WO2023038477A1 - Anticorps spécifique d'une protéine de nucléocapside de coronavirus et utilisation associée - Google Patents

Anticorps spécifique d'une protéine de nucléocapside de coronavirus et utilisation associée Download PDF

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WO2023038477A1
WO2023038477A1 PCT/KR2022/013565 KR2022013565W WO2023038477A1 WO 2023038477 A1 WO2023038477 A1 WO 2023038477A1 KR 2022013565 W KR2022013565 W KR 2022013565W WO 2023038477 A1 WO2023038477 A1 WO 2023038477A1
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seq
antibody
variable region
chain variable
antigen
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부하령
조은위
허창규
양지현
임원희
곽채원
조유정
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한국생명공학연구원
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    • 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
    • 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
    • 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]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to antibodies specific for coronavirus nucleocapsid proteins and uses thereof.
  • Coronavirus disease 2019 (COVID-19) is a severe disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a human coronavirus first reported in December 2019. It is an acute respiratory disease. Coronavirus was first discovered in chickens in the late 1920s, then in animals such as dogs, pigs and birds, and in 1965 in humans. Human coronaviruses are known to be transmitted when droplets from an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, or mouth. However, SARS, which occurred in 2009, which caused fatal respiratory diseases, and Middle East Respiratory Syndrome (MERS), which occurred in 2012, are also known infectious diseases caused by human coronaviruses.
  • SARS severe acute respiratory syndrome coronavirus 2
  • MERS Middle East Respiratory Syndrome
  • COVID-19 caused by SARS-CoV-2 infection spread around the world due to its high fatality rate and rapid spread, and was declared a pandemic by the World Health Organization (WHO) in March 2020, and some The country is making all-out efforts to prevent the spread by controlling entry from countries with a large number of confirmed cases.
  • WHO World Health Organization
  • the SARS-CoV-2 mutant virus emerges in many parts of the world, diagnosing and controlling COVID-19 is more difficult.
  • the most essential method for controlling infectious diseases is virus infection diagnosis, and the most important factors in diagnosis are accuracy and speed. Accordingly, it is necessary to develop a method capable of quickly and accurately diagnosing whether an object to be analyzed is infected with the coronavirus.
  • the present inventors developed an antibody that specifically binds to the protein of nucleocapsid (NP), which is a region with low variation in gene sequence among antigens expressed by coronaviruses, and confirmed that it can be used for diagnosis of coronavirus.
  • NP nucleocapsid
  • An object of the present invention is to provide an antibody that specifically binds to a coronavirus nucleocapsid (NP) protein or an antigen-binding fragment that specifically binds to the nucleocapsid protein.
  • NP coronavirus nucleocapsid
  • Another object of the present invention is to provide a polynucleotide encoding the antibody or antigen-binding fragment, an expression vector containing the same, and a host cell containing the same.
  • Another object of the present invention is to provide a composition for diagnosing coronavirus infection comprising the antibody or the antigen-binding fragment, and a kit including the same.
  • Another object of the present invention is to provide an information providing method for diagnosing coronavirus infection, comprising the step of detecting coronavirus present in a biological sample using the antibody or the antigen-binding fragment.
  • the antibody of the present invention By using the antibody of the present invention, a much smaller amount of coronavirus can be detected compared to conventional coronavirus detection antibodies, so the antibody of the present invention can be usefully used for detecting and diagnosing coronavirus.
  • FIG. 1 shows the results of confirming the reactivity of antibodies produced by mouse-derived B cell hybridomas immunized through inoculation with inactivated SARS-CoV-2 to the SARS-CoV-2 NP antigen by ELISA.
  • the spleen of the immunized mouse was collected to collect B lymphocytes, and a B cell hybridoma cell population was constructed by fusing with mouse myeloma cells.
  • Anti-NP antibody-secreting B cell hybridomas were selected by ELISA to confirm reactivity to NP protein for the antibodies secreted by each hybridoma, and subcloning by sequentially diluting the hybridoma parent cells. was performed several times to finally select 3E10, 3F10, 5B6, 5B6.8 and 1G6 anti-NP antibodies.
  • NP Nucleocapsid
  • S1 and S2 proteins of SARS-CoV-2 spike protein S1 and S2 proteins of SARS-CoV-2 spike protein
  • RBD receptor binding domain
  • An anti-NP antibody purified with Protein G resin was added to the coated well, and the bound antibody was detected with an anti-mouse IgG-HRP (CST) antibody.
  • CST anti-mouse IgG-HRP
  • Figure 3 shows SARS-CoV-2 NP antigens using 5 new antibodies (3E10, 3F10, 5B6, 5B6.8, 1G6) and 2 commercially available anti-SARS-CoV-2 NP antibodies 4B21 and B46F. This is an ELISA result comparing reactivity.
  • Anti-NP antibodies were serially diluted and added to wells coated with SARS-CoV-2 NP antigen, and bound antibodies were detected with an anti-mouse IgG-HRP antibody.
  • Figure 4 is a SARS-CoV NP antigen (SARS-CoV NP antigen) using 5 novel antibodies (3E10, 3F10, 5B6, 5B6.8, 1G6) and 2 commercially available anti-SARS-CoV-2 NP antibodies 4B21 and B46F. -CoV-2 NP and amino acid sequence are 91% identical) This is an ELISA result comparing reactivity.
  • Anti-NP antibodies were serially diluted and added to wells coated with SARS-CoV NP antigen protein, and the bound antibodies were detected with an anti-mouse IgG-HRP antibody.
  • Figure 5 shows inactivation of SARS-CoV using two previously reported anti-SARS-CoV-2 NP antibodies 4B21 and B46F and five new purified antibodies (3E10, 3F10, 5B6, 5B6.8, 1G6) -2 This is an ELISA result comparing the reactivity to the virus lysate.
  • the anti-NP antibodies were serially diluted and added to the wells coated with the inactivated SARS-CoV-2 virus lysate, and the bound antibodies were detected with an anti-mouse IgG-HRP antibody.
  • FIG. 6 is a Western blot (WB) result performed to investigate whether the novel antibody recognizes a linear or conformational epitope of the SARS-CoV-2 NP antigen protein.
  • the SARS-CoV-2 NP recombinant antigen protein was developed on reducing 12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, and then 3E10, 3F10, 5B6, 5B6.8, and 1G6 antibodies were used as primary antibodies and 2 Anti-mouse-IgG-HRP antibody was treated as secondary antibody.
  • PVDF polyvinylidene fluoride
  • 3E10, 3F10, 5B6, 5B6.8, and 1G6 antibodies were used as primary antibodies and 2 Anti-mouse-IgG-HRP antibody was treated as secondary antibody.
  • NP developed on SDS-PAGE was confirmed to be slightly above the predicted molecular weight of 47.33 kDa, and it was confirmed that a protein band was detected
  • Figure 7 is a Western blot result confirming that the anti-NP antibody specifically binds to the intracellularly expressed SARS-CoV-2 NP antigen protein.
  • Human embryonic kidney cells HEK293T
  • HEK293T Human embryonic kidney cells
  • the cell lysate was developed on reducing 12% SDS-PAGE and then transferred to a PVDF membrane to form 3E10, 3F10 , 5B6, 5B6.8, 1G6 antibodies to detect specific proteins Western blot results.
