WO2022050709A1 - Sars-coronavirus infection diagnosis binding molecule that binds to spike protein on surface of sars-coronavirus-2 - Google Patents
Sars-coronavirus infection diagnosis binding molecule that binds to spike protein on surface of sars-coronavirus-2 Download PDFInfo
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- WO2022050709A1 WO2022050709A1 PCT/KR2021/011827 KR2021011827W WO2022050709A1 WO 2022050709 A1 WO2022050709 A1 WO 2022050709A1 KR 2021011827 W KR2021011827 W KR 2021011827W WO 2022050709 A1 WO2022050709 A1 WO 2022050709A1
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- 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
- G01N33/56983—Viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Definitions
- the present invention relates to a binding molecule for diagnosis of SARS-coronavirus infection that binds to a spike protein on the surface of SARS-coronavirus-2.
- SARS-coronavirus-2 severe acute respiratory syndrome coronavirus 2, SARS-CoV-2
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- SARS-CoV-2 may have mild to severe symptoms such as fever, cough, shortness of breath, and diarrhea. People with complications or diseases and the elderly are more likely to die.
- the Corona 19 Central Clinical Task Force prepared the treatment principle for COVID-19 on February 13, 2020, and as the first-line treatment, AIDS treatment Kaletra, malaria treatment chloroquine and hydroxychloroquine ( Hydroxychloroquine) is recommended, and ribavirin and interferon are not recommended as first-line treatment due to concerns about side effects.
- AIDS treatment Kaletra malaria treatment chloroquine and hydroxychloroquine ( Hydroxychloroquine) is recommended, and ribavirin and interferon are not recommended as first-line treatment due to concerns about side effects.
- AIDS treatment Kaletra malaria treatment chloroquine and hydroxychloroquine ( Hydroxychloroquine) is recommended, and ribavirin and interferon are not recommended as first-line treatment due to concerns about side effects.
- ribavirin and interferon are not recommended as first-line treatment due to concerns about side effects.
- mild or young patients if 10 days have passed since the onset of the disease, it was judge
- COVID-19 is not a seasonal virus but can be indigenous and cause infection like MERS; ii) the virus spreads to the community at some point this year or next year, evidence that the coronavirus is dormant in real communities However, it mentioned the need to strengthen monitoring so that data-based conclusions can be drawn (February 13, 2020).
- the Korea Centers for Disease Control and Prevention (KCDC) i) determined that COVID-19 could spread like influenza for a long time and announced that it would be included in the surveillance system like influenza, and ii) Coronaviruses (four types) that are prevalent among humans also occur in winter and spring. As it is prevalent, the possibility that COVID-19 may become indigenous (2020.2.17) remains open.
- RDT Rapid diagnostic test
- the main components are immunochromatographic including a support, a sample pad, a conjugate pad, a signal detection pad, and an absorption pad.
- Rapid diagnostic test is a method that can qualitatively and quantitatively test an analyte in a short time by using the property of specific attachment of biological or chemical substances to each other.
- An immunochromatographic kit in which the same immunochromatographic strip is mounted inside a plastic housing is used. When simply using an immunochromatographic strip, a separate container for the sample is required, but the immunochromatography kit built into the housing is easy to use because it does not require a separate test container by directly inserting the sample into the inlet prepared in the housing.
- Rapid diagnostic test is one of the most advanced analysis kits among detection methods developed recently in terms of simplicity and speed, and is usefully used to diagnose various disease-causing substances such as antigens or antibodies of infectious pathogens, cancer factors, and cardiac markers.
- an immunochromatographic strip or an immunochromatographic kit comprising the same, using samples such as human or animal whole blood, plasma, serum, tears, saliva, urine, runny nose, and body fluid, SARS, MERS , influenza virus, avian influenza virus, rotavirus, hepatitis A, hepatitis B, hepatitis C, AIDS, syphilis, chlamydia, malaria, typhoid, gastric ulcer causative bacteria, tuberculosis, dengue fever, leprosy, etc. can be quickly tested and diagnosed.
- samples such as human or animal whole blood, plasma, serum, tears, saliva, urine, runny nose, and body fluid, SARS, MERS , influenza virus, avian influenza virus, rotavirus, hepatitis A, hepatitis B, hepatitis C, AIDS, syphilis, chlamydia, malaria, typhoid, gastric ulcer causative bacteria, tuber
- SARS-CoV-2 does not yet have a diagnostic antibody and diagnostic kit specific for this virus. Accordingly, the present inventors attempted to develop an antibody specific for SARS-CoV-2. As a result of repeated research to develop an antibody having excellent binding ability and diagnostic effect, the present invention was completed.
- the present inventors have developed a binding molecule having the ability to bind to the S protein of SARS-CoV-2, and confirmed that this binding molecule has an excellent diagnostic effect on SARS-CoV-2. completed.
- An object of the present invention is to provide a binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2).
- Another problem to be solved by the present invention is to provide a composition for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule.
- Another problem to be solved by the present invention is to provide a kit for diagnosing SARS-coronavirus infection (COVID-19).
- SARS-CoV-2 SARS-coronavirus-2
- Another problem to be solved by the present invention is to provide a method for diagnosing SARS-coronavirus infection (COVID-19).
- the present invention provides a binding molecule that binds to the spike protein (S protein) on the surface of SARS-coronavirus-2 (SARS-CoV-2).
- the present invention provides an immunoconjugate in which one or more tags are additionally bound to the binding molecule.
- the present invention also provides a nucleic acid molecule encoding the binding molecule.
- the present invention provides an expression vector into which the nucleic acid molecule is inserted.
- the present invention provides a cell line transformed with the expression vector.
- the present invention provides a composition for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule.
- the present invention provides a strip for immunochromatographic analysis comprising the binding molecule.
- the present invention also provides a kit for diagnosis of SARS-coronavirus infection (COVID-19), comprising the binding molecule.
- the present invention provides a method of detecting SARS-coronavirus-2 (SARS-CoV-2) using the diagnostic kit.
