Anti-sars-cov-2 murine monoclonal antibodies and uses thereof

Abstract

The present application relates to a mouse-derived anti-SARS-CoV-2 monoclonal antibody R58 and its application, the R58 monoclonal antibody comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown as SEQ ID NO:1, and LCDR1, LCDR2 and LCR3 contained in the light chain variable region shown as SEQ ID NO:3, and has high and medium neutralization activity to SARS-CoV-2 live virus.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and virology, and relates to SARS-CoV-2 virus monoclonal antibodies and their applications. Background Technology

[0002] The COVID-19 pandemic, caused by SARS-CoV-2, has been ongoing globally for a year, infecting over 100 million people and causing more than 3 million deaths worldwide. The SARS-CoV-2 virus is highly contagious, and severe illness and death are more likely to occur in elderly individuals with underlying health conditions. SARS-CoV-2 seriously endangers human life and health and has had a profound impact on global economic activity and human life.

[0003] Neutralizing antibodies are antibodies that can eliminate the infectivity of a virus after binding to it. Besides their use in infection diagnosis or the development of antigen detection kits, murine neutralizing antibodies, after humanization, can be used for the clinical treatment of patients infected with SARS-CoV-2. The R58 antibody of this invention has high neutralizing activity (IC50) against live SARS-CoV-2 virus. 50 With a value of 0.02 μg / ml, it has great potential for clinical development. Summary of the Invention

[0004] Specifically, the present invention relates to the following aspects.

[0005] 1. An anti-SARS-CoV-2 antibody or its antigen-binding fragment, comprising HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:1, and LCDR1, LCDR2 and LCR3 contained in the light chain variable region as shown in SEQ ID NO:3;

[0006] Preferably, according to the IMGT numbering system, the antibody comprises:

[0007] HCDR1, comprising the sequence shown in SEQ ID NO:5, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting thereof; HCDR2, comprising SEQ ID NO:5. The sequence shown in NO:6, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with the sequence described therein, or an amino acid sequence having one or more (preferably 1, 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence described therein, or consisting of the sequence described therein, and

[0008] HCDR3, comprising the sequence shown in SEQ ID NO:7, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or composed of said sequence, and the antibody further comprising:

[0009] LCDR1, comprising the amino acid shown in SEQ ID NO:8, or a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with said sequence, or an amino acid sequence having one or more (preferably 1, 2, or 3) conserved amino acid mutations (preferably substitutions, insertions, or deletions) compared to said sequence, or consisting thereof; LCDR2, comprising SEQ ID NO:8. The amino acid sequence shown in NO:9, having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with the sequence described therein, or an amino acid sequence having one or more (preferably 1, 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence described therein, or consisting of the sequence described therein, and

[0010] LCDR3 comprises the sequence shown in SEQ ID NO:10, a sequence having at least 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or is composed of said sequence.

[0011] 2. The anti-SARS-CoV-2 antibody or its antigen-binding fragment described in Project 1, wherein the antibody comprises:

[0012] Heavy chain variable regions, which contain or consist of the following sequences:

[0013] The amino acid sequence shown in SEQ ID NO:1, or

[0014] A sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with the sequence shown in SEQ ID NO:1, or

[0015] An amino acid sequence having one or more (preferably 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, or 30) conserved amino acid mutations (preferably substitutions, insertions, or deletions) compared to the amino acid sequence shown in SEQ ID NO:1.

[0016] The light chain variable region contains or is composed of the following sequences:

[0017] The amino acid sequence shown in SEQ ID NO:3, or

[0018] A sequence having at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with the sequence shown in SEQ ID NO:3, or

[0019] An amino acid sequence having one or more (preferably 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 or 30) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO:3.

[0020] 3. The antibody or antigen-binding fragment thereof described in any one of items 1-2, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, preferably, the heavy chain constant region sequence is shown in SEQ ID NO:17 and the light chain constant region sequence is shown in SEQ ID NO:15.

[0021] 4. The antibody or antigen-binding fragment thereof described in any one of items 1-3, wherein the antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, or a single-chain antibody, and preferably the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, complementarity-determining region (CDR) fragment, single-chain antibody (e.g., scFv), bivalent antibody, or domain antibody.

[0022] 5. A polynucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region or a light chain variable region of an antibody or an antigen-binding fragment thereof described in any one of items 1-4.

[0023] 6. A carrier comprising the polynucleotide molecule described in item 5.

[0024] 7. A host cell comprising the polynucleotide molecule described in item 5, or the vector described in item 6.

[0025] 8. A method for preparing the antibody or antigen-binding fragment thereof according to any one of items 1-4, comprising culturing the host cells of item 7 under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

[0026] 9. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof as described in any one of items 1-4, and a coupling portion conjugated to the antibody or antigen-binding fragment thereof, wherein the coupling portion is a purification tag (such as a His tag), a cytotoxic agent, or a detectable label. Preferably, the coupling portion is a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

[0027] 10. A multispecific antibody, preferably a bispecific antibody, comprising the antibody or antigen-binding fragment thereof described in any one of items 1-4, and an antibody or antigen-binding fragment against other antigens and / or other antigenic epitopes.

[0028] 11. A fusion protein comprising an antibody or an antigen-binding fragment thereof as described in any one of items 1-4.

[0029] 12. A kit comprising the antibody or antigen-binding fragment thereof described in any one of items 1-4, or comprising the antibody conjugate described in item 9, the multispecific antibody described in item 10, or the fusion protein described in item 11.

[0030] 13. The kit described in Item 12, wherein the kit further comprises a second antibody that specifically recognizes the antibody or its antigen-binding fragment; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

[0031] 14. The use of the antibody or antigen-binding fragment thereof described in any one of items 1-4, the antibody-drug conjugate described in item 9, the multispecific antibody described in item 10, or the fusion protein described in item 11 in detecting the presence or level of SARS-CoV-2 in a sample, or in preparing a kit for detecting the presence or level of human SARS-CoV-2 in a sample.