  • the NP-His protein was expressed at the predicted molecular weight of 47.33 kDa through post-translational modification in cells, and 3E10, 3F10, 5B6, 5B6 It was confirmed that both .8 and 1G6 antibodies bind to the protein at the same position as the anti-His antibody.
  • Figure 8 is a commercial analysis using the specific reactivity of the 3E10, 3F10, 5B6, 5B6.8 antibodies to the NP protein expressed in cells in Figure 7 to confirm whether the novel anti-NP antibody is also applicable to immunoprecipitation.
  • Human embryonic kidney cells HEK293T
  • SARS-CoV-2 NP-His/pCMV3 plasmid which is used as a protein, and the cell lysate was mixed with the new antibody, and then the antibody was captured with Protein G resin. This is the result of confirming that antigens are detected together.
  • FIG. 9 is a schematic diagram showing the constituent parts of recombinant proteins cloned with NP fragment variants in which a specific amino acid sequence is missing in order to determine the precise epitope of an anti-SARS-CoV-2 NP antibody.
  • a total of two types of partially defective recombinant proteins were prepared, NP (1-174 AA)-p1-His (sequence 1-174 amino acid partial defect protein), NP (1-248 AA) -p2-His (SEQ ID NO: 1 partially deleted protein with ⁇ 248 amino acids).
  • NP (1-174 AA)-p1-His NP sequence 1-174 amino acids
  • NP (1-248 AA)-p2-His including the N-terminal region and SR-rich portion
  • the 1st to 174th regions of the NP protein were epitope-determining regions in the four types of 3E10, 3F10, 5B6, and 5B6.8 antibodies, and the 1G6 antibody was not the 1st to 248th regions of the NP protein. It was confirmed that the C-terminal region was the epitope of these antibodies. Correct expression of the recombinant protein was confirmed using an anti-His antibody as a positive control.
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • VL light chain variable region
  • cDNA Complementary DNA
  • a polymerase chain reaction is performed using heavy chain variable region complementarity determining region primers and light chain variable region complementarity determining region primers. reaction, PCR), and then ligated to a T-vector to prepare a recombinant plasmid.
  • the recombinant plasmid was amplified in host cells, extracted, and the corresponding region of the complementarity determining region was sequenced.
  • the protein sequence was confirmed from the analyzed nucleotide sequence, and the CDR sequence was determined based on the KaBat CDR definition.
  • 13 is a schematic diagram of the SARS-CoV-2 NP antigen protein and fragment protein and the results of additional analysis to more precisely determine the epitope.
  • 13A is a schematic diagram of the structure of SARS-CoV-2 NPs, full-length NPs numbered 1-419; NP-NTD numbers 47-174; NP-CTD numbers 248-364; NP-p1 is 1-174; NP-p2 is numbered 1-248; NP-p3 is numbered 1-305; 101-419 for NP-p4; NP-p5 is a partially defective recombinant protein having amino acids 248-419.
  • FIG. 13B shows Western Blot results.
  • Recombinant proteins were developed on reducing 12% SDS-PAGE and transferred to a PVDF membrane, and it was confirmed whether a specific NP partially defective protein containing an epitope was detected with an anti-NP antibody. Based on these results, it was confirmed that the epitope of the 3F10 antibody was the 1st to 47th region of the NP protein, and the 3E10 and 5B6 antibodies confirmed that the 47th to 101st region of the NP protein was their epitope. It was confirmed that the 1G6 antibody was the epitope of these antibodies at positions 364 to 419 of the NP protein.
  • FIG. 14 is a result of comparative analysis of SARS-CoV-2 NP and SARS-CoV-2 viral lysate recognition in a sandwich ELISA method loaded with a novel anti-NP antibody pair.
  • the anti-NP antibodies 1G6 or 3E10, which has the strongest binding force to SARS-CoV-2 NP, was coated on the bottom of the ELISA plate as a capture antibody, and then washed.
  • SARS-CoV-2 NP recombinant protein was added, washed, and then detected with an HRP-conjugated anti-NP antibody.
  • 14A and B show that the most effective capture and detection antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 NP antigen protein are 1G6 and 3E10, which have strong binding affinity to NP and have different NP epitopes.
  • 14C and D show that the most effective capture and detection antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 virus lysates are 1G6 and 3F10, and 1G6 and 3E10.
  • the left side is a schematic diagram showing the positions of amino acid mutations in the NP protein of SARS-CoV-2 virus variants, and mutations are indicated in red.
  • the right side is the result of ELISA using the antibody of the present invention.
  • Anti-NP antibodies were serially diluted and added to wells coated with SARS-CoV-2 mutant virus NP antigen, and the bound antibody was anti-mouse IgG-HRP antibody was detected with It was confirmed that all four anti-NP antibodies (1G6, 3E10, 3F10, 5B6) had binding ability to NP of alpha, beta, and gamma mutant SARS-CoV-2 viruses.
  • SARS-CoV-2 variant antigen NP recombinant protein was added to an ELISA plate coated with 3E10 capture antibody, and then detected with HRP-conjugated 1G6 antibody. It was confirmed that all NPs of alpha, beta, and gamma mutant SARS-CoV-2 viruses were detected in a sandwich ELISA loaded with 3E10 and 1G6 antibody pairs. It was confirmed that it was not detected when using the influenza NP antibody bound to HRP, a negative control.
  • One aspect of the present invention provides an antibody that specifically binds to a coronavirus nucleocapsid (NP) protein or an antigen-binding fragment that specifically binds to said protein.
  • NP coronavirus nucleocapsid
  • coronavirus refers to a positive-sense single-stranded RNA virus (ssRNA) belonging to the family Coronaviridae and having a spherical outer membrane and having a size of about 80-220 nm.
  • Coronaviruses are the outermost spike (S) protein, hemagglutinin-esterase (HE) protein, membrane (M) protein, envelope (E) protein and nucleosomes. It is known to contain five structural proteins, including the capsid (nucleocapsid, NP) protein (Lai and Homes, 2001. Fields Virology).
  • SARS-CoV SARS-CoV-1, SARS-coronavirus
  • SARS-CoV SARS-CoV-1, SARS-coronavirus
  • SARS-CoV has a genome of about 29,727 nucleotides, and is the largest genome among RNA viruses known to date.
  • SARS-CoV The genome of SARS-CoV has 11 open reading frames (ORFs) and is known to encode 23 proteins. It was found that the major structural proteins of SARS-coronavirus are nucleocapsid (NP), spike (S), membrane (M), and envelope (E) proteins, like other coronaviruses. As a result of sequencing of these proteins, it was found that SARS-coronavirus has a very low sequence homology of about 40 to 50% compared to other coronaviruses. According to the phylogenetic classification according to the antigenicity of the coronavirus, SARS coronavirus was found to have a high affinity with group II among groups I, II and III.
  • SARS-CoV-2 SARS-coronavirus-2
  • COVID-19 coronavirus infection-19
  • SARS-CoV-2 is taxonomically considered to be a strain of SARS-CoV, has zoonotic origins, and has close genetic similarities to bat coronaviruses.
