WO2023046057A1 - 抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体及其应用 - Google Patents

抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体及其应用 Download PDF

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WO2023046057A1
WO2023046057A1 PCT/CN2022/120799 CN2022120799W WO2023046057A1 WO 2023046057 A1 WO2023046057 A1 WO 2023046057A1 CN 2022120799 W CN2022120799 W CN 2022120799W WO 2023046057 A1 WO2023046057 A1 WO 2023046057A1
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amino acid
seq
acid sequence
variable region
chain variable
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PCT/CN2022/120799
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French (fr)
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丁莉丹
钱永洪
史敏龙
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南京金斯瑞生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

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  • the invention belongs to the field of virus detection, diagnosis and treatment, and relates to a monoclonal antibody against SARS-CoV-2 L452R spike protein.
  • the present invention also relates to the application of the monoclonal antibody against SARS-CoV-2 L452R spike protein.
  • the new coronavirus is also called severe acute respiratory syndrome coronavirus-2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), which is composed of RNA nucleic acid and protein, etc.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, SARS-CoV-2
  • the disease caused by the virus is called new coronavirus disease 2019 (coronavirus disease, 2019 COVID-19).
  • 2019 COVID-19 coronavirus disease
  • the mortality rate of COVID-19 is significantly lower than that of SARS-CoV infection, the human-to-human transmission rate of SARS-CoV-2 is higher, and the WHO declared on March 11, 2020 that COVID-19 has caused a global pandemic.
  • SARS-CoV-2 also uses its highly glycosylated spike protein (Spike protein, S protein, S protein) to complete host cell receptor binding and virus infection in the form of a trimer.
  • ECD extra cellular domain
  • S protein has two subunits——S1 and S2.
  • the RBD (receptor-binding domain) region of the S1 subunit can recognize and bind to the angiotensin-converting enzyme 2 (human angiotensin-converting enzyme 2, hACE2) of the host cell, and the S2 subunit mediates the membrane fusion between the virus and the host cell.
  • angiotensin-converting enzyme 2 human angiotensin-converting enzyme 2, hACE2
  • the mutated virus strains that have received more attention include Alpha (B.1.1.7), Beta (B.1.351, B.1.351.2, B.1.351.3), Delta (B.1.617.2, AY.1, AY.2, AY.3) and Gamma (P.1, P.1.1, P.1.2) mutant strains.
  • Alpha B.1.1.7
  • Beta B.1.351, B.1.351.2, B.1.351.3
  • Delta B.1.617.2, AY.1, AY.2, AY.3
  • Gamma P.1, P.1.1, P.1.2
  • Each mutant strain is mainly involved in the S protein.
  • the mutation sites are shown in Table 1 below. Mutations at these sites lead to changes in how the virus binds to receptors, less neutralizing and therapeutic efficacy of antibodies raised against previous infections or vaccinations.
  • the variation of viral genetic material also has a potential impact on disease diagnosis, prediction of infectivity and judgment of disease severity.
  • the virus strains containing the L452R mutation site include Iota (B.1.526), B.1.427, B.1.429, Kappa (B.1.617.1), B.1.617.3 and Delta (B.1.617.2, AY.1, AY.2, AY.3).
  • Iota B.1.526
  • B.1.427 B.1.429
  • Kappa B.1.617.1
  • B.1.617.3 B.1.617.2, AY.1, AY.2, AY.3
  • Delta B.1.617.2, AY.1, AY.2, AY.3
  • the present invention provides a monoclonal antibody or functional fragment thereof against SARS-CoV-2 L452R spike protein, said antibody or functional fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3,
  • the HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 15, 21, 27, 33 or 39 or a variant having at most three (for example, one, two or three) amino acid mutations with the amino acid sequence shown ;
  • the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 16, 22, 28, 34 or 40 or a variant having at most three amino acid mutations with the indicated amino acid sequence;
  • the HCDR3 comprises a variant selected from SEQ ID NO: : the amino acid sequence shown in 17, 23, 29, 35 or 41 or a variant with up to three amino acid mutations from the amino acid sequence shown; and
  • said light chain variable region comprises LCDR1, LCDR2 and LCDR3,
  • the LCDR1 sequence comprises an amino acid sequence selected from SEQ ID NO: 18, 24, 30, 36 or 42 or a variant having at most three amino acid mutations with the indicated amino acid sequence;
  • the LCDR2 sequence comprises a sequence selected from SEQ ID The amino acid sequence shown in NO: 19, 25, 31, 37 or 43 or a variant with at most three amino acid mutations with the shown amino acid sequence;
  • the LCDR3 sequence comprises a sequence selected from SEQ ID NO: 20, 26, 32, 38 Or the amino acid sequence shown in 44 or a variant with up to three amino acid mutations from the amino acid sequence shown.
  • the HCDR1 sequence comprises an amino acid sequence selected from SEQ ID NO: 15, 21, 27, 33 or 39
  • the HCDR2 sequence comprises an amino acid sequence selected from SEQ ID NO: 16, 22, 28, 34 or the amino acid sequence shown in 40
  • the HCDR3 sequence comprises an amino acid sequence selected from SEQ ID NO: 17, 23, 29, 35 or 41
  • the LCDR1 sequence comprises a sequence selected from SEQ ID NO: 18, 24, The amino acid sequence shown in 30, 36 or 42
  • the LCDR2 sequence comprises an amino acid sequence selected from SEQ ID NO: 19, 25, 31, 37 or 43
  • the LCDR3 sequence comprises a sequence selected from SEQ ID NO: 20, The amino acid sequence shown in 26, 32, 38 or 44.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following sequences:
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 15, 16 and 17 or variants with up to three amino acid mutations respectively with the shown amino acid sequence; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NO: 18, 19 and 20 or variants with up to three amino acid mutations respectively with the shown amino acid sequences;
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 21, 22 and 23 or variants with at most three amino acid mutations respectively with the shown amino acid sequence; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NO: 24, 25 and 26 or variants with up to three amino acid mutations respectively with the shown amino acid sequences;
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 27, 28 and 29 or variants with at most three amino acid mutations respectively with the shown amino acid sequence; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NO: 30, 31 and 32 or variants with up to three amino acid mutations respectively with the shown amino acid sequences;
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 33, 34 and 35 or variants with up to three amino acid mutations respectively with the shown amino acid sequence; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences set forth in SEQ ID NO: 36, 37 and 38 or variants having up to three amino acid mutations each from the set forth amino acid sequences; or
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 39, 40 and 41 or variants with at most three amino acid mutations respectively with the shown amino acid sequence; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NO: 42, 43 and 44 or variants each having up to three amino acid mutations from the shown amino acid sequences.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following sequences:
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:15, 16 and 17 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:18, 19 and 20;
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:21, 22 and 23 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:24, 25 and 26;
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:27, 28 and 29 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:30, 31 and 32;
  • said HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:33, 34 and 35 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:36, 37 and 38; or
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:39, 40 and 41 and the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:42, 43 and 44.
