark for 2 min.

[0267] (8) After the reaction is complete, use a pipette to repeatedly blow and aspirate the liquid in each well 6 to 8 times to ensure complete cell lysis. Then, aspirate 150 μl of liquid from each well and transfer it to the corresponding 96-well chemiluminescence detection plate. Use a chemiluminescence detector (Perkinelmer EnSight multi-microplate reader) to read the luminescence value.

[0268] (9) Calculate the neutralization inhibition rate:

[0269] Suppression rate = [1 - (mean luminescence intensity of experimental wells - mean luminescence intensity of CC wells) / (mean luminescence intensity of VV wells - mean luminescence intensity of CC wells)] × 100%.

[0270] (10) Based on the results of the neutralization inhibition rate, the IC50 of the antibody to be tested was calculated using the Reed-Muench method.

[0271] Table 5 lists the IC50 values ​​of the exemplary antigen-binding units in this paper for the interaction with SARS-CoV-2 pseudoviruses, with each antigen-binding unit having an IC50 value less than 1 μg / ml.

[0272] Table 5. IC50 of the exemplary antigen-binding unit in this paper for neutralizing SARS-CoV-2 pseudoviruses.

[0273] ABU No. IC50 (μg / ml) ABU-174 ＜0.1 ABU-175 ＜0.1 ABU-190 ＜0.1 ABU-207 ＜0.5 ABU-208 ＜0.5 ABU-257 ＜0.5 ABU-290 ＜0.1 ABU-291 ＜0.5 ABU-296 ＜0.1 ABU-297 ＜0.1 ABU-308 ＜0.5

[0274] ABU-322 ＜0.1 ABU-340 ＜0.5 ABU-341 ＜0.1 ABU-344 ＜1 ABU-349 ＜0.1 ABU-351 ＜0.1 ABU-352 ＜0.1 ABU-354 ＜0.1 ABU-355 ＜0.1 ABU-356 ＜0.1 ABU-357 ＜1 ABU-358 ＜0.1 ABU-359 ＜0.1 ABU-360 ＜0.1 ABU-361 ＜0.5 ABU-362 ＜0.5 ABU-365 ＜0.1 ABU-367 ＜0.1 ABU-368 ＜0.5 ABU-369 ＜0.1 ABU-371 ＜1 ABU-372 ＜0.5 ABU-373 ＜0.5 ABU-375 ＜0.1 ABU-376 ＜0.1 ABU-377 ＜0.5 ABU-379 ＜0.5 ABU-380 ＜0.1 ABU-381 ＜0.1 ABU-382 ＜0.1 ABU-386 ＜0.1 ABU-391 ＜1 ABU-392 ＜0.1 ABU-395 ＜0.1

[0275] Figures 3A-3C Further exemplary examples demonstrate the neutralizing activity of ABU-174, ABU-175, and ABU190 against SARS-CoV-2 pseudoviruses. (By...) Figures 3A-3C As can be seen, ABU-174, ABU-175 and ABU190 all have good neutralizing activity, with IC50 values ​​of 0.026 μg / ml (ABU-174), 0.0086 μg / ml (ABU-175) and 0.039 μg / ml (ABU190), respectively.

[0276] Example 6: Evaluation of the ability of the antigen-binding unit in this paper to neutralize SARS-CoV-2 evovirus

[0277] In this embodiment, the neutralizing activity of the test antibodies was assessed using cytopathic effect (CPE) assay and plaque reduction neutralization assay (PRNT). The SARS-CoV-2 virus used was provided by the Academy of Military Medical Sciences, with a titer (TCID50) of 10. 5 / ml, and all experimental procedures were performed in the BSL-3 laboratory.

[0278] 6.1 Cytopathic Effect (CPE) Assay

[0279] (1) Using 5×10 4 At a concentration of / ml, 100μl of Vero E6 cells were added to each well of a 96-well culture plate and cultured at 37°C and 5% CO2 for 24 hours.