  • the SARS-CoV-2 is mainly transmitted through close contact between people or through droplets generated during coughing or sneezing, and mainly binds to the receptor angiotensin-converting enzyme 2 (ACE2) present on the surface of human cells known to enter human cells.
  • ACE2 receptor angiotensin-converting enzyme 2
  • SARS-CoV-2 is RNA-dependent RNA polymerase (RNA-dependent RNA polymerase), spike (S), nucleocapsid (NP), membrane (M) protein, envelope (E) and protein and / or encoding said protein It may contain a gene that Among the structural proteins of SARS-CoV-2, the nucleocapsid (NP) protein surrounds the genetic material ssRNA and is involved in RNA replication.
  • Spike (S) protein exists on the surface of virus particles and is involved in host cell invasion, and is divided into S1 and S2.
  • the S1 protein interacts with the host cell receptor, and the receptor-binding domain (RBD) present in the S1 protein is known as a key site that binds to the ACE2 receptor present on the host cell surface.
  • RBD receptor-binding domain
  • the S2 protein is involved in fusion with the cell membrane of the host cell.
  • the structure is described in Cascella, M., Rajnik, M., Cuomo, A., Dulebohn, S. C., & Di Napoli, R. (2020).
  • the SARS-CoV-2 of the present invention includes all mutant forms that can be classified as SARS-CoV-2 without limitation, and examples thereof include alpha (B.1.1.7 strain) and beta (B.1.351 strain: B. 1.351.2, B.1.351.3), delta (B.1.617.2 and AY strains: AY.1, AY.2, AY.3), gamma (P.1 strain: P.1.1, P.1.2) It may include a mutation, referred to as a mutation. Mutant SARS-CoV-2 may include specific substitutions or substitution combinations in the spike protein, examples of which include L452R, E484K, K417N/T, E484K, and N501Y.
  • the antibody or antigen-binding fragment thereof provided in the present invention may specifically bind to the nucleocapsid (NP) protein (also referred to as “N protein”) of the coronavirus.
  • NP nucleocapsid
  • the antibody or antigen-binding fragment thereof provided in the present invention may have an epitope included in any one region selected from the following;
  • the antibody or antigen-binding fragment thereof thereof
  • a heavy chain variable region comprising HCDR1 of SEQ ID NO: 56, HCDR2 of SEQ ID NO: 58, and HCDR3 of SEQ ID NO: 60; and a light chain variable region comprising LCDR1 of SEQ ID NO: 64, LCDR2 of SEQ ID NO: 66, and LCDR3 of SEQ ID NO: 68; or
  • a heavy chain variable region comprising HCDR1 of SEQ ID NO: 74, HCDR2 of SEQ ID NO: 76, and HCDR3 of SEQ ID NO: 78; and a light chain variable region including LCDR1 of SEQ ID NO: 82, LCDR2 of SEQ ID NO: 84, and LCDR3 of SEQ ID NO: 86.
  • the antibody or antigen-binding fragment thereof thereof
  • the antibody or antigen-binding fragment thereof may further include a heavy chain tail domain of any one of SEQ ID NOs: 8, 26, 44, 62, and 80.
  • the antibody or antigen-binding fragment thereof may further include a light chain tail domain of any one of SEQ ID NOs: 16, 34, 52, 70, and 88.
  • the antibody or antigen-binding fragment thereof thereof
  • (v) may include a heavy chain variable region of SEQ ID NO: 89 and a light chain variable region of SEQ ID NO: 90.
  • the antibody or antigen-binding fragment thereof provided in the present invention may bind to the N-terminus of the nucleocapsid (NP) protein of the coronavirus.
  • the binding site of the antibody or antigen-binding fragment thereof provided in the present invention is 1 to 174 from the N-terminus of the nucleocapsid (NP) protein of the coronavirus shown in FIG. 12 (SEQ ID NO: 91) It can be included within the sequence corresponding to the amino acid. More specifically, the binding site of the antibody or antigen-binding fragment thereof provided in the present invention is 1 to 47 from the N-terminus of the nucleocapsid (NP) protein of the coronavirus shown in FIG. 12 (SEQ ID NO: 91). It may be included in a sequence corresponding to amino acid 1, or a sequence corresponding to amino acids 47 to 101.
  • the antibody 3F10 of the present invention has a binding site in the sequence corresponding to amino acids 1 to 47 from the N-terminus of the nucleocapsid (NP) protein of coronavirus, 3E10 and 5B6 It was confirmed to have a binding site in the sequence corresponding to amino acids 47 to 101 from the N-terminus of the nucleocapsid (NP) protein of this coronavirus.
  • the antibody or fragment thereof provided in the present invention may bind to the C-terminus of the nucleocapsid (NP) protein of the coronavirus.
  • the binding site of the antibody or antigen-binding fragment thereof provided in the present invention is amino acids 248 to 419 from the N-terminus of the nucleocapsid (NP) protein of the coronavirus shown in FIG. 12 (SEQ ID NO: 91) It can be included in the sequence corresponding to. More specifically, the binding site of the antibody or fragment thereof provided in the present invention is located at amino acids 364 to 419 from the N-terminus of the nucleocapsid (NP) protein of the coronavirus shown in FIG. 12 (SEQ ID NO: 91). can be included within the corresponding sequence.
  • the antibody 1G6 of the present invention has a binding site within the sequence corresponding to amino acids 364 to 419 from the N-terminus of the nucleocapsid (NP) protein of coronavirus.
  • sequence of FIG. 12 is a representative example of the nucleocapsid protein of coronavirus, and as a reference sequence for indicating the binding position of the antibody or binding fragment thereof provided in the present invention, A person skilled in the art can align any sequence with the sequence of SEQ ID NO: 91 shown in FIG. 12 to identify a position corresponding to a specific residue number or a sequence region corresponding to a specific residue number from the N-terminus of SEQ ID NO: 91 in any sequence. there is.
  • the term "antibody” refers to a protein molecule that serves as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, Both whole antibodies and antibody fragments are included.
  • the term also includes chimeric antibodies (eg humanized murine antibodies) and bivalent or bispecific molecules (eg bispecific antibodies), diabodies, triabodies and tetrabodies.
  • the term further includes single-chain antibodies, scabs, derivatives of antibody constant regions and artificial antibodies based on protein scaffolds that have a binding function to FcRn (neonatal Fc receptor).
  • a whole antibody has a structure having two full-length light chains (LC) and two full-length heavy chains (HC), and each light chain is connected to the heavy chain by a disulfide bond.
  • the total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.
  • the antibody provided in the present invention may be an IgG antibody.
  • fragment binding fragment of a polypeptide
  • antibody fragment refers to any fragment of an antibody of the invention that retains the antigen-binding activity of the antibody.
  • Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv or scFv.
  • the Fd refers to the heavy chain part included in the Fab fragment.
  • the Fab has a structure having light and heavy chain variable regions, a light chain constant region (framework region, FR), and a heavy chain first constant region (CH1 domain), and has one antigen binding site.
  • Fab' is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'.
  • Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and the light chain variable region.