  • the heavy chain variable region sequence comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 5, 7, 9, 11 or 13; and the light chain variable The region sequence comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 6, 8, 10, 12 or 14.
  • the heavy chain variable region sequence comprises at least 80%, 81%, 82%, 83%, 84%, Amino acid sequences that are 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical;
  • the light chain variable region sequence comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, Amino acid sequences that are 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.
  • the heavy chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 5, 7, 9, 11 or 13; the light chain variable region sequence comprises SEQ ID NO: 6, 8, The amino acid sequence shown in 10, 12 or 14.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO:5 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 6
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • said heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO:7 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 8
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • said heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 9 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO:
  • the sequence shown in 10 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 11 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO:
  • the sequence shown in 12 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences; or
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 13 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO:
  • the sequence shown in 14 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:6;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the present invention provides an isolated polynucleotide encoding the above-mentioned monoclonal antibody against SARS-CoV-2 L452R spike protein or a functional fragment thereof.
  • the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the above-mentioned monoclonal antibody or a functional fragment thereof, and a light chain variable region encoding the monoclonal antibody or a functional fragment thereof the nucleotide sequence.
  • the present invention provides expression vectors comprising said polynucleotides.
  • the present invention provides a host cell or cell-free expression system comprising the expression vector.
  • the present invention provides a pharmaceutical composition, which comprises the monoclonal antibody or its functional fragment and a pharmaceutically acceptable carrier.
  • the present invention provides the application of the monoclonal antibody or its functional fragments in coronavirus detection and diagnosis products.
  • the present invention provides the application of the monoclonal antibody or its functional fragment in the preparation of a drug for treating coronavirus.
  • the present invention provides a coronavirus detection kit, which comprises the monoclonal antibody or a functional fragment thereof.
  • the coronavirus is selected from SARS-CoV, MERS-CoV, SARS-CoV-2 or a mutant strain of SARS-CoV-2; preferably a mutant strain of SARS-CoV-2. In other embodiments, the coronavirus is a mutant strain of SARS-CoV-2 comprising the L452R spike protein.
  • the present invention provides a method for preparing a monoclonal antibody or functional fragment thereof against SARS-CoV-2 L452R spike protein, comprising:
  • variable region coding sequence Use the variable region coding sequence to carry out recombinant antibody production to obtain a functional monoclonal antibody against the SARS-CoV-2 L452R spike protein.
  • the monoclonal antibody is a murine, chimeric, humanized or human antibody. In some preferred embodiments, the monoclonal antibodies are of murine origin. In other preferred embodiments, said monoclonal antibody is humanized.
  • the monoclonal antibody may be IgG1 or IgG2.
  • the antibody comprises an IgG1 heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO:45.
  • the antibody comprises an IgG2a heavy chain constant region sequence, and the IgG2a heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:46.
  • the monoclonal antibody comprises a kappa light chain constant region.
  • the Kappa light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO:47.
  • the monoclonal antibody against SARS-CoV-2 L452R spike protein developed by the present invention can effectively block the binding of S protein and ACE2 protein, and prevent the virus from infecting human cells.
  • the variation of viral genetic material also has a potential impact on disease diagnosis, prediction of infectivity and judgment of disease severity.
  • the anti-SARS-CoV-2 L452R spike protein monoclonal antibody developed by the present invention can specifically detect whether the strain is a SARS-CoV-2 mutant strain containing a spike protein L452R specific mutation site, such as Kappa, Delta or B.1.617.3 mutant strain, for timely and accurate diagnosis and detection of infection with SARS-CoV-2 mutant strain is the key to providing appropriate treatment to patients and limiting the further spread of the virus and ultimately eliminating the virus from human society .
  • antibodies with specific neutralizing effects on mutant strains can also provide options for disease treatment of mutant strains.
  • Fig. 1 is the rat serum titer test result figure after immunization
  • Fig. 2 is a graph showing the affinity detection results of the monoclonal antibody and the RBD protein containing the L452R mutation site.
  • the present invention relates to a monoclonal antibody against SARS-CoV-2 L452R spike protein, and the embodiments of the present invention will be described in detail below in conjunction with examples. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • novel coronavirus SARS-CoV-2
  • 2019-nCoV refers to a new type of viral infection that began to appear and spread in 2019. Oval, often pleomorphic, with a diameter of 60-140nm. Its genetic characteristics are significantly different from those of SARSr-CoV and MERSr-CoV. Studies have shown that it has more than 85% homology with bat SARS-like coronavirus (bat-SL-CoVZC45). When isolated and cultured in vitro, 2019-nCoV can be found in human respiratory epithelial cells in about 96 hours, while it takes about 6 days to isolate and culture Vero E6 and Huh-7 cell lines.
  • antibody is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains (of which two heavy (H) and two light (L) chains are interconnected by disulfide bonds (i.e. a "complete antibody molecule")) , and multimers thereof (such as IgM) or antigen-binding fragments thereof.
  • Each heavy chain is composed of a heavy chain variable region ("HCVR” or “VH”) and a heavy chain constant region (composed of domains CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (“LCVR” or "VL”) and a light chain constant region (CL).
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to hydroxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the antibody may be identical to human germline sequences or may be naturally or artificially modified.
  • the term "monoclonal antibody” refers to a homogeneous antibody directed only against a specific antigenic epitope. Each monoclonal antibody is directed against a single antigenic determinant on the antigen, in contrast to typical common polyclonal antibody preparations that include different antibodies directed against different antigenic determinants (epitopes).
  • the modifier "monoclonal” indicates the homogeneous character of the antibody and is not to be construed as requiring that the antibody be produced by any particular method.
  • the monoclonal antibodies of the invention are preferably produced by recombinant DNA methods, or obtained by screening methods as described elsewhere herein.
  • mutants refers to the alteration of one or more (several) amino acids comprising one or more (several) positions, ie substitutions, insertions and/or deletions.
  • a substitution refers to replacing an amino acid occupying a position with a different amino acid;
  • a deletion refers to removing an amino acid occupying a position; and
  • an insertion refers to adding 1-3 amino acids adjacent to and after the amino acid occupying a position.
  • L452R spike protein refers to the mutant protein in which the leucine (Leu or L) at position 452 of the spike protein is replaced by arginine (Arg or R);
  • E484Q spike protein refers to the spike protein A mutant protein in which glutamic acid (Glu or E) at position 484 of the spike protein is replaced by glutamine (Gln or Q).
  • + plus signs
  • L452R+E484Q indicates substitution of glutamic acid (E) with glutamine (Q) at position 484 and leuco with arginine (R) at position 452 amino acid (L).
  • isolated polynucleotide refers to a polynucleotide that does not exist naturally in nature, including polynucleotides isolated from nature (including organisms) through biological techniques, and artificially synthesized polynucleotides.