- the present invention provides a method for diagnosing SARS-coronavirus infection (COVID-19) using the diagnostic kit.
- the present invention relates to a binding molecule for diagnosis of SARS-coronavirus infection (COVID-19) that binds to a spike protein (S protein) on the surface of SARS-CoV-2.
- COVID-19 SARS-coronavirus infection
- S protein spike protein
- the binding molecule may bind to the receptor binding domain (RBD) region of the spike protein on the SARS-coronavirus-2 surface.
- RBD receptor binding domain
- SARS-Coronavirus-2 is classified into six types of coronavirus by the World Health Organization (WHO) based on amino acid changes caused by differences in gene sequence. First, it was classified into S and L types, then again into L, V, and G types, and as G was divided into GH and GR, it is classified into a total of six types: S, L, V, G, GH, and GR. S and V types were prevalent in Asia, including Wuhan, China, in the early stages of the outbreak of COVID-19, and after that, different types were discovered for each continent. Among them, it has been reported that the GH type has the potential to appear high in transmission power.
- WHO World Health Organization
- type G virus in which amino acid 614 of the spike protein, which plays an important role in virus invasion, is changed from aspartic acid (D) to glycine (G), has increased rapidly in Europe and the United States since March, and is now almost It appears in most areas.
- the binding molecule of the present invention is a SARS-coronavirus-2 strain isolated to date, for example, UNKNOWN-LR757996 strain (Strain), SARS-CoV-2/Hu/ DP/Kng/19-027 strain; Wuhan-Hu-1 strain isolated from China in December 2019; BetaCoV/Wuhan/IPBCAMS-WH-01/2019 strain first isolated in China on December 23, 2019; BetaCoV/Wuhan/IPBCAMS-WH-02/2019 strain, BetaCoV/Wuhan/IPBCAMS-WH-03/2019 strain, BetaCoV/Wuhan/IPBCAMS-WH-04/2019 strain, WIV02 isolated on December 30, 2019 in China strain, WIV04 strain, WIV05 strain, WIV06 strain, WIV07 strain; 2019-nCoV/Japan/TY/WK-521/2020 strain isolated from Japan in January 2020, 2019-nCoV/Japan/TY/WK-501/2020 strain, 2019-nCoV/Japan/TY/WK-012/ 2020 strain, 2019-n
- the binding molecule may have any one or more of the following properties:
- a) binds to the spike protein on the surface of SARS-coronavirus-2 with an equilibrium dissociation constant (K D ) of 1 x 10 -8 M or less;
- b) binds to the spike protein on the surface of SARS-coronavirus-2 with a binding constant (Ka) greater than or equal to 1 x 10 4 1/Ms; or
- the binding molecule of the present invention binds to the spike protein on the surface of SARS-coronavirus-2 with a binding affinity (K D ) of 1.0 ⁇ 10 -8 M or less or 3.0 ⁇ 10 -9 M or less can do.
- the binding molecule of the present invention binds to the spike protein on the surface of SARS-coronavirus-2 with a binding constant (Ka) of 1 x 10 4 1/Ms or more or 1 x 10 5 1/Ms or more.
- Ka binding constant
- the binding molecule of the present invention has a dissociation constant (Kd) of 1 x 10 -2 1/s or less or 5 x 10 -3 1/s or less to the spike protein on the surface of SARS-coronavirus-2 can be combined with
- the binding molecule may be any one binding molecule selected from the group consisting of binding molecules shown in Table 1 below. In Table 1 below, No. means the number of each binding molecule.
- the CDRs of the variable region 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)]). Although the Kabat method was used for CDR numbering used in the present invention, binding molecules comprising CDRs determined according to other methods such as the IMGT method, Chothia method, and AbM method are also included in the present invention.
- the binding molecule may be any one binding molecule selected from the group consisting of binding molecules shown in Table 2 below. In Table 2 below, No. means the number of each binding molecule.
- the binding molecule may be a scFv fragment, an scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody, but is not limited thereto.
- One embodiment of the present invention provides a scFv-Fc fragment that binds to the SARS-CoV-2 S protein.
- another embodiment of the present invention provides a fully human antibody (Full IgG) that binds to the SARS-CoV-2 S protein.
- the term 'antibody' is used in the broadest sense, specifically, an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more intact antibodies (eg, a bispecific antibody); and antibody fragments exhibiting the desired biological activity.
- Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. In terms of their structure, antibodies usually have a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody mainly has two regions: a variable region and a constant region. The variable region located in the distal portion of the arm of Y binds and interacts with the target antigen.
- variable region comprises a complementarity determining region (CDR) that recognizes and binds a specific binding site on a specific antigen.
- CDR complementarity determining region
- the constant region located at the tail of Y is recognized and interacted with by the immune system.
- Target antigens have multiple binding sites, called epitopes, which are generally recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.
- the present invention also includes functional variants of the binding molecule.
- Binding molecules are capable of competing with a binding molecule of the present invention for specific binding to SARS-CoV-2 or its S protein, and retain the ability to bind to SARS-CoV-2. It is considered a functional variant.
- Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, and include, for example, in vitro or in vivo modifications, chemicals and/or biochemicals. and they are not found in the parental monoclonal antibodies of the present invention.
- Such modifications include, for example, acetylation, acylation, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis. and phosphorylation.
- a functional variant may be an antibody comprising an amino acid sequence optionally containing one or more amino acid substitutions, insertions, deletions or combinations thereof compared to the amino acid sequence of the parent antibody.
- functional variants may include truncated forms of the amino acid sequence at either or both the amino terminus or the carboxy terminus.
- Functional variants of the invention may have the same, different, higher or lower binding affinity compared to the parent antibody of the invention, but still be capable of binding SARS-CoV-2 or its S protein.
- the amino acid sequence of a variable region including, but not limited to, a framework structure, a hypervariable region, in particular, a complementarity-determining region (CDR) of a light or heavy chain may be modified.