  • dsFv double disulfide Fv
  • scFv single chain Fv
  • the heavy chain variable region and light chain variable region are generally covalently linked through a peptide linker. .
  • Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), For example, it can be produced through genetic recombination technology.
  • proteolytic enzymes for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin
  • the antibody provided in the present invention may be a monoclonal antibody.
  • the term "monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such a monoclonal antibody has a single binding specificity and affinity for a specific epitope. indicates the figure.
  • an immunoglobulin has a heavy chain and a light chain, each comprising a constant region and a variable region (the regions are also known as domains).
  • the light chain and heavy chain variable regions include three variable regions and four structural regions, which are referred to as complementarity-determining regions (CDRs).
  • the CDR mainly serves to bind to an epitope of an antigen.
  • the CDRs of each chain are typically labeled CDR1, CDR2, and CDR3 sequentially, starting from the N-terminus, and are also identified by the chain in which a particular CDR is located.
  • the complementarity-determining regions are located between regions that are relatively conserved, called constant regions (FR).
  • Each heavy chain variable region (VH) and light chain variable region (VL) consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigen.
  • the CDRs of the heavy chain variable region are HCDR1, HCDR2, and HCDR3, the CDRs of the light chain variable region are LCDR1, LCDR2, and LCDR3, the FRs of the heavy chain variable region are HFR1, HFR2, HFR3, and HFR4, and the FRs of the light chain variable region are LFR1 and LFR2 , LFR3, LFR4.
  • the antibody specifically binding to the coronavirus nucleocapsid (NP) protein of the present invention or the antigen-binding fragment specifically binding to the protein is N of SEQ ID NO: 91 -A heavy chain variable region having an epitope included in the amino acid region at positions 1 to 47 from the end and including HCDR1 of SEQ ID NO: 20, HCDR2 of SEQ ID NO: 22, and HCDR3 of SEQ ID NO: 24; and a light chain variable region including LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 30, and LCDR3 of SEQ ID NO: 32.
  • the antibody or antigen-binding fragment thereof having an epitope included in the region of amino acids 1 to 47 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 19 a heavy chain variable region comprising HFR1 of SEQ ID NO: 21, HFR2 of SEQ ID NO: 23, HFR3 of SEQ ID NO: 23, and HFR4 of SEQ ID NO: 25; and a light chain variable region comprising LFR1 of SEQ ID NO: 27, LFR2 of SEQ ID NO: 29, LFR3 of SEQ ID NO: 31, and LFR4 of SEQ ID NO: 33.
  • the antibody or antigen-binding fragment thereof having an epitope included in the amino acid region at positions 1 to 47 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 26 It may further include a heavy chain tail domain of.
  • the antibody or antigen-binding fragment thereof having an epitope included in the region of amino acids 1 to 47 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 34 It may further include a light chain tail domain of.
  • the antibody its or antigen-binding fragment having an epitope included in the amino acid region at positions 1 to 47 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 35 It may include a heavy chain variable region of and a light chain variable region of SEQ ID NO: 36.
  • the antibody specifically binding to the coronavirus nucleocapsid (NP) protein of the present invention or the antigen-binding fragment specifically binding to the protein is N of SEQ ID NO: 91 -Has an epitope included in the amino acid region at positions 47 to 101 from the end, and has a heavy chain variable region including HCDR1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 4, and HCDR3 of SEQ ID NO: 6, and SEQ ID NO: 10 or a light chain variable region comprising LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 14, or LCDR3 of SEQ ID NO: 14; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 38, HCDR2 of SEQ ID NO: 40, and HCDR3 of SEQ ID NO: 42, and a light chain variable region comprising LCDR1 of SEQ ID NO: 46, LCDR2 of SEQ ID NO: 48, and LCDR3 of
  • the antibody or antigen-binding fragment thereof having an epitope included in the region of amino acids 47 to 101 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 1 HFR1 of SEQ ID NO: 3, HFR3 of SEQ ID NO: 5, HFR4 of SEQ ID NO: 7, and LFR1 of SEQ ID NO: 9, LFR2 of SEQ ID NO: 11, LFR3 of SEQ ID NO: 13, SEQ ID NO: 15 or a light chain variable region including LFR4; HFR1 of SEQ ID NO: 37, HFR2 of SEQ ID NO: 39, HFR3 of SEQ ID NO: 41, a heavy chain variable region comprising HFR4 of SEQ ID NO: 43, and LFR1 of SEQ ID NO: 45, LFR2 of SEQ ID NO: 47, LFR3 of SEQ ID NO: 49, sequence It may be one containing a light chain variable region containing LFR4 of No. 51.
  • the antibody or antigen-binding fragment thereof having an epitope included in the region of amino acids 47 to 101 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 8 Alternatively, it may further include a heavy chain tail domain of 44.
  • the antibody or antigen-binding fragment thereof having an epitope included in the region of amino acids 47 to 101 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 16 Alternatively, it may further include 52 light chain tail domains.
  • the antibody or antigen-binding fragment thereof having an epitope included in the region of amino acids 47 to 101 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 17
  • the antibody specifically binding to the coronavirus nucleocapsid (NP) protein of the present invention or the antigen-binding fragment specifically binding to the protein is N of SEQ ID NO: 91 -A heavy chain variable region having an epitope included in the amino acid region at positions 364 to 419 from the end and including HCDR1 of SEQ ID NO: 74, HCDR2 of SEQ ID NO: 76, and HCDR3 of SEQ ID NO: 78; and a light chain variable region including LCDR1 of SEQ ID NO: 82, LCDR2 of SEQ ID NO: 84, and LCDR3 of SEQ ID NO: 86.
  • the antibody or antigen-binding fragment thereof having an epitope included in the amino acid region at positions 364 to 419 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 73 a heavy chain variable region comprising HFR1 of SEQ ID NO: 75, HFR2 of SEQ ID NO: 77, HFR3 of SEQ ID NO: 79, and HFR4 of SEQ ID NO: 79; and a light chain variable region comprising LFR1 of SEQ ID NO: 81, LFR2 of SEQ ID NO: 83, LFR3 of SEQ ID NO: 85, and LFR4 of SEQ ID NO: 87.
  • the antibody or antigen-binding fragment thereof having an epitope included in the amino acid region at positions 364 to 419 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 80 It may further include a heavy chain tail domain of.
  • the antibody or antigen-binding fragment thereof having an epitope included in the amino acid region at positions 364 to 419 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 88 It may further include a light chain tail domain of.
  • the antibody or antigen-binding fragment thereof having an epitope included in the amino acid region at positions 364 to 419 from the N-terminus of SEQ ID NO: 91 is SEQ ID NO: 89 It may include a heavy chain variable region of and a light chain variable region of SEQ ID NO: 90.
  • the antibody specifically binding to the N-terminus of the coronavirus nucleocapsid (NP) protein of the present invention or the antigen-binding fragment specifically binding to the N-terminus is ,
  • a heavy chain variable region comprising HCDR1 of SEQ ID NO: 56, HCDR2 of SEQ ID NO: 58, and HCDR3 of SEQ ID NO: 60; and a light chain variable region including LCDR1 of SEQ ID NO: 64, LCDR2 of SEQ ID NO: 66, and LCDR3 of SEQ ID NO: 68.