  • An isolated polynucleotide can be genomic DNA, cDNA, mRNA or other synthetic RNA, or a combination thereof. It should be pointed out that, based on the amino acid sequences of the heavy chain variable region and the light chain variable region provided herein, those skilled in the art can design the provided nucleotide sequences based on codon degeneracy. nucleotide sequence, but all encode the same amino acid sequence. These altered nucleotide sequences are also included within the scope of the present invention.
  • vector refers to any molecule (eg, nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide-encoded information into a host cell.
  • expression vector or “expression cassette” refers to a vector suitable for expressing a target gene (nucleotide sequence to be expressed) in a host cell, usually including target gene, promoter, terminator, marker gene and other parts.
  • host cell refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby express a selected gene of interest.
  • the term includes progeny of a parental cell, whether or not the progeny is identical in morphology or genetic makeup to the original parental cell, so long as the selected gene of interest is present in the progeny.
  • Commonly used host cells include bacteria, yeast, mammalian cells, and the like.
  • antibody functional fragment refers to antigen-binding fragments of antibodies and antibody analogs, which typically include at least part of the antigen-binding or variable region (eg, one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody.
  • antibody fragments capable of binding to the coronavirus spike (S) protein or a portion thereof including but not limited to sdAb (single domain antibody), Fab (eg, antibody digested with papain), F(ab')2( For example, obtained by pepsin digestion), Fv or scFv (obtained, for example, by molecular biological techniques).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and sustained release agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in standard references in the latest edition of Remington's Pharmaceutical Sciences, which is hereby incorporated by reference in its entirety. Examples of suitable carriers or diluents include, but are not limited to, water, saline solution, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and hydrophobic-hydrophobic vehicles such as fixed oils may also be used. The use of media and agents for pharmaceutically active substances is well known in the art. Except for those conventional media or agents that are incompatible with the active ingredient, its use in the composition can achieve the desired effect.
  • amino acid substitution refers to the replacement of existing amino acid residues with different amino acid residues in a predetermined (initial) amino acid sequence.
  • amino acid substitutions are preferably made in accordance with the substitutions shown below:
  • Percent (%) amino acid sequence identity with respect to a peptide or polypeptide sequence is defined as after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity, the candidate The percentage of amino acid residues in a sequence that are identical to those in a particular peptide or polypeptide sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Embodiment 1 the acquisition of the monoclonal antibody hybridoma cell line of anti-SARS-CoV-2 L452R spike protein and the preparation of monoclonal antibody
  • Animal immunization antigen adopts recombinant protein S-RBD L452R+E484Q -His (SEQ ID NO: 1).
  • Female Balb/c was subcutaneously immunized with protein containing 5 ⁇ g of S-RBD L452R+E484Q -His (SEQ ID NO: 1). Subsequently, the mice were boosted by repeating immunizations every other day for a total of 6 times. The serum titers of 5 mice all reached above 10 5 after 6 immunizations ( FIG. 1 ). Lymph nodes from 5 mice were pooled for fusion 4 days after the last immunization.
  • Splenocytes were fused with SP2/0 mouse myeloma cells using electrofusion.
  • Fused cells were resuspended in DMEM/10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine and aminopterin, and pipetted into a 96-well plate in a volume of 100 ⁇ l. Incubate the plate at 37 °C in 5% CO2 . After 7 days of incubation, testing for the presence of antibodies specific to the L452R mutein was initiated using the ELISA combination described below.
  • Indirect ELISA was used to evaluate the binding ability of the antibody in the supernatant to S-RBD L452R+E484Q -His (SEQ ID NO:1) protein.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-RBD L452R+E484Q -His (SEQ ID NO: 1 ) protein at 1 ⁇ g/ml in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween), and blocked with 150 ⁇ l/well of 1% BSA in PBST for 1 hour at 37°C.
  • RBD WT amino acid sequence (SEQ ID NO: 4)
  • Subcloning was performed using limiting dilution.
  • Cell numbers were determined using a hemocytometer and serially diluting cells in DMEM/10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine, and aminopterin until a cell density of 5-15 cells/ml.
  • Example 2 Confirmation detection of monoclonal antibody binding to L452R mutant protein based on hybridoma cells
  • Example 3 Binding of monoclonal antibodies in the supernatant to viral S-RBD L452R+E484Q -His protein
  • Indirect ELISA was used to evaluate the binding ability of the monoclonal antibody in the supernatant to S-RBD L452R+E484Q -His protein.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-RBD L452R+E484Q -His protein at 0.5 ⁇ g/ml in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween), and blocked with 250 ⁇ l/well of 1% BSA in PBST for 2 hours at 37°C.
  • Murine immunoglobulin heavy and light chain V-region fragments were subsequently amplified by RACE PCR (GenScript), and the resulting PCR fragments were subcloned into empty vectors and the inserts were sequenced using vector-specific primers. Finally, the unique V-region protein amino acid sequences of L7A3R, L20E10R, L10F10R, L16B9R and L18D11R were obtained.
  • L7A3R heavy chain variable region amino acid sequence (SEQ ID NO: 5):
  • L7A3R light chain variable region amino acid sequence (SEQ ID NO: 6):
  • L20E10R heavy chain variable region amino acid sequence (SEQ ID NO: 7):
  • L20E10R light chain variable region amino acid sequence (SEQ ID NO: 8):
  • L10F10R heavy chain variable region amino acid sequence (SEQ ID NO: 9):
  • L10F10R light chain variable region amino acid sequence (SEQ ID NO: 10):
  • IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 45)
  • IgG2a heavy chain constant region amino acid sequence (SEQ ID NO: 46)
  • Embodiment 5 ELISA competition detection method
  • S-RBD L452R+E484Q -His (SEQ ID NO: 1) protein and ACE2 protein were coated with 100 ⁇ l/well of recombinant S-RBD L452R+E484Q -His (SEQ ID NO: 1 ) protein at 1 ⁇ g/ml in PBS overnight at 4°C. Plates were washed with PBST (0.05% Tween) and blocked with 250 ⁇ l/well of 1% BSA in PBST for 2 hours at 37°C.
  • variable region gene in Example 4 was synthesized into an expression vector containing the corresponding subtype constant region sequence (SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47), and CHO-S cells were used for transfection Antibody after recombinant expression was obtained.