[0032] 15. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of items 1-4, an antibody-drug conjugate as described in item 9, a multispecific antibody as described in item 10, or a fusion protein as described in item 11; optionally, it further comprising a pharmaceutically acceptable carrier and / or excipient.

[0033] 16. The antibody or antigen-binding fragment thereof described in any one of Items 1-4, the antibody-drug conjugate described in Item 9, the multispecific antibody described in Item 10, or the fusion protein described in Item 11, for the treatment of diseases caused by SARS-CoV-2 infection, or for use in the preparation of a medicament for the treatment of diseases caused by SARS-CoV-2.

[0034] 17. The application described in item 16, wherein the drug is in an injectable form, preferably in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0035] 18. A method for treating disease caused by SARS-CoV-2 infection, comprising the step of administering to a subject in need an effective amount of cells containing an antibody or antigen-binding fragment thereof as described in any one of items 1-4, an antibody-drug conjugate as described in item 9, a multispecific antibody as described in item 10, or a fusion protein as described in item 11.

[0036] definition:

[0037] It should be noted that entity qualifiers without a specific quantity should refer to one or more of the entity; for example, "bispecific antibody" should be understood to mean one or more bispecific antibodies. Similarly, the terms "one or more" and "at least one" without a specific quantity are used interchangeably herein.

[0038] "Homology," "identity," or "similarity" refers to the degree of sequence similarity between two peptide molecules or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, and can be compared by alignment. When the same base or amino acid is present at a position in the compared sequences, the molecules at that position are homologous. The degree of homology between multiple sequences is a function of the number of common pairing or homologous sites among these sequences. An "irrelevant" or "non-homologous" sequence has less than 40% homology with one of the sequences of this application, but preferably less than 25%.

[0039] "Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that, at the time of alignment, that percentage of bases (or amino acids) are identical when the two sequences are compared. This alignment and percentage homology or sequence identity can be determined using software programs known in the art, for example, those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Preferably, default parameters are used for alignment. BLAST is an alignment program that uses default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Detailed information about these programs is available at: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi (last accessed May 21, 2008). Biologically equivalent polynucleotides are those with the specific percentage of homology mentioned above that encode polypeptides with the same or similar biological activities.

[0040] The term "encoding," when applied to polynucleotides, refers to a polynucleotide that is believed to "encode" a particular polypeptide, which, when manipulated in its natural state or by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA and / or fragments of that polypeptide. The antisense strand is the complement of this nucleic acid from which the coding sequence can be deduced.

[0041] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, Fv, dAb, Fab / c, complementarity-determining region (CDR) fragments, disulfide-linked Fvs (sdFv), single-chain antibodies (e.g., scFv), bivalent antibodies, or domain antibodies. The term "antibody fragment" also includes any synthetic or genetically modified protein that, like an antibody, can bind to a specific antigen to form a complex.

[0042] "Single-chain variable fragment" or "scFv" refers to a fusion protein of variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some respects, these regions are linked by short linker peptides of 10 to approximately 25 amino acids. This linker may be glycine-rich for flexibility, or contain serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, and vice versa. The protein retains the properties of the original immunoglobulin, except that the constant regions have been removed and linkers have been introduced. ScFv molecules are known in the art and are described in U.S. Patent 5,892,019.

[0043] The antibodies, antigen-binding peptides, their variants or derivatives, as described in this application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. The immunoglobulin molecules or antibody molecules described in this application may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0044] Those skilled in the art can readily identify the amino acids in the CDR and framework regions for any given heavy or light chain variable region, as they have been well defined (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entirety).

[0045] Where a term has two or more definitions as used and / or where acceptable within the scope of this art, the definition of the term used herein is intended to encompass all meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe a discontinuous antigen-binding site present in the variable regions of both heavy and light chain polypeptides. Such a specific region is described by Kabat et al. in the U.S. Department of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al. in J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety. According to the definition by Kabat and Chothia, a CDR comprises overlapping amino acid residues, or amino acid substructures, when compared with each other. However, the application of each definition of a CDR for an antibody or its variant will be within the scope of the terminology defined and used herein. The appropriate amino acid residues containing a CDR as defined in each of the references cited above are listed in the table below for comparison. The exact number of residues containing a particular CDR will vary depending on the sequence and size of that CDR. Given the amino acid sequence of the variable region of the antibody, those skilled in the art can typically determine which residues contain a specific CDR.

[0046] [Table 1] Definition of antibody variable region

[0047] Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

[0048] Kabat et al. also defined a numbering system for variable domain sequences, applicable to any antibody. Those skilled in the art can undoubtedly use the "Kabat numbering" system for any variable domain sequence without relying on any experimental data outside of the sequence itself. The "Kabat numbering" used herein refers to the numbering system described by Kabat et al., as documented in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0049] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), consists of about 5-7 amino acids, and terminates at the next tryptophan residue. CDR-H2 begins 15 residues after the end of CDR-H1, consists of about 16-19 amino acids, and terminates at the next arginine or lysine residue. CDR-H3 begins approximately 33 amino acid residues after the end of CDR-H2; consists of 3-25 amino acids; and terminates at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue); consists of about 10-17 residues; and terminates at the next tryptophan residue. CDR-L2 begins approximately 16 residues after the end of CDR-L1 and consists of about 7 residues. CDR-L3 begins at approximately the 33rd residue after the end of CDR-L2 (i.e., after the cysteine ​​residue); it comprises approximately 7–11 residues and terminates at sequence F or WGXG, where X is any amino acid.

[0050] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide used in this application may comprise a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain having both a CH1 and CH3 domains; a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this application comprises a polypeptide chain having a CH3 domain. Additionally, antibodies used in this application may lack at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). As described above, but those skilled in the art should understand that the heavy chain constant regions may be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0051] A "light chain-heavy chain pair" refers to a combination of light and heavy chains that can form a dimer through disulfide bonds between the CL and CH1 domains of the light chain.