  • the antibody or antigen-binding fragment thereof thereof
  • a heavy chain variable region comprising HFR1 of SEQ ID NO: 55, HFR2 of SEQ ID NO: 57, HFR3 of SEQ ID NO: 59, and HFR4 of SEQ ID NO: 61; and a light chain variable region comprising LFR1 of SEQ ID NO: 63, LFR2 of SEQ ID NO: 65, LFR3 of SEQ ID NO: 67, and LFR4 of SEQ ID NO: 69.
  • the antibody or antigen-binding fragment thereof may further include a heavy chain tail domain of any one of SEQ ID NOs: 8, 26, 44, and 62.
  • the antibody or antigen-binding fragment thereof may further include a light chain tail domain of any one of SEQ ID NOs: 16, 34, 52, and 70.
  • the antibody or antigen-binding fragment thereof thereof
  • (iv) may include the heavy chain variable region of SEQ ID NO: 71 and the light chain variable region of SEQ ID NO: 72.
  • NP coronavirus nucleocapsid
  • the antibody specifically binding to the C-terminus of the coronavirus nucleocapsid (NP) protein of the present invention or the antigen-binding fragment specifically binding to the C-terminus is HCDR1 of SEQ ID NO: 74, a heavy chain variable region comprising HCDR2 of SEQ ID NO: 76 and HCDR3 of SEQ ID NO: 78; and a light chain variable region including LCDR1 of SEQ ID NO: 82, LCDR2 of SEQ ID NO: 84, and LCDR3 of SEQ ID NO: 86.
  • the antibody or antigen-binding fragment thereof comprises HFR (heavy chain FR)1 of SEQ ID NO: 73, HFR2 of SEQ ID NO: 75, HFR3 of SEQ ID NO: 77, and HFR4 of SEQ ID NO: 79.
  • Heavy chain variable region containing; and a light chain variable region comprising LFR (light chain FR)1 of SEQ ID NO: 81, LFR2 of SEQ ID NO: 83, LFR3 of SEQ ID NO: 85, and LFR4 of SEQ ID NO: 87.
  • the antibody or antigen-binding fragment thereof may further include a heavy chain tail domain of SEQ ID NO: 80.
  • the antibody or antigen-binding fragment thereof may further include a light chain tail domain of SEQ ID NO: 88.
  • the antibody or antigen-binding fragment thereof may include a heavy chain variable region of SEQ ID NO: 89 and a light chain variable region of SEQ ID NO: 90.
  • antibody 1G6 that specifically binds to the C-terminus of the coronavirus nucleocapsid (NP) protein was developed, and it was confirmed that the developed antibody has CDR and FR sequences as shown in FIG. 11 Thus, the invention was completed.
  • the CDRs of the variable regions were determined by a conventional method according to the system devised by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda, MD. (1991)) CDR numbering used in the present invention was performed using the Kabat method, but antibodies containing CDRs determined according to other methods such as the IMGT method, the Chothia method, and the AbM method are also included in the scope of the present invention.
  • Another aspect of the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment thereof provided in the present invention.
  • Polynucleotides encoding the antibodies of the present invention can be readily isolated and sequenced using conventional procedures.
  • oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from the phage template DNA can be used.
  • the polynucleotide Once the polynucleotide has been isolated, it can be placed into an expression vector, which can then be introduced into a suitable host cell to produce the desired monoclonal antibody from the transformed host cell (i.e., transformant).
  • Another aspect of the present invention provides an expression vector containing the antibody or antigen-binding fragment thereof provided in the present invention and a host cell into which the vector is introduced.
  • the antibody is as described above.
  • the expression vector containing the polynucleotide encoding the antibody provided in the present invention is not particularly limited thereto, but is mammalian cell (eg, human, monkey, rabbit, rat, hamster, mouse cell, etc.), plant cell, yeast It can be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including cells, insect cells, or bacterial cells (eg, E. coli, etc.), specifically, the nucleotide in the host cell It can be a vector operably linked to an appropriate promoter for expression and containing at least one selectable marker.
  • the polynucleotide may be introduced into a phage, plasmid, cosmid, mini-chromosome, virus or retroviral vector.
  • the expression vector including the polynucleotide encoding the antibody may be an expression vector each including a polynucleotide encoding the heavy chain or light chain of the antibody, or an expression vector including both polynucleotides encoding the heavy chain or light chain of the antibody.
  • Host cells into which the expression vector provided in the present invention is introduced are not particularly limited thereto, but bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium transformed by the introduction of the expression vector; yeast cells; fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells, chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), Bowes melanoma cells, HT-1080 , animal cells such as BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), and PER.C6 (human retinal cells); or plant cells.
  • bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium transformed by the introduction of the expression vector
  • transduction refers to a method of delivering a vector containing a polynucleotide encoding the antibody to a host cell.
  • introduction is carried out by calcium phosphate-DNA co-precipitation method, DEAE-dextran-mediated transfection method, polybrene-mediated transfection method, electroporation method, microinjection method, liposome fusion method, lipofectamine and protoplast fusion method, etc. It can be performed by several methods known in the art.
  • transduction means the transfer of a target into a cell using virus particles as a means of infection.
  • vectors can be introduced into host cells by gene bombardment or the like. In the present invention, introduction may be used in combination with transfection and transformation.
  • Another aspect of the present invention provides a composition for diagnosing coronavirus infection, comprising the antibody or antigen-binding fragment thereof provided in the present invention.
  • Another aspect of the present invention provides a kit for diagnosing coronavirus infection, including the antibody or antigen-binding fragment thereof provided in the present invention.
  • Another aspect of the present invention is an antibody or antigen-binding fragment thereof provided by the present invention; Or a composition for diagnosing infection comprising the same; Alternatively, a method for diagnosing a coronavirus infection or providing information for diagnosis, including detecting a coronavirus present in a biological sample isolated from a subject suspected of having a coronavirus infection, using a kit containing the composition is provided.
  • diagnosis refers to determining an individual's susceptibility to a specific disease or disorder, determining whether an individual currently has a specific disease or disorder, or providing information on treatment efficacy. This includes monitoring the health of the object for
  • the diagnosis may be to determine whether or not a coronavirus infectious disease has occurred.
  • the "coronavirus infectious disease” refers to a disease caused by an infection in which a coronavirus invades the body of an organism that is a host.
  • the coronavirus is SARS-CoV-2 (SARS-coronavirus-2 ) can be
  • the coronavirus infectious disease may be COVID-19, but is not limited thereto.
  • composition for diagnosing coronavirus infection of the present invention may include other antibodies or antigen-binding fragments thereof other than the antibodies or antigen-binding fragments thereof of the present invention.
  • materials necessary for the binding reaction between the antibody or antigen-binding fragment thereof of the present invention and the antigen for example, reagents may be further included.
  • a substance necessary for detecting this binding, a substance for stabilizing the antibody or antigen-binding fragment thereof of the present invention, and the like may be further included.
  • the antibody or antigen-binding fragment thereof included in the diagnostic composition of the present invention may be labeled for detection.
  • a variety of methods available for molecular labeling are well known to those skilled in the art and are contemplated within the scope of the present invention.