Abstract

提供了抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体、其重链可变区和轻链可变区的氨基酸序列。提供的抗SARS-CoV-2 L452R刺突蛋白单克隆抗体能够特异性地与SARS-CoV-2 L452R刺突蛋白突变体结合，部分抗体能够有效地阻断SARS-CoV-2刺突蛋白L452R突变蛋白与ACE2蛋白的结合。提供的抗SARS-CoV-2 L452R刺突蛋白单克隆抗体及潜在医学用途，为包含刺突蛋白L452R突变位点的SARS-CoV-2病毒突变株的治疗和检测提供了可能和便利。

Description

抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体及其应用
交叉引用
本申请要求于2021年9月24日提交的中国专利申请202111120463.8的优先权，其全部内容通过引用并入全文。
技术领域
本发明属于病毒检测诊断、治疗领域，涉及一种抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体。本发明还涉及该抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体的应用。
背景技术
新型冠状病毒也称严重急性呼吸综合征冠状病毒-2(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)，由RNA核酸和蛋白等组成，由该病毒引起的疾病称为新型冠状病毒疾病2019(coronavirus disease，2019 COVID-19)。尽管COVID-19的死亡率明显低于SARS-CoV感染的死亡率，但SARS-CoV-2的人际传播率更高，WHO于2020年3月11日宣布COVID-19已经引起了全球大流行。
与SARS-CoV相似，SARS-CoV-2也是利用其高度糖基化的刺突蛋白(Spike protein,S protein，S蛋白)，以三聚体形式完成宿主细胞受体结合和病毒侵染。ECD(extra cellular domain)是S蛋白的膜外区，S蛋白有两个亚基——S1和S2。S1亚基的RBD(receptor-binding domain)区可以识别并结合宿主细胞的血管紧张素转化酶2(human angiotensin-converting enzyme 2，hACE2)，S2亚基介导病毒与宿主细胞的膜融合。
在整个COVID-19大流行期间，SARS-CoV-2的遗传变异已经出现并在世界各地传播。病毒的突变和变异可以通过测序、实验室研究和流行病学调查进行常规监测。为了协助公众讨论变体，世卫组织建议使用由希腊字母组成的标签，例如Alpha、Beta、Delta、Gamma，作为非科学受众讨论变体的实用方式。
目前比较受关注的变异病毒毒株有Alpha(B.1.1.7),Beta(B.1.351,B.1.351.2,B.1.351.3),Delta(B.1.617.2,AY.1,AY.2,AY.3)和Gamma(P.1,P.1.1,P.1.2)突变株。除此之外，还有一些毒株也引起了大家的关注和兴趣，比如Kappa(B.1.617.1)和没有希腊字母名称的B.1.617.3，各突变毒株主要涉及的S蛋白上的突变位点如下表1所示。这些位点的突变会导致病毒与受体结合的变化，针对先前感染或疫苗接种产生的抗体的中和作用降低、治疗效果降低。同时，病毒遗传物质的变异对疾病诊断，传染性的预测及疾病严重程度的判断也存在潜在的影响。
表1突变毒株涉及到的主要突变位点汇总
Figure PCTCN2022120799-appb-000001
Figure PCTCN2022120799-appb-000002
目前包含有L452R突变位点的病毒毒株有Iota(B.1.526),B.1.427,B.1.429,Kappa(B.1.617.1),B.1.617.3和Delta(B.1.617.2,AY.1,AY.2,AY.3)。已有研究表明，该位点的突变会降低病毒对bamlanivimab和etesevimab单克隆抗体联合治疗的敏感性，导致恢复期和疫苗接种后血清的中和作用降低，同时会增强病毒的传染性。
及时，准确地诊断SARS-CoV-2突变毒株的感染是为患者提供适当治疗，限制病毒进一步传播并最终从人类社会消除病毒的关键。同时，对于突变体毒株有特异性中和作用的抗体也能够为突变毒株的疾病治疗提供选择。综上，开发出特异性识别或者中和SARS-CoV-2 L452R突变毒株的抗体是迫在眉睫的研究内容。
发明内容
在一方面，本发明提供了一种抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体或其功能片段，所述抗体或其功能片段包含重链可变区和轻链可变区，其中
(a)所述重链可变区包含HCDR1、HCDR2和HCDR3，
所述HCDR1包含选自SEQ ID NO:15、21、27、33或39所示的氨基酸序列或与所示氨基酸序列具有至多三个(例如，一个、二个或三个)氨基酸突变的变体；所述HCDR2包含选自SEQ ID NO:16、22、28、34或40所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述HCDR3包含选自SEQ ID NO:17、23、29、35或41所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；以及
(b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，
所述LCDR1序列包含选自SEQ ID NO:18、24、30、36或42所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR2序列包含选自SEQ ID NO:19、25、31、37或43所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR3序列包含选自SEQ ID NO:20、26、32、38或44所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的 变体。
在一些实施方案中，所述HCDR1序列包含选自SEQ ID NO:15、21、27、33或39所示的氨基酸序列，所述HCDR2序列包含选自SEQ ID NO:16、22、28、34或40所示的氨基酸序列，所述HCDR3序列包含选自SEQ ID NO:17、23、29、35或41所示的氨基酸序列；以及所述LCDR1序列包含选自SEQ ID NO:18、24、30、36或42所示的氨基酸序列，所述LCDR2序列包含选自SEQ ID NO:19、25、31、37或43所示的氨基酸序列，所述LCDR3序列包含选自SEQ ID NO:20、26、32、38或44所示的氨基酸序列。
在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
(a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:15、16和17所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:18、19和20所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:21、22和23所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:24、25和26所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(c)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:27、28和29所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:30、31和32所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:33、34和35所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:36、37和38所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；或
(e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:39、40和41所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:42、43和44所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体。
在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
(a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:15、16和17所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:18、19和20所示的氨基酸序列；
(b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:21、22和23所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:24、25和26所示的氨基酸序列；