[0280] (2) Dilute the antibody to be tested to 10 concentrations: 1 / 10 μg / μl, 1 / 30 μg / μl, 1 / 90 μg / μl, 1 / 270 μg / μl, 1 / 810 μg / μl, 1 / 2430 μg / μl, 1 / 7290 μg / μl, 1 / 21870 μg / μl, 1 / 65610 μg / μl, and 1 / 196830 μg / μl. Take 100 μl of the antibody to be tested at the specified concentration, add an equal volume of SARS-CoV-2 true virus (100 TCID50), and incubate at 37°C and 5% CO2 for 1 h.

[0281] (3) After the culture in step (1) is completed, discard the cell culture medium in the 96-well culture plate and add the mixture (200 μl) containing the antibody to be tested and the real virus prepared in step (2) as the experimental group. After incubation for 1 h, aspirate the supernatant from the well and add 200 μl of DMEM medium (containing 2% antibiotics and 16 μg / ml trypsin) to each well.

[0282] During the experiment, a cell control group and a virus control group were set up in parallel. In the cell control group (4 replicates), after discarding the cell culture medium in the wells, 200 μl of DMEM medium (containing 2% antibiotics and 16 μg / ml trypsin) was added to each well. In the virus control group (3 replicates), after discarding the cell culture medium in the wells, 100 TCID50 of true virus (100 μl) was added to each well, and the mixture was incubated at 37°C for 1 h. After incubation, the supernatant was aspirated from the wells, and 200 μl of DMEM medium (containing 2% antibiotics and 16 μg / ml trypsin) was added to each well.

[0283] (4) Culture the cells at 37℃ and 5% CO2 for 4-5 days.

[0284] (5) Observe cytopathic effect (CPE) under an optical microscope and evaluate the inhibitory activity of different concentrations of monoclonal antibody on CPE based on the cytopathic effect.

[0285] The detection results of antigen-binding unit ABU-174 are shown in Table 6 below. The results show that antigen-binding unit ABU-174 has an inhibitory effect on the virus on cells, and the neutralizing antibody titer is 1.6 ng / μl.

[0286] Table 6. Neutralizing activity of antigen-binding unit ABU-174 against SARS-CoV-2

[0287]

[0288] "+" indicates that the cell has CPE changes, and "-" indicates that the cell has no CPE changes or normal cell morphology.

[0289] The detection results of the antigen-binding unit ABU-175 are shown in Table 7 below. Figure 4 As shown, the results indicate that the antigen-binding unit ABU-175 has an inhibitory effect on the virus on cells, with a neutralizing antibody titer of 0.7 ng / μl.

[0290] Table 7. Neutralizing activity of antigen-binding unit ABU-175 against SARS-CoV-2

[0291]

[0292] "+" indicates that the cell has CPE changes, and "-" indicates that the cell has no CPE changes or normal cell morphology.

[0293] 6.2 Plaque Reduction and Neutralization Test (PRNT):

[0294] (1) Using 5×10 4 At a concentration of / ml, 100μl of Vero E6 cells were added to each well of a 96-well culture plate and cultured at 37°C and 5% CO2 for 24 hours.

[0295] (2) Dilute the antibody to be tested to five concentrations: 50 μg / ml, 10 μg / ml, 2 μg / ml, 0.4 μg / ml, and 0.08 μg / ml.

[0296] (3) After the culture in step (1) is completed, discard the cell culture medium in the 96-well culture plate and add the mixture (200 μl) containing the antibody to be tested and the real virus prepared in step (2) as the experimental group. After incubation for 1 h, aspirate the supernatant from the well and add 200 μl of DMEM medium (containing 2% antibiotics and 16 μg / ml trypsin) to each well.

[0297] During the experiment, a cell control group and a virus control group were set up in parallel. In the cell control group, after discarding the cell culture medium in the wells, 200 μl of DMEM medium (containing 2% antibiotics and 16 μg / ml trypsin) was added to each well. In the virus control group (4 replicates), after discarding the cell culture medium in the wells, 100 μl of 100 TCID50 of true virus was added to each well, and the mixture was incubated at 37°C for 1 h. After incubation, the supernatant was aspirated from the wells, and 200 μl of DMEM medium (containing 2% antibiotics and 16 μg / ml trypsin) was added to each well.

[0298] (4) Culture the cells at 37°C and 5% CO2 for 4 days.