- CDR complementarity-determining region
- a light or heavy chain region comprises three hypervariable regions, comprising three CDR regions, and a more conserved region, namely a framework region (FR).
- a hypervariable region comprises amino acid residues from the CDRs and amino acid residues from the hypervariable loops.
- Functional variants within the scope of the present invention include about 50%-99%, about 60%-99%, about 80%-99%, about 90%-99%, about 95%-99%, or about 97%-99% amino acid sequence identity.
- the Gap or Bestfit of computer algorithms known to those skilled in the art can be used.
- a functional variant may be obtained by organic synthesis or by changing the parent antibody or a part thereof by known general molecular biological methods including PCR, mutagenesis using oligomeric nucleotides, and partial mutagenesis, but is not limited thereto.
- the present invention provides an immunoconjugate in which one or more tags are additionally bound to the binding molecule.
- a drug may be further attached to the binding molecule.
- the binding molecule according to the present invention may be used in the form of an antibody-drug conjugate to which a drug is bound.
- ADCs antibody-drug conjugates
- immunoconjugates for local delivery of drugs allows for targeted delivery of the drug moiety to infected cells, which when administered unconjugated to normal cells. This is because unacceptable levels of toxicity can result. Maximal efficacy and minimal toxicity of ADCs can be improved by increasing drug-connectivity and drug-releasing properties, as well as selectivity of polyclonal and monoclonal antibodies (mAbs).
- Another embodiment of the present invention provides a nucleic acid molecule encoding the binding molecule.
- Nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of the antibody provided in the present invention is translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by an open reading frame (ORF), and all of these are also included in the nucleic acid molecule of the present invention.
- ORF open reading frame
- another embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.
- Examples of the expression vector include the MarEx vector (refer to Korean Patent Registration No. 10-1076602), which is Celltrion's own expression vector, and the widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmid; phage such as lambda, lambdoid, M13, Mu, p1 P22, Q ⁇ , T-even, T2, T3, T7;
- An expression vector selected from any one selected from the group consisting of plant viruses may be used, but the present invention is not limited thereto, and any expression vector known to those skilled in the art can be used in the present invention.
- the vector contains one or more selectable markers, but is not limited thereto, and a vector that does not contain a selectable marker may be used to select depending on whether a product is produced.
- the selection of the selection marker is selected by the host cell of interest, and the present invention is not limited thereto since it uses a method already known to those skilled in the art.
- a tag sequence may be inserted and fused onto an expression vector.
- the tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
- the present invention provides a cell line transformed with the expression vector.
- the expression vector is transformed into a host cell to provide a cell line producing a binding molecule having the ability to bind to SARS-CoV-2.
- the cell line may include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin.
- the mammalian cells include any one selected from the group consisting of CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, A549 cells, HEK 293 cells and HEK293T cells.
- the present invention provides a composition for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule.
- the composition of the present invention may include a pharmaceutically acceptable excipient in addition to the binding molecule.
- Pharmaceutically acceptable excipients are well known to those skilled in the art.
- the composition may be a sterile injection solution, a lyophilized formulation, a pre-filled syringe solution, an oral formulation, an external formulation, or a suppository, but is not limited thereto. .
- composition of the present invention may further comprise at least one other diagnostic agent.
- it may further include a binding molecule that binds to a nucleocapsid protein (N protein) on the surface of SARS-CoV-2 together with the binding molecule.
- N protein nucleocapsid protein
- the present invention provides an immunochromatographic analysis comprising a binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2) strips are provided.
- the immunochromatographic analysis strip may further include a binding molecule that binds to the nucleocapsid protein (N protein) of the coronavirus.
- Said coronavirus is SARS-coronavirus-2 (SARS-CoV-2), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), It may be any one selected from the group consisting of human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), but is not limited thereto.
- the strip for immunochromatographic analysis is
- a sample pad for receiving a sample to be analyzed and having a buffer input unit and a sample input unit;
- a signal detection pad including a signal detection unit for detecting whether or not the coronavirus is present in the sample and a control unit for checking whether the sample has moved to the absorbent pad regardless of the presence or absence of an analyte;
- the sample pad may be made of cellulose, acrylic fiber, rayon, polyester fiber, glass fiber, wool fiber, silk fiber, cotton yarn, flax fiber or pulp, or a mixture of one or more types.
- the conjugate pad may be made of glass fiber, polyethylene fiber, or the like.
- the signal detection pad is composed of a porous membrane pad, and may be made of nitrocellulose, cellulose, polyethylene, polyethersulfone, nylon, or the like. One or more signal detectors may be included.
- the absorbent pad may be any pad capable of accelerating the movement of the buffer, and may be made of, for example, pulp, cellulose, or cotton fiber.
- the strip may include, in a conjugate pad and a signal detection pad, a binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2, respectively.
- S protein spike protein
- the SARS-CoV-2 S protein binding molecule included in the conjugate pad and the signal detection pad may be the same or different.
- the SARS-CoV-2 S protein binding molecule included in the conjugate pad and the signal detection pad may be a binding molecule including the above-described sequence.
- the binding molecule contained in the conjugate pad may be labeled with a metal particle, a latex particle, a fluorescent substance, or an enzyme.
- the metal particles may be gold particles.
- the binding molecule of the present invention may be detectably labeled on the conjugate pad of the strip for immunochromatographic analysis.
- the various methods available for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention.
- Examples of label types that can be used in the present invention include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds.
- markers include fluorescent substances (eg, fluorescein, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, beta-galactosidase, alkaline phosphatase), radioactive isotopes (eg, 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (eg, dioxetane, luminol or acridinium). Labeling methods such as covalent bonding of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are well known in the art.
- fluorescent substances eg, fluorescein, rhodamine, Texas red, etc.
- enzymes eg, horseradish peroxidase, beta-galactosidase, alkaline phosphatase
- radioactive isotopes eg, 32P or 125I
- Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, and the like.