(c)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:27、28和29所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:30、31和32所示的氨基酸序列；
(d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:33、34和35所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:36、37和38所示的氨基酸序列；或
(e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:39、40和41所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:42、43和44所示的氨基酸序列。
在一些实施方案中，所述重链可变区序列包含与SEQ ID NO:5、7、9、11或13所示氨基酸序列具有至少80％一致性的氨基酸序列；以及所述轻链可变区序列包含与SEQ ID NO:6、8、10、12或14所示氨基酸序列具有至少80％一致性的氨基酸序列。在一些实施方案中，所述重链可变区序列包含与SEQ ID NO:5、7、9、11或13所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；所述轻链可变区序列包含与SEQ ID NO:6、8、10、12或14所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列。
在一些实施方案中，所述重链可变区序列包含SEQ ID NO:5、7、9、11或13所示氨基酸序列；所述轻链可变区序列包含SEQ ID NO:6、8、10、12或14所示氨基酸序列。
在一些实施方案中，所述重链可变区和轻链可变区选自如下序列：
(a)所述重链可变区包含与SEQ ID NO:5所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:6所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(b)所述重链可变区包含与SEQ ID NO:7所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:8所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(c)所述重链可变区包含与SEQ ID NO:9所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:10所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(d)所述重链可变区包含与SEQ ID NO:11所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:12所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；或
(e)所述重链可变区包含与SEQ ID NO:13所示序列具有至少80％、81％、82％、83％、84％、 85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:14所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列。
在一些实施方案中，所述重链可变区和轻链可变区选自如下序列：
(a)所述重链可变区包含SEQ ID NO:5所示的氨基酸序列，所述轻链可变区包含SEQ ID NO:6所示的氨基酸序列；
(b)所述重链可变区包含SEQ ID NO:7所示的氨基酸序列，所述轻链可变区包含SEQ ID NO:8所示的氨基酸序列；
(c)所述重链可变区包含SEQ ID NO:9所示的氨基酸序列，所述轻链可变区包含SEQ ID NO:10所示的氨基酸序列；
(d)所述重链可变区包含SEQ ID NO:11所示的氨基酸序列，所述轻链可变区包含SEQ ID NO:12所示的氨基酸序列；或
(e)所述重链可变区包含如SEQ ID NO:13所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:14所示的氨基酸序列。
在另一方面，本发明提供了编码上述的抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体或其功能片段的分离的多核苷酸。
在一些实施方案中，所述多核苷酸包含编码上述单克隆抗体或其功能片段的重链可变区的核苷酸序列，和编码所述单克隆抗体或其功能片段的轻链可变区的核苷酸序列。
在另一方面，本发明提供了包含所述多核苷酸的表达载体。
在另一方面，本发明提供了包含所述表达载体的宿主细胞或无细胞表达系统。
在另一方面，本发明提供了一种药物组合物，所述药物组合物包含所述的单克隆抗体或其功能片段和药学上可接受的载体。
在另一方面，本发明提供了所述的单克隆抗体或其功能片段在冠状病毒检测和诊断产品中的应用。
在另一方面，本发明提供了所述单克隆抗体或其功能片段在制备治疗冠状病毒药物中的应用。
在另一方面，本发明提供了一种检测冠状病毒的试剂盒，所述试剂盒中包含所述的单克隆抗体或其功能片段。
在一些实施方案中，所述冠状病毒选自SARS-CoV、MERS-CoV、SARS-CoV-2或SARS-CoV-2突变株；优选为SARS-CoV-2突变株。在另一些实施方案中，所述冠状病毒为包含L452R刺突蛋白的SARS-CoV-2突变株。
在另一方面，本发明提供了一种制备抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体或其功能片段的方法，包括：
1)以SARS-CoV-2刺突蛋白S-RBDL452R/E484Q-His免疫动物，在所述动物中产生针对该抗 原的免疫反应；
2)取所述动物的淋巴结细胞与骨髓瘤细胞融合获得的杂交瘤细胞，并进行筛选，得到特异性识别SARS-CoV-2刺突蛋白L452R突变位点的阳性克隆；
3)对所述阳性母克隆进行亚克隆，以获得稳定的杂交瘤细胞株；
4)对所述杂交瘤细胞株进行基因测序，获得抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体的重链和轻链的可变区编码序列；以及
5)用所述可变区编码序列进行重组抗体生产，获得功能性抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体。
在一些实施方案中，所述的单克隆抗体是鼠源、嵌合的、人源化的或人的抗体。在一些优选实施方案中，所述的单克隆抗体是鼠源的。在另一些优选实施方案中，所述的单克隆抗体是人源化的。
在一些实施方案中，所述单克隆抗体可以是IgG1或IgG2。在一些具体实施方案中，所述抗体包含IgG1重链恒定区，所述IgG1重链恒定区包含SEQ ID NO:45所示的氨基酸序列。在另一些具体实施方案中，所述抗体包含IgG2a重链恒定区序列，所述IgG2a重链恒定区包含SEQ ID NO:46所示的氨基酸序列。在一些实施方案中，所述单克隆抗体包含Kappa轻链恒定区。在一些具体实施方案中，所述抗体的Kappa轻链恒定区包含SEQ IDNO:47所示的氨基酸序列。
有益效果：
本发明开发的抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体能够有效的阻断S蛋白与ACE2蛋白的结合，阻止病毒感染人的细胞。病毒遗传物质的变异对疾病诊断、传染性的预测及疾病严重程度的判断也存在潜在的影响。本发明开发的抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体能够特异性的检测出毒株是否为包含刺突蛋白L452R特异性突变位点的SARS-CoV-2突变株，例如Kappa、Delta或B.1.617.3突变株，为能及时、准确地诊断和检测SARS-CoV-2突变毒株的感染是为患者提供适当治疗，也是限制病毒进一步传播并最终从人类社会消除病毒的关键。同时，对于突变体毒株有特异性中和作用的抗体也能够为突变毒株的疾病治疗提供选择。
附图说明
图1为免疫之后的鼠血清效价检测结果图；
图2为单克隆抗体与含有L452R突变位点的RBD蛋白的亲和力检测结果图。
具体实施方式
本发明涉及一种抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体，下面将结合实施例对本发明的实施方案进行详细描述。除非另有说明，本发明所用的技术和科学术语与本发明所属领域的普通技术员通常所理解的含义相同。
术语“新型冠状病毒”(SARS-CoV-2)，亦称为2019-nCoV，是指2019年开始出现并蔓延的新型病毒感染，其属于β属冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。其基因特征与SARSr-CoV和MERSr-CoV有明显区别。研究显示，其与蝙蝠SARS样冠状病毒(bat- SL-CoVZC45)同源性达85％以上。体外分离培养时，2019-nCoV 96个小时左右即可在人呼吸道上皮细胞内发现，而在Vero E6和Huh-7细胞系中分离培养需约6天。
术语“抗体”意在指由四条多肽链组成的免疫球蛋白分子(其中两条重链(H)和两条轻链(L)通过二硫键相互连接(即“完整的抗体分子”))，以及其多聚体(例如IgM)或其抗原结合片段。每条重链由重链可变区(“HCVR”或“VH”)和重链恒定区(由结构域CH1、CH2和CH3组成)组成。每条轻链由轻链可变区(“LCVR”或“VL”)和轻链恒定区(CL)组成。VH和VL区可进一步细分为称为互补决定区(CDR)的高变区，其间插有更保守的区称为框架区(FR)。每个VH和VL由三个CDR和四个FR组成，以下列顺序从氨基末端至羟基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。在本发明的一些实施方案中，抗体(或其抗原结合片段)的FR可与人种系序列相同或可经天然或人工修饰。