  • labels examples include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds.
  • Commonly used labels include fluorescent substances (eg, flurescin, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, beta-galactosidase, alkaline phosphatase), radioactive isotopes (eg, 32 P or 125 I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds such as dioxetane, luminol or acridinium. Labeling methods such as covalent bonding of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are also well known.
  • the kit of the present invention may further include reagents, instruments, etc. necessary for detecting the binding of the antibody or antigen-binding fragment thereof of the present invention and an antigen.
  • a solid carrier may be included in the kit container of the present invention.
  • An antibody of the present invention may be attached to a solid carrier, and such a solid carrier may be porous or non-porous, planar or non-planar.
  • the kit may include a solid carrier; a sample pad accommodating a sample to be analyzed and having a buffer input unit and a sample input unit; a conjugate pad containing an antibody that specifically binds to the coronavirus contained in the sample introduced from the sample pad; a signal detection pad including a signal detection unit for detecting whether or not coronavirus is present in the sample and a control unit for determining whether the sample has moved to the absorbent pad regardless of the presence or absence of an analyte; and an absorbent pad for absorbing the specimen after the signal detection reaction is completed, but is not limited thereto.
  • the kit of the present invention may be in the form of an enzyme-linked immunosorbent assay (ELISA) kit.
  • ELISA enzyme-linked immunosorbent assay
  • the term "ELISA” of the present invention also referred to as an enzyme immunoassay method, is a method of quantification using absorbance through a reaction between an enzyme and a substrate by binding an enzyme to an antibody to form an antigen-antibody complex.
  • the ELISA includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of attached antibody and antigen, followed by reaction with another antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support and then recognizing this antibody Indirect sandwich ELISA using labeled secondary antibodies and the like.
  • the kit of the present invention may be a sandwich ELISA kit.
  • HCDR1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 4, HCDR3 of SEQ ID NO: 6, and LCDR1 of SEQ ID NO: 10 SEQ ID NO: An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR2 of SEQ ID NO: 12 and LCDR3 of SEQ ID NO: 14; And a heavy chain variable region comprising HCDR1 of SEQ ID NO: 74, HCDR2 of SEQ ID NO: 76, and HCDR3 of SEQ ID NO: 78, and a light chain variable region comprising LCDR1 of SEQ ID NO: 82, LCDR2 of SEQ ID NO: 84, and LCDR3 of SEQ ID NO: 86.
  • the sandwich ELISA kit of the present invention includes a heavy chain variable region comprising HCDR1 of SEQ ID NO: 20, HCDR2 of SEQ ID NO: 22, and HCDR3 of SEQ ID NO: 24 as a capture antibody and a detection antibody; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 30, and LCDR3 of SEQ ID NO: 32; And a heavy chain variable region comprising HCDR1 of SEQ ID NO: 74, HCDR2 of SEQ ID NO: 76, and HCDR3 of SEQ ID NO: 78, and a light chain variable region comprising LCDR1 of SEQ ID NO: 82, LCDR2 of SEQ ID NO: 84, and LCDR3 of SEQ ID NO: 86.
  • An antibody or antigen-binding fragment thereof comprising a may be included.
  • sandwich ELISA using the antibody of the present invention can be usefully used for detecting SARS-CoV-2.
  • the antibody or fragment thereof of the present invention can be used to detect the presence or absence of a coronavirus in a biological sample by contacting it with a biological sample and confirming the reaction.
  • the biological sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, mucus of lung tissue, respiratory tissue and saliva of the subject, but is not limited thereto, and is prepared by a conventional method known to those skilled in the art possible.
  • the information providing method for diagnosing coronavirus infection of the present invention includes (a) contacting the antibody or antigen-binding fragment thereof provided in the present invention with a biological sample; and (b) detecting a complex of the antibody or antigen-binding fragment thereof of the present invention and the coronavirus NP protein formed by the contact.
  • the term "antigen-antibody complex” refers to a combination of a corresponding protein antigen in a sample and an antibody recognizing it. Detection of the antigen-antibody complex can be performed using methods known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, spectroscopic, chemical and other methods.
  • Example 1 Preparation of mouse-derived anti-SARS-CoV-2 NP monoclonal antibody
  • an inactivated SARS-CoV-2 virus was used as an antigen.
  • Six-week-old female BALB/c mice were purchased from Orient Bio (Gyeonggi-do, Korea) and reared in sterile facilities. TiterMax Gold adjuvant (Sigma -Aldrich) and immunized with 10 ⁇ g virus a total of three times. Two weeks after the final immunization, immune cells were isolated from lymph nodes and used to prepare B cell hybridomas producing anti-SARS-CoV-2 NP antibodies.
  • Fusion with follicular (F0) cells which are mouse myeloma cells, was performed according to a general B cell hybridoma preparation method and HAT (hypoxanthine-aminopterin-thymidine medium) media selection method.
  • the antigen (antigen, Ag) to be attached to the bottom of a MaxiSorp 96-well plate is a SARS-CoV-2 NP (sequence 1 to 419 amino acids) recombinant protein (Sino Biological) in phosphate-buffered PBS (PBS). saline), treated with 100 ng per well, and attached for 16 hours at 4°C.
  • PBS phosphate-buffered PBS
  • PBST phosphate-buffered PBS
  • the plate was washed three times with PBS (PBST) containing 0.05% Tween-20, and then treated with PBS containing 1% bovine serum albumin (BSA) at 37 ° C for 1 hour to obtain non-specific Prepared so that the reaction does not occur.
  • PBS phosphate-buffered PBS
  • BSA bovine serum albumin
  • the culture supernatant of the primary clone was incubated at 37°C for 2 hours, washed three times with PBST to remove the residue, and then horse-radish peroxidase (HRP)-attached anti-mouse IgG (1:5000 dilution, CST g) and reacted at 37 ° C for 1 hour.
  • HRP horse-radish peroxidase
  • CST g horse-radish peroxidase
  • Optical density (OD) was measured at 450 nm using a VICTOR Nivo plate reader (PerkinElmer). A monoclonal hybridoma selection process was performed by selecting clones having a high optical density (OD) value among a total of 503 initial clones (FIG. 1).
  • the hybridoma culture was diluted 1/50 in TBS (Tris-buffered saline) on an ELISA plate coated with anti-mouse capture antibody specific for mouse antibody subtypes (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM). 50 ⁇ l per well was treated, and anti-mouse-IgG+IgA+IgM-HRP antibody was simultaneously treated to induce a reaction for 1 hour, followed by a color reaction using TMB.
  • TBS Tris-buffered saline
  • 1G6, 3F10 and 5B6 were subtypes of IgG2a kappa chain
  • 3E10 was subtype of IgG2b kappa chain
  • 5B6.8 was subtype of IgG3 kappa chain.
  • SARS-CoV-2 structural proteins SARS-CoV-2 structural proteins, nucleocapsid (NP, amino acids SEQ ID NO: 1 to 419), spike ( S) S1 (spike sequence 1-685 amino acids) and S2 (spike sequence 686-1273 amino acids) constituting proteins, and RBD (spike sequence 319-541 amino acids) present in S1 to recombinant proteins (Sino Biological Co.) It was confirmed by comparing the reactivity to The S1, S2, RBD, and NP recombinant protein antigens to be attached to the bottom of the MaxiSorp 96-well plate were used at 100 ng per well, and the specific experimental method is the same as in Example 1.