- Commonly used detection assays include radioactive isotope or non-radioactive isotope methods. These include, among others, Western blotting, overlay-assay, Radioimmuno Assay (RIA) and ImmuneRadioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (FIA) and Chemioluminescent Immune (CLIA). Assay).
- the detection may be read by visual, optical, electrochemical, or electrical conductivity, but is not limited thereto.
- the present invention provides a kit for diagnosis of SARS-coronavirus infection (COVID-19), comprising the strip for immunochromatographic analysis.
- COVID-19 SARS-coronavirus infection
- the present invention provides a kit for diagnosis of SARS-coronavirus infection (COVID-19), comprising the binding molecule.
- the diagnostic kit may further include a binding molecule that binds to the nucleocapsid protein (N protein) of the coronavirus.
- Said coronavirus is SARS-coronavirus-2 (SARS-CoV-2), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), It may be any one selected from the group consisting of human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), but is not limited thereto.
- the diagnostic kit of the present invention may be used to detect the presence or absence of SARS-CoV-2 by contacting a sample with the binding molecule and then confirming a reaction.
- the sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, lung tissue mucus, respiratory tissue, and saliva, but is not limited thereto, and the sample can be prepared by a conventional method known to those skilled in the art. possible.
- Kit for diagnosing diseases caused by SARS-CoV-2 comprising:
- the kit container may contain a solid carrier.
- Antibodies of the invention may be attached to a solid carrier, which may be porous or non-porous, planar or non-planar.
- the present invention provides a method for detecting SARS-coronavirus-2 (SARS-CoV-2) using the diagnostic kit.
- the present invention provides a method for diagnosing SARS-coronavirus infection (COVID-19) using the diagnostic kit.
- binding molecule refers to an intact immunoglobulin, including a monoclonal antibody such as a chimeric, humanized or human monoclonal antibody, or an antigen-binding immunoglobulin that binds to an antigen. Includes fragments. For example, in binding to the spike protein of SARS-CoV-2, it refers to a variable domain, an enzyme, a receptor, or a protein comprising an immunoglobulin fragment that competes with an intact immunoglobulin. Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact immunoglobulin.
- An antigen-binding fragment comprises at least two contiguous groups of the amino acid sequence of the antibody, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, At least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, 150 a peptide or polypeptide comprising an amino acid sequence of at least two contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.
- Antigen-binding fragments are inter alia Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv-Fc), bivalent ( bivalent) single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind a particular antigen to the polypeptide, and the like.
- the fragment may be produced synthetically or by enzymatic or chemical digestion of complete immunoglobulins, or may be genetically engineered by recombinant DNA technology. Methods of production are well known in the art.
- pharmaceutically acceptable excipient refers to an inert substance that is combined into an active molecule, such as a drug, agent, or antibody, to prepare an acceptable or convenient dosage form.
- Pharmaceutically acceptable excipients are excipients which are non-toxic, or at least toxic, acceptable for their intended use in recipients at the dosages and concentrations employed, and with the other ingredients of the formulation, including the drug, agent, or binding agent; compatible.
- the binding molecule of the present invention exhibits an excellent diagnostic effect by having an excellent binding ability to the S protein of SARS-CoV-2, it is very useful for rapid diagnosis of SARS-coronavirus infection (COVID-19).
- 1 is a schematic diagram illustrating the principle of operation of a strip for immunochromatographic analysis of a diagnostic kit according to an embodiment of the present invention. 1 is a description of a strip for immunochromatographic analysis loaded with the antibody Anti-S that binds to the surface protein S of SARS-CoV-2.
- Anti-S an antibody that binds to the surface protein S of SARS-CoV-2
- Anti-N an antibody that binds to protein N present in SARS-CoV-2. explanation.
- 3a to 3r show the present invention antibody No. according to one embodiment of the present invention.
- 1 to No. 18 shows the results of antigen-antibody binding affinity measurement using Surface Plasmon Resonance technology, respectively.
- Example 1 Isolation of PBMCs from the blood of patients recovering from SARS-CoV-2
- the donor of blood was people who were confirmed to have been infected with SARS-CoV-2 in 2020 and no longer had the virus detected through treatment. .
- PBMCs peripheral blood mononuclear cells
- variable regions of the light and heavy chains of the antibody are amplified by PCR (polymerase chain reaction) method using high fidelity Taq polymerase (Roche) and degenerative primer set (IDT) from the synthesized cDNA.
- the scFv-type gene is created and amplified by the overlap PCR method, cut with restriction enzymes, and 1% agarose gel electrophoresis and scFv was isolated using the gel extraction kit (Qiagen) method.
- the phage vector was also digested with the same restriction enzyme and separated, mixed with the scFv gene, added with T4 DNA ligase (New England Biolab), and reacted at 16° C. for more than 12 hours.
- the reaction solution was mixed with ER2738 competent cells and transformed by electroporation. Transformed ER2738 was cultured with shaking, followed by VCSM13 helper phage (Agilent Technologies) and incubated for more than 12 hours.
- the phage library culture medium prepared in Example 2 was centrifuged to remove host cells, 4% PEG and 0.5 M NaCl were added thereto, centrifuged to settle the phage, and the supernatant was removed.
- the precipitated phage was diluted in 1% BSA/TBS to obtain a phage library, and then, the binding and dissociation reactions for various SARS-CoV-2 Spike proteins (hereinafter, S protein) were independently performed for panning to SARS.
- S protein SARS-CoV-2 Spike proteins
- the phage library on an ELISA plate to which the receptor binding domain (RBD) region (residues N331 to V524 on S1 glycoprotein), a part of the SARS-CoV-2 S protein, is bound, and react at room temperature for 2 hours. made it After removing the reaction solution, the ELISA plate was washed with PBS containing 0.05% tween 20, and 60 ⁇ l of 0.1M glycine-HCl (pH 2.2) was added to remove the antigen-bound scFv-phage, and 2M Tris (pH 9.1) was added. was used for neutralization.
- RBD receptor binding domain
- helper phage was added and cultured to be used for the next panning. A portion of the infected ER2738 was plated on an LB plate before adding auxiliary phages, and colonies were obtained the next day.