术语“单克隆抗体”指均一的仅针对某一特定抗原表位的抗体。与典型的包括针对不同抗原决定簇(表位)的不同抗体的普通多克隆抗体制剂相比，每种单克隆抗体针对抗原上的单个抗原决定簇。修饰语“单克隆”表示抗体的均一特征，不解释为需要通过任何特定方法产生的抗体。本发明的单克隆抗体优选通过重组DNA方法产生，或通过本发明其他地方描述的筛选方法获得。
术语“突变”是指包含一个或多个(数个)位置的一个或多个(数个)氨基酸的变更，即取代、插入和/或缺失。取代是指用不同的氨基酸替代占据某位置的氨基酸；缺失是指除去占据某位置的氨基酸；而插入是指在占据某位置的氨基酸邻接处且在之后添加1-3个氨基酸。本发明描述SARS-CoV-2刺突蛋白突变体时，使用了下述命名法便于参考。在所有情况下，使用了公认的IUPAC单字符或三字符氨基酸缩写。对于氨基酸的取代，使用了下列命名法：原始氨基酸、位置、取代氨基酸。例如，“L452R刺突蛋白”指的是刺突蛋白第452位的亮氨酸(Leu或L)被精氨酸(Arg或R)取代的突变蛋白；“E484Q刺突蛋白”指的是刺突蛋白第484位的谷氨酸(Glu或E)被谷氨酰胺(Gln或Q)取代的突变蛋白。通过加号(“+”)来分开多个突变，例如“L452R+E484Q”表示在位置484用谷氨酰胺(Q)取代谷氨酸(E)和位置452用精氨酸(R)取代亮氨酸(L)。
术语“分离的多核苷酸”指非自然界中天然存在状态的多核苷酸，包括通过生物学技术从自然界(包括生物体内)分离出的多核苷酸，也包括人工合成的多核苷酸。分离的多核苷酸可以是基因组DNA、cDNA、mRNA或合成的其他RNA，或者它们的组合。需要指出的是，本领域技术人员可以根据本文所提供的重链可变区和轻链可变区的氨基酸序列，基于密码子简并性，设计出提供的核苷酸序列不完全相同的核苷酸序列，但都编码相同的氨基酸序列。这些经改动的核苷酸序列也包括在本发明的范围内。
当涉及多核苷酸时，术语“载体”指用于将核苷酸编码信息转移到宿主细胞内的任一种分子(例如核酸、质粒或病毒等)。术语“表达载体”或“表达盒”指适于在宿主细胞内表达目的基因(待表达核苷酸序列)的载体，通常包括目的基因、启动子、终止子、标记基因等部分。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选 目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
术语“抗体功能片段”意即抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。例如，能够结合冠状病刺突(S)蛋白或其部分的抗体片段，包括但不限于sdAb(单域抗体)、Fab(例如，抗体经木瓜蛋白酶消化而得到)、F(ab’)2(例如，通过胃蛋白酶消化得到)、Fv或scFv(例如通过分子生物学技术得到)。
术语“药学上可接受的载体”包括与药物给药相容的任何和所有溶剂，分散剂，包被物，抗细菌和抗真菌药剂，等渗和缓释剂，及其类似物。合适的载体在Remington’s Pharmaceutical Sciences最新版中的标准参考文件中有所叙述，其通过在此引述而全部合并于本文。合适的载体或稀释液例子包括，但不局限于，水，盐溶液，ringer’s液，葡萄糖溶液，和5％人血清白蛋白。也可以使用脂质体和疏-水介质如不挥发油。药物活性物质的介质和药剂的使用在本领域中是熟知的。除了那些对于活性成分不相容的常规介质或试剂以外，其在成分中的使用都可以达到预期效果。
术语“氨基酸替换”，指在预先确定的(初始)氨基酸序列中，用不同的氨基酸残基代替现有的氨基酸残基。一般而言，本领域技术人员公认在多肽非必需区的单个氨基酸取代基本上不改变生物学活性(参见例如Watson等，Molecular Biology of the Gene(基因的分子生物学)，The Benjamin/Cummings Pub.Co.，第224页(第四版，1987)。这样的例示性取代优选依照以下所示的取代来进行：
示例性保守氨基酸取代
原残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His
Asp(D) Glu；Asn
Cys(C) Ser；Ala
Gln(Q) Asn
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
关于肽或多肽序列的“百分比(％)氨基酸序列一致性”定义为对比序列并在必要时引入缺口以 获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
除非另外特别说明，否则单数的使用包括复数。除非另外特别说明，否则词语“一个(a)”或“一个(an)”意指“至少一个”。除非另外说明，否则“或”的使用意指“和/或”。短语“至少一个”的含义等同于短语“一个或多个”的含义。此外，术语“包括(including)”以及其他形式诸如“包括(includes)”和“包括(included)”的使用不是限制性的。此外，除非另外特别说明，否则术语诸如“要素”或“组分”包括包含一个单元的元素或组分以及包含多于一个单元的元素和组分。
除非另有说明，下文描述的实施例的方法和材料均为可以通过市场购买获得的常规产品。本发明所属领域技术员将会理解，下文描述的方法和材料，仅是示例性的，而不应视为限定本发明的范围。
实施例1：抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体杂交瘤细胞株的获得以及单克隆抗体的制备
1)动物免疫抗原采用重组蛋白S-RBD L452R+E484Q-His(SEQ ID NO:1)。用含5μg S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白皮下免疫雌性Balb/c。随后，每隔一天重复免疫，从而对小鼠进行加强免疫，共6次。5只小鼠血清效价在6次免疫之后均达到10 5以上(图1)。5只小鼠在最后一次免疫后4天取淋巴结混合进行融合。
2)杂交瘤融合和筛选
提取小鼠淋巴结并进行均质化以产生单细胞悬液，同时准备骨髓瘤细胞(SP2/0)单细胞悬液。使用电融合将脾细胞与SP2/0小鼠骨髓瘤细胞进行融合。将融合的细胞重悬于含杂交瘤细胞选择剂胸腺核苷嘧啶，次黄嘌呤和氨基喋呤的DMEM/10％FBS培养基中，并用移液管以100μl的体积移至96孔板中。将板在37℃下在5％CO 2中孵育。7天的孵育之后，开始使用下文所述的ELISA结合来测试特异性针对L452R突变蛋白的抗体的存在情况。
ELISA结合检测方法：
间接ELISA用于评估上清液中抗体对于S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白的结合能力。将ELISA板用100μl/孔的PBS中1μg/ml的重组S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用150μl/孔的含1％BSA的PBST在37℃封闭1小时。随后弃去封闭液，向每个板加入100μl杂交瘤细胞培养上清液，然后在37℃孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗小鼠IgG(Fc-特异性)二抗(Jackson，115-035-071)37℃孵育0.5小时。将板用PBST洗涤五次，然后加入TMB显色液并在室温下在黑暗中孵育13分钟。通过加入50μl的1M HCl终止液(国药，10011018)终止反应。使用酶标仪在450nm下读板。同时，为了排除非L452R突变位点的S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白其他表位的非特异性抗体，我们同时使用了S1 E484Q+L452R(SEQ ID NO:2)作为正向确认筛选材料，RBD L452R-His(SEQ ID NO:3)作为正向 筛选材料，RBD WT(SEQ ID NO:4)作为反向筛选材料，L452R多肽(SEQ ID NO:48)作为正向筛选材料，L452R-WT多肽(SEQ ID NO:49)作为反向筛选材料。我们选择正向筛选材料OD值大于1.0，反向筛选材料OD值小于0.01的阳性孔进行下一步实验。
S-RBD L452R+E484Q-His氨基酸序列(SEQ ID NO:1)
Figure PCTCN2022120799-appb-000003
S1 E484Q+L452R氨基酸序列(SEQ ID NO:2)
Figure PCTCN2022120799-appb-000004
RBD L452R-His氨基酸序列(SEQ ID NO:3)
Figure PCTCN2022120799-appb-000005
RBD WT氨基酸序列(SEQ ID NO:4)
Figure PCTCN2022120799-appb-000006
L452R多肽氨基酸序列(SEQ ID NO:48)
Figure PCTCN2022120799-appb-000007
L452R-WT多肽氨基酸序列(SEQ ID NO:49)
Figure PCTCN2022120799-appb-000008
3)杂交瘤亚克隆