[0052] As used herein, the term "chimeric antibody" will be used to refer to any antibody whose immune-reactive region or site is derived from or originates from a first species and whose constant region (which may be complete, partial, or modified according to this application) is derived from a second species. In some embodiments, the target-binding region or site will be derived from a non-human source (e.g., mouse or primate) and the constant region will be derived from a human.

[0053] The "percentage humanization" used in this paper is calculated as follows: the number of framework amino acid differences (i.e., non-CDR differences) between humanized and germline domains is determined, this number is subtracted from the total number of amino acids, then divided by the total number of amino acids, and then multiplied by 100.

[0054] As used herein, the term "treatment" refers to therapeutic treatment and preventative or preventative measures that prevent or slow (mitigate) adverse physiological changes or diseases, such as the development of cancer, in a subject. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of the severity of the disease, stabilization (e.g., preventing it from worsening) of the disease state, delay or slowing of disease progression, improvement or mitigation of the disease state, and relief (whether partial or complete), whether detectable or not. "Treatment" may also refer to an extension of survival compared to the expected survival without treatment. Conditions requiring treatment include those already having a condition or symptoms, those prone to having a condition or symptoms, and those where prevention of a condition or symptoms will be undertaken.

[0055] Any of the antibodies or peptides described above may also include additional peptides, forming conjugates or fusion proteins, such as peptides encoded as described herein, signal peptides at the N-terminus of antibodies that guide secretion, or other heterologous peptides as described herein.

[0056] Those skilled in the art should also understand that the antibodies described herein can be modified so that their amino acid sequences differ from those of naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity with the starting sequence, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0057] In addition, nucleotide or amino acid substitutions, deletions, or insertions can be made to perform conserved substitutions or alterations in "non-essential" amino acid regions. For example, the polypeptide or amino acid sequence from the specified protein may be identical to the starting sequence, except for one or more independent amino acid substitutions, insertions, or deletions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more independent amino acid substitutions, insertions, or deletions. In some embodiments, the polypeptide or amino acid sequence from the specified protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 independent amino acid substitutions, insertions, or deletions relative to the starting sequence.

[0058] In other embodiments, the antigen-binding polypeptide of this application may contain conserved amino acid substitutions.

[0059] "Conservative amino acid substitution" refers to the substitution of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues of immunoglobulin polypeptides are preferably substituted with other amino acid residues from the same side chain family. In another embodiment, a string of amino acids may be substituted with a structurally similar string of amino acids that differ in sequence and / or composition of the side chain family.

[0060] The table below provides non-limiting examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates a conserved substitution between the two amino acids.

[0061] [Table 2] Non-restrictive list of conservative amino acid substitutions

[0062] C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0063] Examples of techniques that can be used to generate single-chain FVS (scFVS) and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For certain applications, including the use of antibodies in vivo and in vitro detection assays, chimeric, humanized, or human antibodies may be preferred. Chimeric antibodies are antibodies whose different portions are derived from molecules of different animal species, such as antibodies containing a variable region derived from a mouse monoclonal antibody and a constant region of a human immunoglobulin. Methods for manufacturing chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715; 4,816,567 and 4,816,397, the entire contents of which are incorporated herein by reference.

[0064] Humanized antibodies are antibody molecules derived from non-human species that bind to the desired antigen. These antibody molecules have one or more complementarity-determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Typically, framework residues in the human framework region are altered by substitution of corresponding residues from a CDR donor antibody, preferably to enhance antigen-binding ability. These framework substitutions are identified by methods known in the art, for example, by establishing interaction models of CDRs and framework residues to identify framework residues important for antigen binding and sequence comparison, and to identify aberrant framework residues at specific locations. (See, for example, U.S. Patent No. 5,585,089 to Queen et al.; Nature 332:323 (1988) to Riechmann et al., the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR transplantation (EP 239,400; PCT Publication No. WO 91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and shuffling (U.S. Patent No. US 5,565,332, the entire contents of which are incorporated herein by reference).

[0065] Using conventional recombinant DNA techniques, one or more CDRs of the antigen-binding polypeptide of this application can be inserted into a frame region, for example, into a human frame region to humanize a non-human antibody. This frame region can be a naturally occurring or common frame region, and is preferably a human frame region (see, for example, Chothia et al., J. Mol. Biol. 278:457-479 (1998), a list of human frame regions). Preferably, the combination of the frame region and the CDR results in a polynucleotide encoding a polypeptide that specifically binds to at least one antigenic epitope of the desired polypeptide, for example, LIGHT. Preferably, one or more amino acid substitutions can be made within the frame region, and preferably, these amino acid substitutions enhance the antibody's antigen-binding ability. Furthermore, this method can be used to obtain amino acid substitutions or deletions of one or more variable region cysteine ​​residues (which participate in intrachain disulfide bond formation), thus producing an antibody molecule lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotide are included within the scope of this application and within the scope of the prior art.

[0066] Furthermore, techniques for producing “chimeric antibodies” by splicing genes from mouse antibody molecules (Morrison et al., Proc. Natl. Acad. Sci. USA: 851-855 (1984); Neuberger et al., Nature 372: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)) can be used to combine human antibody molecule genes with appropriate antigen specificity and appropriate biological activity. As used herein, chimeric antibodies are molecules in which different parts are derived from different animal species, such as antibodies containing the variable region from mouse monoclonal antibodies and the constant region of human immunoglobulins.

[0067] However, another efficient method for generating recombinant antibodies is disclosed in Newman, Biotechnology 10:1455-1460 (1992). Specifically, this technique results in the production of primate-like antibodies containing monkey variable domains and human constant sequences. This document is incorporated herein by reference in its entirety. Furthermore, this technique is also described in commonly assigned U.S. Patent Nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference.

[0068] Alternatively, cell lines that produce antibodies can be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in a variety of laboratory manuals and major publications. In this regard, the techniques suitable for use herein are described, for example, as follows in Current Protocols in Immunology, edited by Coligan et al., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety, including supplemental references.