- Colonies formed for each panning were put into a culture solution contained in a 96-well deep well plate (Axygen) and cultured with shaking. The culture medium was centrifuged to remove host cells, and a supernatant containing scFv-phages was prepared.
- the prepared scFv-phage supernatant was diluted 1:1 with 6% BSA/PBS, and then put into each well of a 96-well microtiter plate in which SARS-CoV-2 S proteins were adsorbed and blocked and placed at 37°C for 2 hours. been in politics for a while.
- Each well was washed three times with PBS containing 0.05% Tween 20, and then HRP (mustard peroxidase, horseradish peroxidase)-labeled anti-M13 antibody was added thereto and left at 37°C for 1 hour.
- HRP mustard peroxidase, horseradish peroxidase
- the scFv-phage selected in Example 3 was then cultured with shaking to obtain DNA and then sequenced for the antibody variable region was analyzed. Among them, the selected scFv-phages were cloned into a vector in the form of an scFv antibody fragment (scFv-Fc) in order to evaluate the expression ability in the candidate antibody animal cell line, except for duplicated clones as an amino acid sequence.
- CHO cells were transfected and expressed using a transfection reagent, and the ability of the scFv antibody fragment to bind to two S proteins of SARS-CoV-2 was confirmed by ELISA using the culture medium. Briefly, SARS-CoV-2 S proteins were attached to an ELISA plate and the expressed antibody fragment was added. After washing the unbound antibody with PBS containing 0.05% Tween 20, HRP (horseradish peroxidase)-conjugated anti-human IgG antibody was used to select and evaluate antigen-bound antibody fragments.
- Example 5 Determination of antigen-antibody binding affinity using surface plasmon resonance technology
- the Surface Plasmon Resonance assay determines the binding affinity of an antibody by kinetic measurements of forward and reverse rate constants.
- Example 4 in order to confirm the binding affinity value (K D ) of SARS-CoV-2 for the S RBD protein of the full human antibody (Full IgG) transformed with the scFv-Fc antibody fragment, SPR (surface Plasmon resonance; Surface Plasmon Resonance) analysis was performed. Briefly, SARS-CoV-2 S proteins were attached to a CM5 chip and the antibody fragments were serially diluted using HBS-EP buffer (pH 7.4) to make 5 concentrations. Each concentration was flowed to the CM5 chip to which the SARS-CoV-2 S protein was attached, and then HBS-EP buffer was injected to generate association and dissociation curves. Figure (K D ) was measured (Table 4 and Figures 3A-3R).
- K D Kd/Ka. Binding is recorded as a function of time and reaction rate constants. In Table 4 below, No. refers to the same binding molecule as the No. of each binding molecule shown in Tables 1 and 2.
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Abstract
The present invention relates to a SARS-coronavirus infection diagnosis binding molecule that binds to a spike protein on the surface of SARS-coronavirus-2. More specifically, a binding molecule of the present invention has excellent binding ability to an S protein of SARS-CoV-2 to exhibit excellent diagnosis effects, and thus is very useful in the rapid diagnosis of a SARS-coronavirus infection (COVID-19).
Description
본 발명은 사스-코로나바이러스-2 표면의 스파이크 단백질에 결합하는 사스-코로나바이러스 감염증의 진단용 결합 분자에 관한 것이다. The present invention relates to a binding molecule for diagnosis of SARS-coronavirus infection that binds to a spike protein on the surface of SARS-coronavirus-2.
사스-코로나바이러스-2(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)는 유전적 배열(DNA sequencing)상 전도 기능(Positive sense) 단일 가닥 RNA(single-stranded RNA) 코로나바이러스로서, 인간에게 전염성이 있고 코로나바이러스감염증-19(coronavirus disease 2019, COVID-19)의 원인이다. COVID-19의 최초 발생지는 중국 후베이성의 우한시이다. SARS-coronavirus-2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is a positive sense single-stranded RNA coronavirus on the genetic sequence (DNA sequencing) to humans. It is contagious and is the cause of coronavirus disease 2019 (COVID-19). The first outbreak of COVID-19 was in Wuhan, Hubei Province, China.
SARS-CoV-2에 감염된 사람들은 열, 기침, 호흡 곤란, 설사와 같이 경증에서 중증의 증상을 보일 수 있다. 합병증이나 병을 가진 사람들, 노인은 사망할 가능성이 크다. People infected with SARS-CoV-2 may have mild to severe symptoms such as fever, cough, shortness of breath, and diarrhea. People with complications or diseases and the elderly are more likely to die.
특히 심장질환 및 당뇨병 등의 기저질환 보유자가 감염에 더 취약하며, 합병증이나 장기 손상 등을 겪기 때문에 조기 발견과 치료가 매우 중요하다. 2019년 12월 8일부터 2020년 3월 20일 현재까지 245,550명의 환자가 발생하였고, 그 중 10,049명이 사망하여 치사율은 4.09%에 달한다(WHO). 현재까지 한국을 포함한 177개국에서 발생하였다. In particular, people with underlying diseases such as heart disease and diabetes are more susceptible to infection and suffer complications or organ damage, so early detection and treatment are very important. From December 8, 2019 to March 20, 2020, there were 245,550 cases, of which 10,049 died, resulting in a fatality rate of 4.09% (WHO). So far, it has occurred in 177 countries, including Korea.