使用有限稀释法进行亚克隆。使用血球细胞计数器并在含杂交瘤细胞选择剂胸腺核苷嘧啶，次黄嘌呤和氨基喋呤的DMEM/10％FBS培养基中对细胞进行系列稀释来确定细胞数量，直至细胞密度达到5-15个细胞/ml。对于每个杂交瘤，将200μl的细胞溶液用移液管移至96孔中，密度为1-3个细胞/孔。将培养物在37℃下在5％CO2中培养1周后，挑选单克隆细胞，同时对上清液进行上述ELISA结合,来评估针对S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白的抗体的存在情况，挑选OD值大于1.0的单克隆孔扩增进行后续实验。
实施例2：基于杂交瘤细胞的单克隆抗体结合L452R突变蛋白的确认检测
将上述细胞培养扩增之后，收取上清使用快速ELISA小鼠抗体亚型鉴定试剂盒(Clonotyping System-HRP，Southern Biotech)对单克隆抗体进行亚型鉴定。同时采用实施案例1中的ELISA方法用于L452R特异性验证。使用S-RBD L452R+E484Q-His(SEQ ID NO:1)，S1 E484Q+L452R(SEQ ID NO:2)，RBD L452R-His(SEQ ID NO:3)，L452R多肽作为正向筛选材料，RBD WT(SEQ ID NO:4)以及L452R-WT多肽作为反向筛选材料，OD值大于1.0判断为阳性，用“+”表示，OD值小于0.01判断为阴性，用“-”表示，结果汇总如下表2所示：
表2抗体与L452R突变蛋白的特异性结合检测
Figure PCTCN2022120799-appb-000009
实施例3：上清中单克隆抗体对病毒S-RBD L452R+E484Q-His蛋白的结合
间接ELISA用于评估上清中的单克隆抗体对于S-RBD L452R+E484Q-His蛋白的结合能力。将ELISA板用100μl/孔的PBS中0.5μg/ml的重组S-RBD L452R+E484Q-His蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用250μl/孔的含1％BSA的PBST在37℃封闭2小时。随后弃去封闭液，向首孔加入未稀释的含有抗体的上清100μl，并按照3倍梯度稀释，共计11个测试浓度梯度外加一个空白孔。然后在37℃下孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗鼠IgG(Fc-特异性)二抗(GenScript,A01856)37℃孵育0.5小时。ELISA板用PBST洗涤四次，然后加入TMB显色液(GenScript)并在25℃下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液(国药，10011018)终止反应。使用酶标仪在450nm下读板，各克隆的EC 50检测曲线如图2，EC 50值如表3 所示：
表3抗体对S-RBD L452R+E484Q-His蛋白结合的EC 50值
抗体 L10F10R L16B9R L18D11R L20E10R L7A3R
EC 50(ng/ml) 5.713 9.351 11.630 6.034 5.029
实施例4：单克隆抗体的可变区测序
使用TRIzol(Life Technology，15596-026)从1×10 6～5×10 6个杂交瘤细胞提取总RNA，并利用抗体亚型特异性引物和通用引物(PrimeScript TM 1st Strand cDNA Synthesis Kit，Takara)将其逆转录为cDNA。随后通过RACE PCR(GenScript)扩增鼠免疫球蛋白重链和轻链V-区域片段，并将所得的PCR片段亚克隆至空载体中，并使用载体特异性引物对插入片段进行测序。最终获取了L7A3R，L20E10R，L10F10R，L16B9R和L18D11R的独特V-区域蛋白氨基酸序列。
L7A3R重链可变区氨基酸序列(SEQ ID NO:5)：
Figure PCTCN2022120799-appb-000010
L7A3R轻链可变区氨基酸序列(SEQ ID NO:6)：
Figure PCTCN2022120799-appb-000011
L20E10R重链可变区氨基酸序列(SEQ ID NO:7)：
Figure PCTCN2022120799-appb-000012
L20E10R轻链可变区氨基酸序列(SEQ ID NO:8)：
Figure PCTCN2022120799-appb-000013
L10F10R重链可变区氨基酸序列(SEQ ID NO:9)：
Figure PCTCN2022120799-appb-000014
L10F10R轻链可变区氨基酸序列(SEQ ID NO:10)：
Figure PCTCN2022120799-appb-000015
L16B9R重链可变区氨基酸序列(SEQ ID NO:11)：
Figure PCTCN2022120799-appb-000016
L16B9R轻链可变区氨基酸序列(SEQ ID NO:12)：
Figure PCTCN2022120799-appb-000017
L18D11R重链可变区氨基酸序列(SEQ ID NO:13)：
Figure PCTCN2022120799-appb-000018
L18D11R轻链可变区氨基酸序列(SEQ ID NO:14)：
Figure PCTCN2022120799-appb-000019
IgG1重链恒定区氨基酸序列(SEQ ID NO:45)
Figure PCTCN2022120799-appb-000020
IgG2a重链恒定区氨基酸序列(SEQ ID NO:46)
Figure PCTCN2022120799-appb-000021
Kappa轻链恒定区氨基酸序列(SEQ ID NO:47)
Figure PCTCN2022120799-appb-000022
表4抗体的CDR区序列
Figure PCTCN2022120799-appb-000023
Figure PCTCN2022120799-appb-000024
实施例5：ELISA竞争检测方法
竞争法ELISA被用于评估上述细胞培养上清对于S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白和ACE2蛋白的结合的阻断能力。将ELISA板用100μl/孔的PBS中1μg/ml的重组S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白在4℃下包被过夜。用PBST(0.05％吐温)洗涤板，并将其用250μl/孔的含1％BSA的PBST在37℃封闭2小时。随后弃去封闭液，每个测试孔加入50μl适宜浓度的待测上清液。然后每孔分别添加100ng/ml 50μl重组ACE2-Fc蛋白(GenScript，Z03484)，在37℃孵育1小时。将板用PBST洗涤3次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗小鼠IgG(Fc-特异性)二抗(Jackson，115-035-071)37℃孵育0.5小时。最后将板用PBST洗涤4次，然后加入TMB显色液(GenScript)并在25℃下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液(国药，10011018)终止反应。使用酶标仪在450nm下读板。空白对照组不加抗体，直接反应了ACE2与S-RBD L452R+E484Q-His(SEQ ID NO:4)蛋白的结合能力。如果抗体对S-RBD L452R+E484Q-His(SEQ ID NO:1)蛋白和ACE2的结合有阻断效果，那么检测值比对照低。以对照为基准，计算出来的各克隆的阻断效果，例如L20E10R的阻断效果就是(1.947-0.793/1.947)％＝59.3％。按照同样的计算方式得到的所有抗体的阻断效果如表5所示。
表5各克隆细胞上清对S-RBD L452R+E484Q-His蛋白和ACE2蛋白结合的阻断效果
Figure PCTCN2022120799-appb-000025
Figure PCTCN2022120799-appb-000026
实施例6：单克隆抗体的重组表达
将实施例4中的可变区基因合成到含有对应亚型恒定区序列(SEQ ID NO:45，SEQ ID NO:46，SEQ ID NO:47)的表达载体中，使用CHO-S细胞进行转染得到重组表达后的抗体。

Claims (19)

  1. 一种抗SARS-CoV-2 L452R刺突蛋白的单克隆抗体或其功能片段，所述抗体或其功能片段包含重链可变区和轻链可变区，其中，
    (a)所述重链可变区包含HCDR1、HCDR2和HCDR3，
    所述HCDR1包含选自SEQ ID NO:15、21、27、33或39所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述HCDR2包含选自SEQ ID NO:16、22、28、34或40所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述HCDR3包含选自SEQ ID NO:17、23、29、35或41所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；以及
    (b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，
    所述LCDR1序列包含选自SEQ ID NO:18、24、30、36或42所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR2序列包含选自SEQ ID NO:19、25、31、37或43所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR3序列包含选自SEQ ID NO:20、26、32、38或44所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体。