[0069] Furthermore, standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the antibody of this application, including, but not limited to, site-directed mutagenesis and PCR-mediated mutations, which produce amino acid substitutions. Preferably, the variants (including derivatives) encode fewer than 50, fewer than 40, fewer than 30, fewer than 25, fewer than 20, fewer than 15, fewer than 10, fewer than 5, fewer than 4, fewer than 3, or fewer than 2 amino acid substitutions relative to the reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations can be randomly introduced along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutations that retain activity. Attached Figure Description

[0070] Figure 1 .pHRNT vector map.

[0071] Figure 2 The pHRNT-RBD map of the mRNA transcription template plasmid for RBD was cloned.

[0072] Figure 3 The RBDmRNA vaccine induces high levels of SARS-CoV-2 virus-specific antibodies.

[0073] Figure 4 Determination of neutralizing antibody titers against real viruses

[0074] Figure 5 Memory B cell sorting in RBD group mice

[0075] Figure 6 The affinity of the R58 antibody for SARS-CoV-2 RBD

[0076] Figure 7Neutralizing activity of negative control antibody (irrelevant isotype IgG antibody).

[0077] Figure 8 Neutralizing activity of R58 antibody.

[0078] Figure 9 Neutralizing activity of the positive control antibody CB6. Detailed Implementation

[0079] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions such as those described in *Molecular Cloning: A Laboratory Manual*, edited by J. Sambrook et al. (3rd edition, published by Science Press) or as recommended by the manufacturer. Unless otherwise specified, all reagents used in the experiments are commercially available.

[0080] Example 1: mRNA vaccine preparation

[0081] (1) Acquisition of the target gene: The amino acid sequence of the SARS-CoV-2 receptor-binding domain RBD was obtained from Genebank MN908947, and its amino acid sequence is shown in SEQ ID NO:11. After codon optimization, its nucleotide sequence is shown in SEQ ID NO:12. It was synthesized by Beijing Qingke Xinyue Biotechnology Co., Ltd. and cloned into the mRNA transcription template pHRNT vector (e.g., purchased from Beijing Qingke Xinyue Biotechnology Co., Ltd.). Figure 1 After that, the mRNA transcription template plasmid pHRNT-RBD was obtained. Figure 2 ).

[0082] (2) mRNA transcription

[0083] The mRNA transcription template pHRNT-RBD was linearized by restriction endonuclease BamHI, followed by agarose gel electrophoresis to confirm complete linearization, and then the linearized transcription template was recovered using a gel recovery kit.

[0084] Prepare the mRNA in vitro transcription reaction system according to the table below (enzymes and reagents used were purchased from NEB Corporation, USA):

[0085]

[0086] The total volume of RNase-free water and DNA template is 7.5 μL.

[0087] After reacting at 37°C for 4 hours, add 1 μl of RNase-free DNase I and react at 37°C for 15 minutes.

[0088] Then, RNA is isolated and purified. There are various methods for RNA isolation and purification, such as ammonium acetate precipitation, LiCl precipitation, organic solvent extraction-ammonium acetate precipitation, and RNA binding column purification. The LiCl precipitation method will be used as an example:

[0089] a) Add 7.5M LiCl to the RNA solution to make the final LiCl concentration 2.5M;

[0090] b) Overnight at -20℃;

[0091] c) Centrifuge at 12000 rpm / min for 15 minutes and discard the solution;

[0092] d) Add 75% ethanol pre-cooled to -20℃ to the precipitate, wash the precipitate, then centrifuge at 12000 rpm / min for 1 minute, discard the ethanol solution, and repeat the washing three times;

[0093] e) Air-dry the RNA precipitate at room temperature, then dissolve the RNA in RNase-free water. After determining the RNA concentration using Nanodrop, store at -80°C.

[0094] (2) mRNA capping

[0095] The following method can be used to cap mRNA. 7 Gppp(m 2′-O N1, the specific method is as follows:

[0096] a) The in vitro transcribed mRNA (50-60 μg) was diluted to 67 μl with RNase-free water;

[0097] b) Incubate at 65℃ for 5-10 minutes, then cool on ice;

[0098] c) Prepare the reaction mixture according to the table below (the enzymes and reagents used were purchased from NEB Corporation, USA);

[0099]

[0100] d) Before the reaction begins, add the cooled mRNA from b) to the mixture in c), then add 4 μl of capped enzyme, and react at 37°C for half an hour.

[0101] The capped mRNA was then isolated and purified. The specific method is as described above, and finally, mRNA-RBD was obtained.

[0102] (3) mRNA nanoparticle packaging

[0103] mRNA was packaged into nanoparticles using microfluidic technology. The aqueous phase was an mRNA solution (50 mM sodium acetate buffer, pH 4.0), and the ethanol phase was a lipid mixture prepared by dilinoleyl-methyl-4-dimethylaminobutyrate, distearate, phosphatidylcholine, cholesterol, and PEG2K-DMPE in a molar ratio of 50:10:38.5:1.5. During packaging, the total flow rate of the aqueous and ethanol phases was 12 ml / min, and the volume ratio of the aqueous to ethanol phases was 3:1. After packaging, the buffer solution was replaced with PBS using a dialysis bag to obtain the mRNA vaccine RBD. The concentration of the encapsulated mRNA was then determined using RiboGreen reagent and stored at 4°C for later use.

[0104] Example 2: Evaluation of vaccine-induced antibody levels

[0105] Ten female 6-8 week old BALB / c mice were randomly divided into two groups of five each. One group received an intramuscular injection of a placebo (packaged in the same way as the vaccine group but with lipid nanoparticles encapsulated with polycytidylic acid, purchased from Sigma) and the other received an RBD (15 μg) mRNA vaccine. A booster immunization was administered at week 4 post-immunization. Blood samples were collected at weeks 4 and 8 post-immunization. Serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at -80°C for later use.