[0299] (5) After formaldehyde fixation, cells were labeled with rabbit anti-SARS-CoV serum (Sino Bio) and peroxidase-labeled goat anti-rabbit IgG (Dako). Plaques were observed after TMB (True Blue, KPL) staining, the inhibition rate was calculated, and dose-response curves were plotted.

[0300] Figure 5 The dose-response profiles for the exemplary antigen-binding units ABU-174, ABU-175, and ABU190 described herein are shown. Figure 5 It is evident that the antigen-binding units ABU-174, ABU-175, and ABU190 all exhibit good neutralizing activity against SARS-CoV-2 evovirus, effectively inhibiting viral infection and cell invasion. Their IC50 values ​​are 0.5 μg / ml (ABU-174), 0.3 μg / ml (ABU-175), and 0.8 μg / ml (ABU-190), respectively.

[0301] Example 7. In vivo efficacy of the antigen-binding unit described herein

[0302] SARS-CoV-2 infects cells through interaction with the hACE2 receptor. The neutralizing efficacy of the antigen-binding unit described in this paper against SARS-CoV-2 in vivo was evaluated in two different animal models.

[0303] 7.1 Efficacy of the antigen-binding unit in hACE2 transgenic mice

[0304] In the first model, hACE2 transgenic mice were used as an animal model and treated with two different modes of prophylaxis: pre-exposure prophylaxis and post-exposure prophylaxis. Specifically, hACE2 transgenic mice were intranasally infected with SARS-CoV-2 virus (2019-nCoV Beta CoV / Wuhan / AMMS01 / 2020) at a dose of 10⁵ TCID₅₀.

[0305] In the pre-exposure prophylaxis modality, 20 mg / kg of the antigen-binding unit described herein was injected intraperitoneally into hACE2 transgenic mice 24 hours prior to viral infection, and the efficacy of the antigen-binding unit as a pre-exposure prophylaxis intervention was tested.

[0306] In the post-exposure prophylaxis regimen, mice were injected with an antigen-binding unit at a dose of 20 mg / kg two hours after viral infection. HG1K (an IgG1 antibody against H7N9 virus) was used as a negative control, injected at 20 mg / kg two hours after viral infection. Body weight, reflecting the health status of the infected mice, was recorded daily for five consecutive days.

[0307] 7.2 Efficacy of antigen-binding units in hamsters

[0308] In the second model, hamsters (Mesocricetus auratus) were used as animal models, and treatment was administered in two different modes: pre-exposure prophylaxis and post-exposure prophylaxis. Specifically, similar to hACE2 transgenic mice, hamsters were infected intranasally with the SARS-CoV-2 provirus (SARS-CoV-2 / WH-09 / human / 020 / CHN) at a dose of 10⁵ TCID₅₀.

[0309] In the pre-exposure prophylaxis model for hamsters, hamsters were injected with the antigen-binding unit described in this paper at a dose of 20 mg / kg one day before viral infection. In the control group, animals were injected with PBS two hours after infection.

[0310] In the post-exposure prophylaxis regimen for hamsters, the antigen-binding unit described in this paper was injected intraperitoneally into hamsters 2 hours after infection at different doses (including 20, 10, 5, and 2 mg / kg) based on body weight. Hamsters injected with phosphate-buffered saline (PBS) served as controls. The body weight of infected hamsters was recorded daily for 7 consecutive days. Seven days after infection, the hamsters were euthanized, and their lungs were collected for viral load analysis.

[0311] Sequence information

[0312] Information on some of the sequences involved in this article is shown in Table 8 below.

[0313] Table 8. Sequence List

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391] Although the specific embodiments described herein have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings published, and all such modifications and variations are within the scope of protection described herein. All descriptions herein are given by the appended claims and any equivalents thereof.

Claims

1. An antigen-binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the sequence of the VH is as shown in SEQ ID NO: 3117 and the sequence of the VL is as shown in SEQ ID NO: 3152.

2. The antigen-binding unit as claimed in claim 1, which binds to the receptor-binding domain (RBD) of the novel coronavirus (SARS-CoV-2) S protein with an equilibrium dissociation constant (KD) of less than 100 nM.

3. The antigen-binding unit as described in claim 1, wherein the concentration is less than 20 μg / ml using an IC50 method. 50 Neutralizes the novel coronavirus (SARS-CoV-2).