현재 코로나바이러스감염증-19(coronavirus disease 2019, COVID-19)의 치료제는 없고, 기존 치료제로 환자에게 투여하여 치료 효과를 기대하고 있는 실정이다. 에볼라 치료제 혹은 치료 후보 물질인 항바이러스제 파비피라비르 (favipiravir), 렘데시비르 (remdesivir), 갈리데시비어 (galidesivir)와 C형 간염 치료제인 리바비린 (ribavirin)을 코로나19 치료제로 사용하고 있다. 에볼라 치료제로 쓰이는 약물에 비해 C형 간염 치료제인 리바비린은 빈혈과 같은 부작용이 심할 수 있고, 항바이러스제인 인터페론 (interferon)도 여러 가지 부작용을 우려하여 주의해서 사용할 것을 권고하고 있다. 말라리아치료제 클로로퀸 (Chloroquine)도 코로나19에 효과를 보이는 것으로 나타나 공개 임상시험 진행 중에 있다. Currently, there is no treatment for coronavirus disease 2019 (COVID-19), and the treatment effect is expected by administering the existing treatment to the patient. Antiviral agents favipiravir, remdesivir, and galidesivir, which are Ebola treatment or treatment candidates, and ribavirin, a hepatitis C treatment, are being used as treatments for COVID-19. Compared to the drug used for the treatment of Ebola, ribavirin, a hepatitis C treatment, may have severe side effects such as anemia, and the antiviral drug interferon is also recommended to be used with caution due to concerns about various side effects. Chloroquine, an antimalarial drug, has also been shown to be effective against COVID-19, and is currently undergoing public clinical trials.
그러나 이러한 약물들이 코로나19 환자 치료에 활용되어 효과를 보고 있지만 아직 어떠한 근거에서 효과를 내는지는 아직 명확히 입증되지 않았다. 중국에서 코로나19 회복 환자의 현장을 주입하는 혈장 요법을 시행하여 중증 환자의 치료에 효과를 보였다고 발표하였으나 치료 효과가 불분명하고 불확실성이 크기 때문에 신중해야 한다. However, although these drugs are being used to treat COVID-19 patients and are seeing effects, it has not yet been clearly proven on what basis they are effective. In China, it was announced that it was effective in the treatment of severely ill patients by implementing on-site plasma therapy for patients recovering from COVID-19, but caution should be exercised because the therapeutic effect is unclear and uncertainty is great.
국내의 경우, 코로나19 중앙임상 TF(테스크포스)가 2020년 2월 13일 코로나19의 치료 원칙을 마련하여, 1차 치료제로 에이즈 치료제인 칼레트라 (Kaletra), 말라리아치료제인 클로로퀸과 하이드록시클로로퀸(Hydroxychloroquine)을 권하며, 리바비린과 인터페론은 부작용을 우려해 1차 치료제로 권하지 않기로 발표했다. 경증이거나 젊은 환자, 발병 10일이 지난 경우에는 항바이러스제를 투여하지 않아도 증상이 호전된다고 판단하고, 고령자, 기저질환자, 중증 환자에게는 항바이러스 치료제를 투여하기로 합의했다. In Korea, the Corona 19 Central Clinical Task Force (Task Force) prepared the treatment principle for COVID-19 on February 13, 2020, and as the first-line treatment, AIDS treatment Kaletra, malaria treatment chloroquine and hydroxychloroquine ( Hydroxychloroquine) is recommended, and ribavirin and interferon are not recommended as first-line treatment due to concerns about side effects. In mild or young patients, if 10 days have passed since the onset of the disease, it was judged that the symptoms improved even if antiviral drugs were not administered.
미국 CDC는 i) 코로나19가 계절성 유행 바이러스가 아닌 메르스처럼 토착화되어 감염을 일으킬 수 있다고 발표하였고, ii) 바이러스가 올해 또는 내년 어느 시점에 커뮤니티로 전파, 코로나 바이러스가 실제 커뮤니티에 잠복되어 있다는 증거는 없으나, 데이터 기반으로 결론을 내릴 수 있도록 감시강화 필요성을 언급하였다(2020.2.13).The U.S. CDC has announced that i) COVID-19 is not a seasonal virus but can be indigenous and cause infection like MERS; ii) the virus spreads to the community at some point this year or next year, evidence that the coronavirus is dormant in real communities However, it mentioned the need to strengthen monitoring so that data-based conclusions can be drawn (February 13, 2020).
한국 질병관리본부는 i) 코로나19도 인플루엔자처럼 장기적으로 유행할 수 있다고 판단하여, 인플루엔자와 같이 감시 체계에 포함하겠다고 발표하였고, ii) 사람 사이에 유행하는 코로나바이러스(4종)도 겨울~봄에 유행하고 있어서 코로나19도 토착화될 수 있다는 가능성을 열어두고 있다(2020.2.17).The Korea Centers for Disease Control and Prevention (KCDC) i) determined that COVID-19 could spread like influenza for a long time and announced that it would be included in the surveillance system like influenza, and ii) Coronaviruses (four types) that are prevalent among humans also occur in winter and spring. As it is prevalent, the possibility that COVID-19 may become indigenous (2020.2.17) remains open.
사스나 메르스와는 다르게 코로나19의 세계적 유행 (pandemic) 현실화에 대한 우려가 있지만 봄 이후 (4월) 소강 상태가 될 가능성도 있어, 추이를 보며 신중하게 접근하는 전문가들이 많다. 아직 코로나19에 대한 정보 부족으로 전문가들도 추후 전개 양상에 대해서는 의견이 분분하나, 단시일 내에 해결되리라 전망하는 전문가는 거의 없다. 코로나19의 유행 양상과 특징이 정확히 분석되고 이번 코로나19로 인한 위기 상황이 얼마나 지속되는지에 영향을 받겠지만, 무증상 감염자가 전세계에 퍼지게 될 경우 풍토병화 될 가능성에 대한 우려가 있다. 중국 및 국내에서의 토착화를 통한 국내 코로나19 재발병 가능성에 대한 대응책 마련이 시급하다. Unlike SARS and MERS, there are concerns about the realization of a global pandemic of Corona 19, but there is a possibility that it will be quiet after spring (April), so there are many experts who take a cautious approach while watching the trend. Due to the lack of information on COVID-19, experts also have differing opinions about the future development, but few experts predict that it will be resolved in a short time. Although the epidemic pattern and characteristics of COVID-19 will be accurately analyzed and will be affected by how long the crisis will last, there is concern that asymptomatic infected people may become endemic if they spread around the world. It is urgent to prepare countermeasures against the possibility of a reoccurrence of COVID-19 in China and Korea through indigenization.