[0106] (1) Antigen-specific antibody titer determination

[0107] The SARS-CoV-2 RBD protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was diluted to 2 μg / ml with ELISA coating buffer. 100 μl was added to each well of the ELISA plate and incubated overnight at 4°C. One hour after blocking the ELISA plate the next day, mouse serum was serially diluted 2-fold and added to the ELISA plate. The plate was incubated at 37°C for 1 hour, followed by washing three times with PBS (PBST) containing 0.05% Tween 20. Goat anti-mouse HRP secondary antibody (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was added, and the plate was incubated at 37°C for 1 hour. After washing five times with PBST, TMB chromogenic buffer was added, and the assay was stopped with 2M hydrochloric acid. The OD450 value was read on a microplate reader. ELISA results showed that after one immunization, the RBD mRNA vaccine could induce high levels of SARS-CoV-2 virus-specific antibodies. Figure 3 After booster immunization, antibody levels increased 170-fold. Figure 3 ).

[0108] (2) Determination of neutralizing antibody titer against real virus

[0109] Mouse serum was serially diluted 2-fold and mixed with 100 TCID45. 50Equal volumes of wild-type SARS-CoV-2 true virus (HB01 strain, derived from the P3 laboratory of the Institute of Microbiology, Chinese Academy of Sciences) were mixed and incubated at 37°C for 1 hour. Then, 100 μl of a solution with a density of 1.5 × 10⁻⁶ was added to 100 μl of the mixture. 5 Vero E6 cells were incubated at 37°C for 72 hours. Cell pathogenesis was observed under a microscope. The serum dilution factor required to protect 50% of cells from viral infection was calculated using the Karber method; this was defined as the true virus neutralizing antibody titer (NT). 50 Value. The results showed that the neutralizing antibody titer NT produced by the initial RBD mRNA vaccine administration was [value missing]. 50 The titer of neutralizing antibodies after booster immunization was 263 (NT). 50 Increased by 222 times ( Figure 4 ).

[0110] Example 3: Screening of memory B cells in mice after immunization

[0111] (1) Acquisition of lymphocytes

[0112] Eight weeks after the initial immunization, mice were anesthetized and euthanized, and dissected to extract lymphocytes. Eight lymph nodes were collected from each mouse (one from each side: superficial parotid lymph node, axillary lymph node, subiliac lymph node, and popliteal lymph node). After extraction, the lymph nodes were placed in 1640 medium containing 1% fetal bovine serum, ground, and filtered through a 0.45 μm filter.

[0113] (2) Lymphocyte staining

[0114] Lymphocytes filtered using a refrigerated centrifuge were centrifuged at 400g for 10 minutes at 4°C. The supernatant was removed, and the cells were resuspended in 1 ml of PBS solution containing 0.04% BSA (i.e., staining buffer) and transferred to a 1.5 ml EP tube. The cells were centrifuged again for 10 minutes, the supernatant was removed, and the cells were resuspended in the residual staining buffer at the bottom of the tube. 4 μl of cells were taken, and 400 μl of staining buffer was added. After mixing, the cells were evenly divided into 8 EP tubes, 50 μl per tube. Seven tubes were used as single-positive tubes (stained with FITC anti-mouse GL7 antigen, PE anti-mouse CD138, PE / Cyanine7 anti-mouse CD38, APC anti-mouse CD93, Brilliant Violet 421 anti-mouse CD45R / B220, Brilliant Violet 510 anti-mouse IgD, or strep-BV711 antibody, respectively; all antibodies were purchased from Biolegend). One tube was used as a negative control tube (unstained with any antibody). Resuspend the remaining cells in 250 μl of staining buffer and mix well; use this as a sample tube. Add biotin-labeled SARS-CoV-2 RBD protein (final concentration 400 nM, purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) to the sample tube and the strep-BV711 single-staining tube, and incubate at 4°C in the dark for 30 minutes. Wash the cells twice with staining buffer, add the corresponding antibody to the cells (refer to the manufacturer's instructions for concentration), and incubate at 4°C in the dark for 30 minutes. Wash the cells twice more with staining buffer, resuspend the cells in 2 ml of staining buffer, filter through a 0.45 μm filter, and transfer to a flow cytometry tube, ready for instrumentation.

[0115] (3) Sorting of antigen-specific memory B cells

[0116] Cells that simultaneously meet the following criteria using flow cytometry (BD Biosciences) for gating and sorting are antigen-specific memory B cells: positive for FITC-GL7, negative for PE-CD138, negative or weakly positive for PE / Cy7-CD38, negative for APC-CD93, strongly positive for BV421-B220, negative for BV510-IgD, and positive for strep-BV711.

[0117] The results showed that approximately 20,000 target cells were obtained from the RBD mRNA vaccine fraction. Figure 5 Ultimately, the number of cells that met the viability standard was detected by the instrument, which was approximately 9,000.

[0118] Example 4: High-throughput sequencing and antibody sequence acquisition

[0119] Following the Chromium single-cell 5′ library construction manual from 10×Genomics, monoclonal memory B cell BCR sequencing samples were processed. After being sorted by flow cytometry, the memory B cells were centrifuged and resuspended in PBS buffer containing 3% fetal bovine serum (Sigma-Aldrich). Cell count and quality control were then performed using a cell counter. The processed B cells needed to have a viability greater than 70%. After determining the cell density and quality, the cells were loaded into three channels to ensure approximately 1000-3000 cells per channel. In the 10×Chromium instrument, oil droplets (GEMs) formed by the gel beads and single cells in each channel were collected and subjected to GEM reverse transcription. After demulsification of the GEMs, the GEM-RT products were amplified by cyclic PCR and purified using SPRIselect beads (Beckman Coulter).

[0120] Single-cell BCR V(D)J libraries were prepared according to the 10×Genomics user guide. The next step was performed using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Quantification was then performed using the Kapa Library Quantification Kit (Kapa Biosystems). Finally, the prepared libraries were sequenced using paired-end sequencing on an Illumina NovaSeq system. The BCL data were converted to FASTQ files using Illumina bcl2fastq2.20.