  • Example 2 the five antibodies obtained in Example 1 (3E10, 3F10, 5B6, 5B6.8, 1G6) showed high absorbance only to the NP antigen, but showed no reactivity to the other antigens (S1, S2, RBD). (Fig. 2).
  • ELISA was performed by preparing 5 new purified antibodies at various concentrations.
  • SARS-CoV NP Semo biological antigen
  • 5B6, 3E10, and 1G6 were superior to commercially available antibodies in binding ability to the SARS-CoV NP antigen (FIG. 4).
  • EC50 (effective concentration 50%, effective concentration until the ability to recognize the antigen reaches 50%) values that can quantify the binding ability of the antigen and antibody are shown in Table 2 below.
  • Example 5 SARS-CoV-2 virus diagnosis using anti-SARS-CoV-2 NP monoclonal antibody
  • novel anti-SARS-CoV-2 NP antibody can be used to detect the actual SARS-CoV-2 virus, it was confirmed using an inactivated SARS-CoV-2 lysate.
  • inactivated SARS-CoV-2 lysate (Native Antigen) was attached per well at 4° C. for 16 hours, and then ELISA was performed in the same manner as above.
  • EC50 values were 3E10 (4 ng/mL), 1G6 (6 ng/mL), 5B6 (10 ng/mL), 3F10 (10 ng/mL), 5B6.8 (1056 ng/mL), and B46F (1246 ng/mL). mL), followed by 4B21 (20760 ng/mL) (Table 3).
  • Antibody EC 50 (ng/mL) to SARS-CoV-2 viral lysates 1G6 6 3E10 4 3F10 10 5B6 10 5B6.8 1056 B46F (Commercial mAb) 1246 4B21 (Commercial mAb) 20760
  • SARS-CoV-2 NPs (1 ⁇ g) were developed in reducing 12% SDS-PAGE conditions and transferred to a PVDF membrane.
  • 2 ⁇ g of each of the five developed monoclonal antibodies (3E10, 3F10, 5B6, 5B6.8, 1G6) /mL at 4°C for 12 hours.
  • the membrane was washed 3 times for 10 minutes with PBST and further reacted with HRP-conjugated anti-mouse IgG (1:5000 dilution).
  • Immunoreactive bands were reacted with Enhanced Chemi-Luminescence (ECL) and visualized using an Azure C300 Western blot imager (Azure Biosystems).
  • NP recombinant antigen protein was detected in SDS-PAGE at a position slightly higher than the predicted molecular weight of about 47 kDa, and all 5 antibodies detected NP protein at the position of SARS-CoV-2 NP antigen protein (FIG. 6) . From these results, it can be seen that these antibodies can recognize the linear epitope of SARS-CoV-2 NP protein.
  • NP-10xHis (NP-His) expression vector was transduced into human embryonic kidney cells (HEK293T) using polyethylenimine (PEI). After 72 hours, the cells were collected and dissolved in RIPA buffer [PBS containing 1.0% (v/v) NP40, 0.1% Sodium Dodecyl Sulfate, 0.5% Sodium Deoxycholate, protease inhibitor cocktail (Roche)] and used as a sample for protein analysis. Protein quantification of the cell lysate was performed by the Bradford method. Prepared protein samples were developed in reducing 12% SDS-PAGE conditions by 10 ⁇ g, and transferred to a PVDF membrane.
  • the membrane onto which the protein was transferred was blocked by immersion in a TBS solution containing 5% (w/v) skim milk, and then purified anti-NP antibodies (3E10, 3F10, 5B6, 5B6.8, 1G6) (0.5 ⁇ g/ml).
  • mL) or mouse anti-6xHis antibody (1:5000, Qiagen) was diluted in a blocking solution and treated as a primary antibody. After treatment with the primary antibody, sufficient washing with TBST (TBS containing 0.1% (v/v) tween-20), treatment with anti-mouse IgG-HRP as the secondary antibody, and protein bands bound to the antibody were detected by enhanced chemiluminescence. Confirmed.
  • the NP-10xHis expression vector was transduced into human embryonic kidney cells (HEK293T) using PEI. After 72 hours, cells were collected, dissolved in RIPA buffer, and used as samples for immunoprecipitation protein analysis. The cell lysate was quantified by the Bradford method, and 800 ⁇ g of the cell lysate protein sample was treated with 5 ⁇ g of antibodies (3E10, 3F10, 5B6, 5B6.8, 1G6) or negative control antibodies of the same subtype (IgG2a (1G6, 3F10, 5B6) or IgG2b (3E10) and mixed well for 16 hours at 4° C.
  • antibodies 3E10, 3F10, 5B6, 5B6.8, 1G6
  • negative control antibodies of the same subtype IgG2a (1G6, 3F10, 5B6) or IgG2b (3E10) and mixed well for 16 hours at 4° C.
  • NP one of the structural proteins of SARS-CoV-2, is responsible for packaging the genome of SARS-CoV-2, has 419 amino acids, and has N-arm (sequence 1 to 46 amino acids) RNA binding site N-terminal domain (sequence 47-174 amino acids), a linker with multiple serine and arginine (SEQ ID NOs: 175-247 amino acids), a C-terminal domain that allows NP proteins to form dimers (SEQ ID NOs: 248-364 amino acids), a C-tail (SEQ ID NOs: 365 ⁇ 419).
  • Example 3 As a result of Example 3, it was confirmed that the 1G6, 3E10, 3F10, 5B6, and 5B6.8 antibodies derived from inactivated SARS-CoV-2 immunized mice specifically bind to NP among structural proteins of SARS-CoV-2 , determined a more elaborate epitope.
  • NP-His NP-p1-His
  • NP-p2-His NP recombinant proteins
  • a PCR reaction was performed using ng as a template.
  • 10xHis/pCMV3 and NP-p2-10xHis/pCMV3 expression vectors were transduced into human embryonic kidney cells (HEK293T) using PEI. After 72 hours, cells were collected, dissolved in RIPA buffer, and used as protein analysis samples.
  • Protein quantification of the cell lysate was performed by the Bradford method. Prepared protein samples were developed in reducing 12% SDS-PAGE conditions by 20 ⁇ g, and transferred to a PVDF membrane. The membrane onto which the protein was transferred was blocked by soaking in a TBS solution containing 5% (w/v) skim milk, and then purified anti-NP antibody (0.5 ⁇ g/mL) or mouse anti-6xHis antibody (1 :5000, Qiagen) was diluted in a blocking solution and treated with a primary antibody. After treatment with the primary antibody, it was thoroughly washed with TBST, treated with anti-mouse IgG-HRP as the secondary antibody, and the antibody-reactive protein band was confirmed by enhanced chemiluminescence.