4. The antigen-binding unit according to any one of claims 1-3, wherein the antigen-binding unit further comprises a heavy chain constant region (CH).

5. The antigen-binding unit of claim 4, wherein the sequence of CH is as shown in SEQ ID NO: 1457.

6. The antigen-binding unit according to any one of claims 1-3, wherein the antigen-binding unit further comprises a light chain constant region (CL).

7. The antigen-binding unit of claim 6, wherein the sequence of CL is as shown in SEQ ID NO: 1458.

8. A pharmaceutical composition comprising an antigen-binding unit as described in any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. An isolated nucleic acid encoding an antigen-binding unit as described in any one of claims 1 to 7.

10. A vector comprising a nucleic acid sequence encoding an antigen-binding unit as described in any one of claims 1 to 7.

11. The vector of claim 10, wherein the vector is a cloning vector or an expression vector.

12. The vector of claim 10, wherein the vector is a plasmid, a granule, or a bacteriophage.

13. A host cell that expresses an antigen-binding unit as described in any one of claims 1 to 7.

14. A host cell comprising a nucleic acid encoding an antigen-binding unit as described in any one of claims 1 to 7 or a vector as described in any one of claims 10 to 12.

15. The host cell of claim 14, wherein the host cell is a prokaryotic cell, a eukaryotic cell, or a cell line.

16. The host cell of claim 15, wherein the prokaryotic cell is an Escherichia coli cell or a Bacillus subtilis cell.

17. The host cell of claim 15, wherein the eukaryotic cell is a fungal cell, a plant cell, or an animal cell.

18. The host cell of claim 17, wherein the animal cell is an insect cell, a mouse cell, or a human cell.

19. The host cell of claim 14, wherein the host cell is a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, or HEK293 cell.

20. A method for generating an antigen-binding unit as described in any one of claims 1 to 7, comprising: The host cells as described in any one of claims 13-19 are cultured under conditions suitable for expressing the antigen-binding unit. And to isolate the antigen-binding unit expressed by the host cell.

21. A conjugate comprising an antigen-binding unit as described in any one of claims 1 to 7, wherein the antigen-binding unit is conjugated to a chemically functional portion.

22. The conjugate of claim 21, wherein the chemically functional portion is selected from radioactive isotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compound substrate cofactors, and inhibitors.

23. A composition comprising an antigen-binding unit as described in any one of claims 1 to 7, a nucleic acid encoding the antigen-binding unit as described in any one of claims 1 to 7, a vector as described in any one of claims 10 to 12, or a host cell as described in any one of claims 13 to 19.

24. Use of the antigen-binding unit as described in any one of claims 1 to 7, or a nucleic acid encoding the antigen-binding unit as described in any one of claims 1 to 7, in the preparation of a medicament for the prevention and / or treatment of SARS-CoV-2 infection or SARS-CoV-2 pneumonia in patients in need.

25. Use of the antigen-binding unit as described in any one of claims 1 to 7 in the preparation of a medicament for detecting the presence or level of SARS-CoV-2 or its S protein or the receptor-binding domain of the S protein in a sample.

26. Use of the antigen-binding unit according to any one of claims 1 to 7 in the preparation of a medicament for diagnosing whether a subject is infected with SARS-CoV-2.

27. Use of the antigen-binding unit according to any one of claims 1 to 7 in the preparation of a medicament for neutralizing the virulence of SARS-CoV-2 in a sample.

28. Use of the antigen-binding unit according to any one of claims 1 to 7 in the preparation of a kit for detecting the presence or level of SARS-CoV-2 or its S protein or the receptor-binding domain of the S protein in a sample, or for diagnosing whether a subject is infected with SARS-CoV-2.

29. A kit for detecting SARS-CoV-2, comprising an antigen-binding unit as described in any one of claims 1 to 7.

30. The kit of claim 29, wherein the antigen binding unit further comprises a detectable label.

31. The kit of claim 29, wherein the kit further comprises a second antibody that specifically recognizes the antigen-binding unit of any one of claims 1 to 7.

32. The kit of claim 31, wherein the second antibody further comprises a detectable marker.

33. The kit of claim 32, wherein the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, and enzymes.

34. The kit of claim 33, wherein the enzyme is horseradish peroxidase.

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