[0121] We retained the first 26 bases for reading a 16nt cell barcode and a 10nt unique molecular identifier (UMI). The FASTQ file was then analyzed. Barcode processing and single-cell V(D)J sequence analysis were performed using Cell Ranger Single-Cell Software Suite (version 3.1.0). The Cell Ranger V(D)J pipeline was then used for FASTQ file processing. First, reads were filtered for valid cell barcodes and UMIs. Filtered reads were aligned to the GRCm38 V(D)J reference genome, assembled into contigs, and then the V, D, and J fragments were defined as a single contig. The CDR3 sequence was identified, and based on this data, the readability of the contig was determined, indicating that it likely corresponds to a functional B-cell receptor. Finally, the barcode was identified as the target cell if the following three requirements were met: 1) It must be a readable and reliable contig; if there is only one such contig, there must be at least one UMI supporting its J region. 2) There must be at least three filtered UMIs, each with at least two read pairs. 3) Calculate the N50 value for the number of read pairs for each UMI across all barcodes. If, for a given barcode, the maximum number of read pairs for the filtered UMIs is less than 3% of N50, the barcode should not be considered a single cell. Cell sets with identical readable CDR3 sequences are defined as clonoids through precise nucleotide matching. V(D)J sequences and clonoids in 10×Chromium-generated single-cell 5′ data were analyzed, searched, and visualized using the LoupTem V(D)J browser, and further annotated and analyzed using IgBLAST v1.6.1 to identify variable region gene fragments and somatic mutations.

[0122] The results showed that after the cells were loaded onto the instrument, 4060 cells were detected in the RBD vaccine group, including 3615 heavy chain sequences and 4163 light chain sequences. 3048 cells had matching light and heavy chain sequences. Cells with completely identical CDR3 regions of light and heavy chains were defined as a clone, with a total of 1611 clones.

[0123] Example 5: Antibody construction, expression, and activity assay

[0124] (1) Construction of antibody heavy chain and light chain plasmids

[0125] The top 100 most frequent clones from the RBD vaccine antibody library (corresponding antibody names R1, R2, ..., up to R100) were selected. Nanjing GenScript Biotech Co., Ltd. was commissioned to optimize the codons of the amino acid sequences of the variable regions of the monoclonal antibodies. Then, a signal peptide sequence and the constant region of the mouse IgG2a antibody were added to the 5' and 3' ends of the gene, respectively, before the complete gene was synthesized. The amino acid and nucleotide sequences of the signal peptide are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively; the amino acid and nucleotide sequences of the constant region of the mouse IgG2a light chain are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively; and the amino acid and nucleotide sequences of the constant region of the mouse IgG2a heavy chain are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively. After the complete light and heavy chain genes of the antibody were synthesized, they were constructed into the pCAGGS vector, and finally, complete light and heavy chain expression plasmids of the monoclonal antibody were obtained.

[0126] (2) Determination of antibody concentration in cell expression supernatant

[0127] Heavy and light chain plasmids paired with the same antibody were co-transfected into 293T cells at a ratio of 2:3. Four to six hours after transfection, the cells were washed twice with PBS and cultured in serum-free DMEM medium. Cell supernatant was collected three days post-transfection, centrifuged to remove cell debris, and antibody supernatant was obtained.

[0128] The antibody concentration in the supernatant was determined using the Mouse IgG2a Elisa Kit (purchased from Multi Sciences). First, mouse anti-IgG2a standards or the cell supernatant to be tested were added to a commercially available ELISA plate pre-coated with anti-mouse IgG2a monoclonal antibody and incubated at room temperature for 2 hours. After washing 6 times with PBST, HRP-conjugated detection antibody was added and incubated at room temperature for 1 hour. After 6 washes with PBST, TMB was added for color development, and the reaction was stopped with 2M sulfuric acid. The OD450 value was read on a microplate reader. A standard curve was calculated based on the concentration and reading of the standards, and the antibody concentration in the cell supernatant was calculated based on the standard curve and the absorbance of the sample.

[0129] (3) Evaluation of neutralizing activity of antibody expression supernatant

[0130] Based on the antibody concentration quantification results in the cell supernatant, the antibody was diluted to different concentration ranges (>6 μg / ml, 1-6 μg / ml, 0.1-0.6 μg / ml, and <0.1 μg / ml). Four replicates were performed for each sample and each concentration gradient, with each replicate consisting of 50 μl of antibody and 50 μl of 100 TCID50. 50An equal volume of wild-type SARS-CoV-2 true virus (HB01 strain) was mixed and incubated at 37°C for 1 hour. Then, 100 μl of a solution with a density of 1.5 × 10⁻⁶ was added to the mixture. 5 / mL of Vero E6 cells. Incubated at 37°C for 72 hours, and the cytopathic effects were observed under a microscope. The IC50 of the antibody was defined. 50-100％ The value represents the minimum antibody concentration range required to inhibit cytopathic effects in 50% or more of the cells (i.e., two replicates). If the cytopathic effect rate remains above 50% at antibody concentrations >6 μg / ml, the antibody is defined as having no neutralizing activity. Finally, R58 antibodies with strong neutralizing activity were screened, and their IC50 values ​​were [not specified in the original text]. 50-100％ Less than 0.1 μg / ml. The amino acid and nucleotide sequences of the variable regions of the light and heavy chains of the neutralizing antibody, and the amino acid sequences of the CDR region are shown in SEQ ID NO:1 to SEQ ID NO:10.

[0131] Example 7: Expression, isolation, and purification of R58 antibody

[0132] 14-16 hours before transfection, divide the cells with high cell density into plates (e.g., passage a 10cm culture dish 100% filled with 293T cells at a 1:3 ratio). After 14-16 hours, when the cell density reaches more than 70%, transfection can be performed.

[0133] The heavy and light chain expression plasmids of the R58 antibody from Example 5(1) were co-transfected into 293T cells at a ratio of 2:3. After 4-6 hours of transfection, the cells were washed twice with PBS and cultured in serum-free DMEM medium. Cell supernatants were collected on days 3 and 7 after transfection, respectively. Cell debris was removed by centrifugation, and the antibody supernatants obtained from the two transfections were mixed for subsequent purification of the target protein.