  • the four antibodies (3E10, 3F10, 5B6, 5B6.8) were NP-His (47.33 kDa, SEQ ID NOs: 1 to 419 amino acids), NP-p1-His (20.73 kDa, SEQ ID NOs: 1 to 174 amino acids), NP-p2 -His (28.15 kDa, sequence 1 to 247 amino acids), and based on this, the N-terminus of sequence 1 to 174 amino acids of SARS-CoV-2 NP was determined as the epitope (Fig. 10a).
  • the 1G6 antibody bound only to NP-His (47.33 kDa, amino acid sequence 1 to 419), and based on this, the C-terminus (sequence 248 to 419 amino acid sequence) of SARS-CoV-2 NP was determined as the epitope (Fig. 10b).
  • the complementarity determining region (CDR) sequence was analyzed.
  • RNA extraction kit Novartis RNA extraction kit
  • cDNA was synthesized from 5 ⁇ g of total RNA using a complementary DNA synthesis kit (Promega), and polymerase chain using 1 ⁇ g of synthesized cDNA and mouse heavy chain region primers and mouse light chain region primers in Table 4
  • the CDR-containing regions of the mouse heavy and light chains were amplified by reaction (PCR).
  • the amplified DNA was ligated with pTOP Blunt V2 vector using TOPcloner Blunt kit (Enzynomics). The recombinant plasmid was transformed into E.
  • SARS-CoV-2 NP full-length (amino acids 1-419) protein and 7 partially defective recombinant proteins were cloned, and the defective proteins were NP-NTD at 47-174; NP-CTD numbers 248-364; NP-p1 is numbered 1-174; NP-p2 is numbered 1-248; NP-p3 is numbered 1-305; 101-419 for NP-p4; NP-p5 has amino acids 248-419 (FIG. 13A).
  • NP full-length or partially deleted gene and pCMV3 vector (Sino Biological) were treated with restriction enzymes (HindIII/EcoRV) at 37°C for 15 hours, and then the cut gene and vector were mixed at a certain ratio to ligase (Roche g) was added, and a ligation reaction was performed at 16° C. for 16 hours.
  • the conjugation reaction mixture was transformed into DH5 ⁇ , spread on LB-restricted medium plates containing kanamycin antibiotic, and incubated at 37°C for 15 hours. The colony grown on the plate was selected and cultured in LB-limited liquid medium to isolate the amplified recombinant plasmid.
  • the expression vector was transferred to human embryonic kidney cells (HEK293T) using PEI. transduced. After 72 hours, cells were collected, dissolved in RIPA buffer, and used as protein analysis samples.
  • Protein quantification of the cell lysate was performed by the Bradford method. Prepared protein samples were developed in reducing 12% SDS-PAGE conditions by 20 ⁇ g, and transferred to a PVDF membrane. The membrane onto which the protein was transferred was blocked by soaking in a TBS solution containing 5% (w/v) skim milk, and then purified anti-NP antibody (0.5 ⁇ g/mL) or mouse anti-6xHis antibody (1 :5000, Qiagen) was diluted in a blocking solution and treated with a primary antibody. After treatment with the primary antibody, it was thoroughly washed with TBST, treated with anti-mouse IgG-HRP as the secondary antibody, and the antibody-reactive protein band was confirmed by enhanced chemiluminescence.
  • the epitope of the 3F10 antibody was the 1st to 47th amino acid region of the NP protein, and for 3E10 and 5B6, it was confirmed that the 47th to 101st amino acid region of the NP protein was their epitope. It was confirmed that the 1G6 antibody was the epitope of these antibodies at amino acids 364 to 419 of the NP protein (FIG. 13B).
  • Example 12 Sandwich ELISA and SARS-CoV-2 detection using antibody pairs
  • Sandwich ELISA is a method widely used in the diagnostic industry.
  • An antibody capture antibody, Capture Ab
  • an antigen is bound to the antibody, and then another antigen (detection antibody, Detection Ab) is used. to be detected either directly or indirectly. Since this method uses two antibodies specific to an antigen, an antibody pair having different antigenic determining regions is used, and it is most widely used due to its high sensitivity.
  • 1G6 or 3E10 which has the strongest binding affinity to SARS-CoV-2 NP among anti-NP antibodies, was used as a capture antibody in a MaxiSorp 96-well plate ( Thermo Scientific) was treated with 100 ng per well on the bottom and adhered at 4° C. for 12 hours. In order to remove non-adhered substances, the plate was washed three times with TBS (TBST) containing 0.05% Tween-20, and then treated with 5% BSA-added TBST at 37 ° C for 2 hours to prevent non-specific reactions. did 300 ng of SARS-CoV-2 NP recombinant protein (Fig.
  • 1G6 and 3E10 were the most effective capture and detection antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 NP antigen protein (FIG. 14A, B). It was confirmed that the most effective antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 virus lysate were 1G6 and 3F10, and 1G6 and 3E10 (Fig. 14C, D). No detection was seen when influenza NP-HRP was used as a negative control. Through the above results, it was confirmed that 1G6 and 3E10 were the best antibody pairs for detecting SARS-CoV-2 NP as well as viral lysates in the Sandwich ELISA method.
  • Viruses are prone to genetic mutations, and amino acid mutations have also been reported in the NP protein sequence of the SARS-CoV-2 mutant virus (Fig. 15 left). Therefore, since the binding ability of anti-NP antibodies may change, the binding ability of 3E10, 3F10, 5B6 and 1G6 antibodies to SARS-CoV-2 mutant virus NP was confirmed through ELISA. Anti-NP antibodies were serially diluted and added to wells coated with the SARS-CoV-2 mutant virus NP antigen, and bound antibodies were detected with an anti-mouse IgG-HRP antibody. It was confirmed that all four anti-NP antibodies (1G6, 3E10, 3F10, 5B6) had binding ability to NP of alpha, beta, and gamma mutant SARS-CoV-2 viruses.
  • Example 14 Detection of sandwich ELISA and SARS-CoV-2 mutant virus using antibody pairs
  • Example 13 Based on the results of Example 13, the NP detection ability of the SARS-CoV-2 mutant virus using sandwich ELISA was confirmed.
  • the specific experimental method was the same as in Example 13, and the ELISA plate was coated with 3E10 capture antibody, blocked, and then SARS-CoV-2 mutant recombinant NP protein was added and incubated for 2 hours. After washing the plate, 1G6-HRP was used as a detection antibody. Influenza H1N1 NP and BSA were used as negative controls. The experimental results are shown in FIG. 16 .

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Abstract

La présente invention concerne un anticorps qui se lie spécifiquement à une protéine de nucléocapside (NP) de coronavirus ou un fragment de liaison à l'antigène qui se lie spécifiquement à la protéine NP, qui peut détecter une quantité beaucoup plus petite de coronavirus par comparaison avec des anticorps de détection de coronavirus classiques, et peut ainsi être efficacement utilisé pour détecter et diagnostiquer un coronavirus.
PCT/KR2022/013565 2021-09-10 2022-09-08 Anticorps spécifique d'une protéine de nucléocapside de coronavirus et utilisation associée WO2023038477A1 (fr)

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CN117534750A (zh) * 2023-10-16 2024-02-09 遵义医科大学珠海校区 一种抗新型冠状病毒核衣壳蛋白的抗体或其抗原结合片段及其应用

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