  2. 根据权利要求1所述的单克隆抗体或其功能片段，其中，
    所述HCDR1序列包含选自SEQ ID NO:15、21、27、33或39所示的氨基酸序列；所述HCDR2序列包含选自SEQ ID NO:16、22、28、34或40所示的氨基酸序列；所述HCDR3序列包含选自SEQ ID NO:17、23、29、35或41所示的氨基酸序列；以及
    所述LCDR1序列包含选自SEQ ID NO:18、24、30、36或42所示的氨基酸序列；所述LCDR2序列包含选自SEQ ID NO:19、25、31、37或43所示的氨基酸序列；所述LCDR3序列包含选自SEQ ID NO:20、26、32、38或44所示的氨基酸序列。
  3. 根据权利要求1或2所述的单克隆抗体或其功能片段，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
    (a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:15、16和17所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:18、19和20所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:21、22和23所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:24、25和26所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (c)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:27、28和29所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:30、31和32所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:33、34和35所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:36、37和38所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；或
    (e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:39、40和41所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:42、 43和44所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体。
  4. 根据权利要求3中所述的单克隆抗体或其功能片段，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
    (a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:15、16和17所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:18、19和20所示的氨基酸序列；
    (b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:21、22和23所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:24、25和26所示的氨基酸序列；
    (c)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:27、28和29所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:30、31和32所示的氨基酸序列；
    (d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:33、34和35所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:36、37和38所示的氨基酸序列；或
    (e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:39、40和41所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:42、43和44所示的氨基酸序列。
  5. 根据权利要求1～4中任一项所述的单克隆抗体或其功能片段，其中，所述重链可变区序列包含与SEQ ID NO:5、7、9、11或13所示氨基酸序列具有至少80％一致性的氨基酸序列；以及
    所述轻链可变区序列包含与SEQ ID NO:6、8、10、12或14所示氨基酸序列具有至少80％一致性的氨基酸序列。
  6. 根据权利要求5中所述的单克隆抗体或其功能片段，其中，
    所述重链可变区序列包含SEQ ID NO:5、7、9、11或13所示的氨基酸序列；以及所述轻链可变区序列包含SEQ ID NO:6、8、10、12或14所示的氨基酸序列。
  7. 根据权利要求1～6中所任一项所述单克隆抗体或其功能片段，所述重链可变区和轻链可变区选自如下序列：
    (a)所述重链可变区包含与SEQ ID NO:5所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:6所示序列具有至少80％一致性的氨基酸序列；
    (b)所述重链可变区包含与SEQ ID NO:7所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:8所示序列具有至少80％一致性的氨基酸序列；
    (c)所述重链可变区包含与SEQ ID NO:9所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:10所示序列具有至少80％一致性的氨基酸序列；
    (d)所述重链可变区包含与SEQ ID NO:11所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:12所示序列具有至少80％一致性的氨基酸序列；或
    (e)所述重链可变区包含与SEQ ID NO:13所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:14所示序列具有至少80％一致性的氨基酸序列。
  8. 根据权利要求7所述单克隆抗体或其功能片段，所述重链可变区和轻链可变区选自如下序列：
    (a)所述重链可变区包含SEQ ID NO:5所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:6所示的氨基酸序列；
    (b)所述重链可变区包含如SEQ ID NO:7所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列；
    (c)所述重链可变区包含如SEQ ID NO:9所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:10所示的氨基酸序列；
    (d)所述重链可变区包含如SEQ ID NO:11所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:12所示的氨基酸序列；或
    (e)所述重链可变区包含如SEQ ID NO:13所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:14所示的氨基酸序列。
  9. 编码权利要求1～8中任一项所述的抗SARS-CoV-2 L452R刺突蛋白单克隆抗体或其功能片段的分离的多核苷酸。
  10. 根据权利要求9所述的多核苷酸，其特征在于，所述多核苷酸包含编码所述单克隆抗体或其功能片段的重链可变区的核苷酸序列，和编码所述单克隆抗体或其功能片段的轻链可变区的核苷酸序列。
  11. 包含根据权利要求9或10所述的多核苷酸的表达载体。
  12. 包含根据权利要求11所述表达载体的宿主细胞或无细胞表达系统。
  13. 一种药物组合物，所述药物组合物包含权利要求1～8中任一项所述的单克隆抗体或其功能片段和药学上可接受的载体。
  14. 权利要求1～8中任一项所述的单克隆抗体或其功能片段在冠状病毒检测和诊断产品中的应用。
  15. 权利要求1～8中任一项所述的单克隆抗体或其功能片段在制备治疗冠状病毒药物中的应用。
  16. 根据权利要求14或15所述的应用，所述冠状病毒选自SARS-CoV、MERS-CoV、SARS-CoV-2或SARS-CoV-2突变株；优选为SARS-CoV-2突变株；更优选为包含L452R刺突蛋白的SARS-CoV-2突变株。
  17. 一种检测冠状病毒的试剂盒，所述试剂盒中包含权利要求1～8中任一项所述的单克隆抗体或其功能片段。
  18. 根据权利要求17所述的试剂盒，所述冠状病毒选自SARS-CoV、MERS-CoV、SARS-CoV-2或SARS-CoV-2突变株；优选为SARS-CoV-2突变株；更优选为包含L452R刺突蛋白的SARS-CoV-2突变株。
  19. 根据权利要求1～8中任一项所述的单克隆抗体或其功能片段，其特征在于，所述抗体是鼠源的、嵌合的、人源化的或者人的。
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