[0134] Connect a Protein G (5 ml) HP affinity column (GE) to an AKTA Purifier / Explorer / FPLC / START (GE). The procedure on the instrument is as follows: First, flush the column with water to remove the 20% ethanol. Then, equilibrate the column with 20 mM Na3PO4, pH 7.0 buffer. Once the conductivity reading on the instrument is 4.5% and stable, inject the antibody supernatant into the column using a 10 ml loop at a flow rate of 2 ml / min. After the UV stabilizes, add approximately 0.8 ml of 1 M Tris pH 9.0 buffer (collection volume approximately 3.2 ml) to the subsequent collection tube. Then, change the program to 100% 0.1 M Gly pH 3.0 to elute the antibody adhering to the column. Collect the eluted sample and then replace the antibody buffer with PBS using a concentration and replacement method. The sample can be used directly or aliquoted and stored at -80°C.

[0135] Example 9: R58 antibody affinity assay

[0136] R58 antibody affinity was determined using SPR (surface plasmon resonance) technology, and a Biacore 8k (purchased from GE Healthcare, USA) was used for sample detection. The chip surface was equilibrated with HBS-EP buffer at a flow rate of 10 μl / min for 5 minutes, followed by activation by injecting a 1:1 mixture of "NHS+EDC" at a flow rate of 10 μl / min for 100 seconds. Anti-mouse IgG Fc (purchased from GE Healthcare, USA) diluted in 10 mM sodium acetate buffer was injected at a flow rate of 10 μl / min for approximately 180 seconds for conjugation. Finally, ethanolamine was injected at a flow rate of 10 μl / min for 200 seconds for surface blocking. Three pre-cycles using HBS-EP buffer as the sample were performed to equilibrate the chip and stabilize the baseline. The antibody was captured by injecting R58 antibody (20 μg / mL) at a flow rate of 30 μl / min for 120 seconds. Then, 100 nM of SARS-CoV-2 RBD-his protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was injected at a flow rate of 30 μl / min for 240 seconds for binding. Next, buffer was injected at a flow rate of 30 μl / min for 300 seconds for dissociation. Regeneration was then achieved by injecting 10 mM Gly-HCl (pH 1.7) at a flow rate of 30 μl / min three times, each time for 30 seconds, completing one cycle. The antibody concentration was varied for each gradient until all gradient concentrations (6.125 nM, 12.5 nM, 25 nM, 50 nM, 100 nM) were achieved. Experimental data were double-subtracted (control channel and zero concentration) and fitted to a "1:1 binding" model using Biacore 8K evaluation software (GE), analyzing binding kinetic parameters and calculating the affinity constant (kD). The results showed that the affinity kD value of the R58 antibody for SARS-CoV-2 RBD was 9.74 × 10⁻⁶. -10 M( Figure 6 ).

[0137] Example 10: Neutralizing activity of R58 antibody

[0138] Serially dilute the antibody two-fold, performing eight replicates for each concentration of each antibody sample, with each replicate consisting of 50 μl and 50 μl of 100 TCID50. 50Wild-type SARS-CoV-2 virus (HB01 strain, preserved in the P3 laboratory of the Institute of Microbiology, Chinese Academy of Sciences) was mixed in equal volumes and incubated at 37°C for 1 hour. The positive control antibody was CB6 (Institute of Microbiology, Chinese Academy of Sciences; the light and heavy chain amino acid sequences of CB6 were obtained from the GeneBank database, and the expression codes were MT470196 and MT470197, respectively; the neutralizing activity of CB6 antibody against SARS-CoV-2 virus is described in the article Shi, R., Shan, C., Duan, X. et al. A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2. Nature 584, 120–124 (2020).), and the negative control antibody was an unrelated isotype IgG antibody (Institute of Microbiology, Chinese Academy of Sciences). Then, 100 μl of a 1.5 × 10⁻⁶ ammonia solution was added to the mixture. 5 / mL of Vero E6 cells were incubated at 37℃ for 72 hours, and the cytopathic effect was observed under a microscope. The number of cytopathic effects in parallel sample wells for each concentration was counted. The neutralization inhibition rate was calculated as 100% - (number of cytopathic wells / total number of wells) * 100%. Based on the neutralization inhibition rate results, an antibody concentration-inhibition rate curve was fitted using the biostatistical software Graphpad, and the IC50 was calculated. 50 Value. The results showed that the negative control antibody had no neutralizing activity ( Figure 7 R58 antibody IC 50 The value was 0.02 μg / ml. Figure 8 Its neutralizing activity is stronger than that of the positive control antibody CB6 (IC). 50 The value is 0.4 μg / ml. Figure 9 ).

[0139] sequence list

[0140] R58 antibody heavy chain variable region amino acid sequence SEQ ID NO:1

[0141] QIQLVQSGPELKKPGETVKISCTASVYTFTNYGMNWVKQAPGKGLKWMGWINTYS

[0142] GEPTYSDDFKGRFAFSLETSASTAYLQINNLRNEDTATYFCARGGNWDWFFDVWGA

[0143] GTTVTVSS

[0144] R58 antibody heavy chain variable region nucleotide sequence SEQ ID NO:2

[0145] CAAATTCAGCTGGTGCAGAGTGGGCCAGAGCTCAAGAAACCTGGAGAAACTGT

[0146] CAAAATAAGCTGTACTGCATCTGTCTATACCTTCACCAACTATGGCATGAACTGGG

[0147] TGAAGCAGGCTCCTGGGAAAGGCCTTAAGTGGATGGGCTGGATCAATACTTATTC

[0148] AGGAGAGCCCACCTACAGTGATGACTTCAAGGGGAGGTTTGCCTTTTCTTTGGA

[0149] GACCTCAGCTTCCACAGCCTACCTGCAGATCAACAACCTAAGAAATGAAGACAC

[0150] AGCTACATACTTCTGCGCCCGGGGTGGAAATTGGGACTGGTTCTTTGATGTGTGG

[0151] GGTGCAGGCACCACAGTAACGGTTTCCAGC

[0152] Amino acid sequence of the variable region of the light chain of R58 antibody, SEQ ID NO:3