*신속진단검사(Rapid diagnostic test, RDT)는 면역크로마토그래피 분석, 래피드 키트 분석 등 다양한 명칭으로 불리며, 주된 구성이 지지체, 검체패드, 컨쥬게이트 패드, 신호검출패드, 및 흡수패드를 포함하는 면역 크로마토그래피 스트립에 의한 분석방법으로서, 사용자가 생물학적 또는 화학적 샘플로부터 분석 물질을 특별한 기술이나 장비 없이 1-100 마이크로 리터의 샘플로 2-30분 사이에서 간단하게 검출할 수 있다. 신속진단검사는 생물학적 물질 또는 화학적 물질이 서로 특이적으로 부착하는 성질을 이용하여 분석 물질을 단시간에 정성 및 정량적으로 검사할 수 있는 방법으로, 신속진단검사는 단순히 면역 크로마토그래피 스트립을 사용하거나, 이와 같은 면역 크로마토그래피 스트립을 플라스틱 하우징 내부에 장착한 형태의 면역 크로마토그래피 키트가 사용된다. 단순히 면역 크로마토그래피 스트립을 사용할 때는 시료를 담은 용기가 별도로 필요하지만 하우징에 내장된 면역 크로마토그래피 키트는 하우징에 준비된 투입구에 시료를 직접 투입함으로서 별도의 실험용기가 필요 없어 사용하기 간편하다. *Rapid diagnostic test (RDT) is called by various names such as immunochromatographic analysis and rapid kit analysis, and the main components are immunochromatographic including a support, a sample pad, a conjugate pad, a signal detection pad, and an absorption pad. As an analysis method using a graphic strip, a user can simply detect an analyte from a biological or chemical sample in 2-30 minutes with a sample of 1-100 microliters without special skills or equipment. Rapid diagnostic test is a method that can qualitatively and quantitatively test an analyte in a short time by using the property of specific attachment of biological or chemical substances to each other. An immunochromatographic kit in which the same immunochromatographic strip is mounted inside a plastic housing is used. When simply using an immunochromatographic strip, a separate container for the sample is required, but the immunochromatography kit built into the housing is easy to use because it does not require a separate test container by directly inserting the sample into the inlet prepared in the housing.
신속진단검사는 간편성 및 신속성 측면에서 최근까지 개발된 검출 방법 중 가장 진보된 분석 키트 중 하나로서 감염성 병원체의 항원 또는 항체, 암 인자, 심장 마커 등 다양한 질병원인 물질을 진단하는데 유용하게 사용한다. Rapid diagnostic test is one of the most advanced analysis kits among detection methods developed recently in terms of simplicity and speed, and is usefully used to diagnose various disease-causing substances such as antigens or antibodies of infectious pathogens, cancer factors, and cardiac markers.
이와 같은 면역크로마토그래피 스트립 또는 이를 포함하는 면역크로마토그래피 키트를 이용한 분석을 통해, 사람 또는 동물의 전혈, 혈장, 혈청, 눈물, 침, 소변, 콧물, 체액 등의 검체를 이용하여, 사스, 메르스, 인플루엔자바이러스, 조류독감 바이러스, 로타 바이러스, A형 간염, B형 간염, C형 간염, 에이즈, 매독, 클라미디아, 말라리아, 장티프스, 위궤양 원인균, 결핵, 뎅기열, 나병 등의 원인 병원체 및 항체의 유무 등을 신속하게 검사 및 진단할 수 있다. Through analysis using such an immunochromatographic strip or an immunochromatographic kit comprising the same, using samples such as human or animal whole blood, plasma, serum, tears, saliva, urine, runny nose, and body fluid, SARS, MERS , influenza virus, avian influenza virus, rotavirus, hepatitis A, hepatitis B, hepatitis C, AIDS, syphilis, chlamydia, malaria, typhoid, gastric ulcer causative bacteria, tuberculosis, dengue fever, leprosy, etc. can be quickly tested and diagnosed.
SARS-CoV-2는 아직까지 이 바이러스에 특이적인 진단용 항체 및 진단용 키트가 없다. 이에 따라, 본 발명자들은 SARS-CoV-2에 특이적인 항체를 개발하고자 하였다. 결합력 및 진단 효과가 우수한 항체를 개발하기 위해 지속적인 연구를 거듭한 결과, 본 발명을 완성하였다. SARS-CoV-2 does not yet have a diagnostic antibody and diagnostic kit specific for this virus. Accordingly, the present inventors attempted to develop an antibody specific for SARS-CoV-2. As a result of repeated research to develop an antibody having excellent binding ability and diagnostic effect, the present invention was completed.
본 발명자들은 상기 문제점을 해결하고자 SARS-CoV-2의 S 단백질에 대한 결합 능력을 갖는 결합 분자를 개발하였고, 이 결합 분자가 SARS-CoV-2에 대하여 우수한 진단 효과를 가짐을 확인함으로써 본 발명을 완성하였다. In order to solve the above problems, the present inventors have developed a binding molecule having the ability to bind to the S protein of SARS-CoV-2, and confirmed that this binding molecule has an excellent diagnostic effect on SARS-CoV-2. completed.
본 발명이 해결하고자 하는 과제는 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 결합 분자를 제공하는 것이다. An object of the present invention is to provide a binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2).
또한, 본 발명이 해결하고자 하는 다른 과제는 상기 결합 분자를 포함하는 사스-코로나바이러스 감염증(COVID-19)의 진단용 조성물을 제공하는 것이다. In addition, another problem to be solved by the present invention is to provide a composition for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule.
또한, 본 발명이 해결하고자 하는 다른 과제는 사스-코로나바이러스 감염증(COVID-19) 진단용 키트를 제공하는 것이다. In addition, another problem to be solved by the present invention is to provide a kit for diagnosing SARS-coronavirus infection (COVID-19).