[0153] DVVLTQSPVTLSVTPGDRVSLSCRASQSVSNFLHWYQQKSHESPRLLIKYASQSISGI

[0154] PSRFSGSGSGTDFTLNINSVETEDFGMYFCQQTKTWPLTFGAGTKLELK

[0155] Nucleotide sequence of the variable region of the light chain of R58 antibody, SEQ ID NO:4

[0156] GATGTGGTTTTAACTCAAAGTCCTGTGACTCTTTCAGTCACCCCAGGAGATCGCG<000081​​​​CAGAAGAGCCATGAATCACCGAGGCTGCTCATCAAGTATGCCAGCCAGAGCATTT

[0159] CTGGCATACCCAGCAGATTTTCTGGGAGTGGCTCCGGTACAGACTTCACGCTCAA

[0160] CATCAATTCAGTGGAGACAGAAGACTTTGGCATGTACTTCTGCCAACAGACCAAGACCTGGCCTTTGACATTTGGAGCAGGGACTAAACTGGAGCTGAAA

[0161] R58 antibody heavy chain variable region CDR1 amino acid sequence SEQ ID NO:5

[0162] VYTFTNYG

[0163] R58 antibody heavy chain variable region CDR2 amino acid sequence SEQ ID NO:6

[0164] INTYSGEP

[0165] R58 antibody heavy chain variable region CDR3 amino acid sequence SEQ ID NO:7

[0166] ARGGNWDWFFDV

[0167] The amino acid sequence of the CDR1 variable region of the R58 antibody light chain is SEQ ID NO:8

[0168] QSVSNF

[0169] The amino acid sequence of the CDR2 variable region of the R58 antibody light chain is SEQ ID NO:9

[0170] YAS

[0171] R58 antibody light chain variable region CDR3 amino acid sequence SEQ ID NO:10

[0172] QQTKTWPLT

[0173] The amino acid sequence of the SARS-CoV-2 receptor-binding domain (RBD) is SEQ ID NO:11

[0174]

[0175]

[0176] The nucleotide sequence of the SARS-CoV-2 receptor-binding domain RBD is SEQ ID NO:12

[0177]

[0178] Signal peptide amino acid sequence SEQ ID NO:13

[0179] METDTLLLWV LLLWVPGSTG D

[0180] Signal peptide nucleotide sequence SEQ ID NO:14

[0181] ATGGAGACGG ATACGCTGCT CCTGTGGGTT TTGCTGCTCT GGGTTCCAGG

[0182] TTCCACTGGT GAC

[0183] The amino acid sequence of the constant region of the mouse IgG2a light chain is SEQ ID NO:15

[0184]

[0185] SEQ ID NO:16 nucleotide sequence of the constant region of the mouse IgG2a light chain

[0186]

[0187] The amino acid sequence of the constant region of the mouse IgG2a heavy chain is SEQ ID NO:17

[0188]

[0189] SEQ ID NO:18 of the mouse IgG2a heavy chain constant region nucleotide sequence

[0190]

[0191]

Claims

1. An anti-SARS-CoV-2 antibody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD, characterized in that, The antibody or its antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:1 according to the IMGT numbering system, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:3 according to the IMGT numbering system. According to the IMGT numbering system, the antibody or its antigen-binding fragment comprises: HCDR1, whose sequence is shown in SEQ ID NO:5, HCDR2, whose sequence is shown in SEQ ID NO:6, and HCDR3, the sequence of which is shown in SEQ ID NO:7, and the antibody or its antigen-binding fragment further comprises: LCDR1, whose sequence is shown in SEQ ID NO:

8. LCDR2, whose sequence is shown in SEQ ID NO:9, and LCDR3, the sequence of which is shown in SEQ ID NO:

10. The heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:

3.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody further comprises a heavy chain constant region and a light chain constant region.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain constant region sequence is shown in SEQ ID NO:17 and the light chain constant region sequence is shown in SEQ ID NO:

15.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody is a monoclonal antibody.

5. A polynucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region and a light chain variable region encoding an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-4.

6. A carrier comprising the polynucleotide molecule of claim 5.

7. A host cell comprising the polynucleotide molecule of claim 5 or the vector of claim 6.

8. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-4, comprising culturing the host cell of claim 7 under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

9. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, and a conjugation portion conjugated to the antibody or antigen-binding fragment thereof, said conjugation portion being a purification tag, a cytotoxic agent, or a detectable label.

10. The antibody conjugate according to claim 9, characterized in that, The purification tag is a His tag.

11. The antibody conjugate according to claim 9, characterized in that, The coupling component is a radioactive isotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

12. A fusion protein comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

13. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or comprising the antibody conjugate as described in any one of claims 9-11 or the fusion protein as described in claim 12.

14. The kit of claim 13, further comprising a second antibody that specifically recognizes the antibody or its antigen-binding fragment.

15. The kit of claim 14, wherein the second antibody further comprises a detectable marker.

16. The kit of claim 15, wherein the detectable marker is a radioactive isotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the antibody-drug conjugate according to any one of claims 9-11, or the fusion protein according to claim 12 in the preparation of a kit for detecting the presence or level of human SARS-CoV-2 in a sample.

18. A pharmaceutical composition for treating diseases caused by SARS-CoV-2, comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the antibody-drug conjugate as described in any one of claims 9-11, or the fusion protein as described in claim 12.

19. The pharmaceutical composition for treating diseases caused by SARS-CoV-2 according to claim 18, characterized in that, The pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

20. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the antibody-drug conjugate according to any one of claims 9-11, or the fusion protein according to claim 12 in the preparation of a medicament for treating diseases caused by SARS-CoV-2.

21. The application of claim 20, wherein the drug is in an injectable form.

22. The application of claim 20, wherein the drug is administered via subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

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