또한, 본 발명이 해결하고자 하는 다른 과제는 사스-코로나바이러스-2(SARS-CoV-2) 검출 방법을 제공하는 것이다. In addition, another problem to be solved by the present invention is to provide a method for detecting SARS-coronavirus-2 (SARS-CoV-2).
또한, 본 발명이 해결하고자 하는 다른 과제는 사스-코로나바이러스 감염증(COVID-19) 진단 방법을 제공하는 것이다. In addition, another problem to be solved by the present invention is to provide a method for diagnosing SARS-coronavirus infection (COVID-19).
상기 과제를 해결하고자, 본 발명은 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 결합 분자를 제공한다. In order to solve the above problems, the present invention provides a binding molecule that binds to the spike protein (S protein) on the surface of SARS-coronavirus-2 (SARS-CoV-2).
또한, 본 발명은 상기 결합 분자에 추가적으로 하나 이상의 태그가 결합된 이뮤노컨쥬게이트를 제공한다. In addition, the present invention provides an immunoconjugate in which one or more tags are additionally bound to the binding molecule.
또한, 본 발명은 상기 결합 분자를 암호화하는 핵산 분자를 제공한다. The present invention also provides a nucleic acid molecule encoding the binding molecule.
또한, 본 발명은 상기 핵산 분자가 삽입된 발현 벡터를 제공한다. In addition, the present invention provides an expression vector into which the nucleic acid molecule is inserted.
또한, 본 발명은 상기 발현 벡터로 형질전환된 세포주를 제공한다. In addition, the present invention provides a cell line transformed with the expression vector.
또한, 본 발명은 상기 결합 분자를 포함하는 사스-코로나바이러스 감염증(COVID-19)의 진단용 조성물을 제공한다. In addition, the present invention provides a composition for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule.
또한, 본 발명은 상기 결합 분자를 포함하는 면역크로마토그래피 분석용 스트립을 제공한다. In addition, the present invention provides a strip for immunochromatographic analysis comprising the binding molecule.
또한, 본 발명은 상기 결합 분자를 포함하는, 사스-코로나바이러스 감염증(COVID-19)의 진단용 키트를 제공한다.The present invention also provides a kit for diagnosis of SARS-coronavirus infection (COVID-19), comprising the binding molecule.
또한, 본 발명은 상기 진단용 키트를 이용하여 사스-코로나바이러스-2(SARS-CoV-2)를 검출하는 방법을 제공한다. In addition, the present invention provides a method of detecting SARS-coronavirus-2 (SARS-CoV-2) using the diagnostic kit.
또한, 본 발명은 상기 진단용 키트를 이용하여 사스-코로나바이러스 감염증(COVID-19)을 진단하는 방법을 제공한다. In addition, the present invention provides a method for diagnosing SARS-coronavirus infection (COVID-19) using the diagnostic kit.
이하 본 발명을 더욱 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명은 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 사스-코로나바이러스 감염증(COVID-19)의 진단용 결합 분자에 관한 것이다. The present invention relates to a binding molecule for diagnosis of SARS-coronavirus infection (COVID-19) that binds to a spike protein (S protein) on the surface of SARS-CoV-2.
본 발명의 일 구체예로서, 상기 결합 분자는 사스-코로나바이러스-2 표면의 스파이크 단백질의 RBD(Receptor binding domain) 영역에 결합할 수 있다. In one embodiment of the present invention, the binding molecule may bind to the receptor binding domain (RBD) region of the spike protein on the SARS-coronavirus-2 surface.
사스-코로나바이러스-2는 세계보건기구(WHO)는 유전자 염기서열 차이로 인한 아미노산 변화를 기준으로 코로나 바이러스를 6개 유형으로 분류하고 있다. 먼저 S, L 유형으로 분류되었다가 다시 L, V, G 유형으로 나뉘고 G가 GH와 GR로 나뉘면서 S, L, V, G, GH, GR 의 총 6개 유형으로 분류하고 있다. 코로나19 발생 초기에 중국 우한을 비롯한 아시아 지역에는 S와 V 유형이 유행하였고, 이후 대륙별로 서로 다른 유형이 발견되었다. 이 중 GH 유형이 전파력이 높게 나타날 가능성이 있다고 보고된 바 있다. 국내의 경우 코로나바이러스 감염증 환자에서 채취한 유전자를 분류한 결과, 대부분은 유럽과 미국에서 유행한 G형의 변종인 GH형인 것으로 나타났고, 이 유형은 바이러스 전파력이 높은 것으로 알려져 있다. 이 중 바이러스의 세포 내 침입 시 중요한 역할을 하는 스파이크 단백질의 614번 아미노산을 아스파트산 (D)에서 글리신 (G)로 바뀐 G형의 바이러스는 3월 이후 유럽과 미국에서 급격히 증가해 현재는 거의 대부분 지역에서 나타나고 있다. 최근 보고된 바에 따르면 70여개 넘는 코로나바이러스 변이가 발생한 것으로 확인되었고, 전파력이 증가된 변이가 8개 (D614G 등), 중화항체를 회피하는 변이가 10개 (A841V 등), 혈장치료 효과가 낮은 변이 17개 (I472V 등)가 확인되었다. SARS-Coronavirus-2 is classified into six types of coronavirus by the World Health Organization (WHO) based on amino acid changes caused by differences in gene sequence. First, it was classified into S and L types, then again into L, V, and G types, and as G was divided into GH and GR, it is classified into a total of six types: S, L, V, G, GH, and GR. S and V types were prevalent in Asia, including Wuhan, China, in the early stages of the outbreak of COVID-19, and after that, different types were discovered for each continent. Among them, it has been reported that the GH type has the potential to appear high in transmission power. In Korea, as a result of classifying the genes collected from patients with coronavirus infection, most of them were found to be the GH type, a variant of the G type prevalent in Europe and the United States, and this type is known to have high virus transmission power. Among these, type G virus, in which amino acid 614 of the spike protein, which plays an important role in virus invasion, is changed from aspartic acid (D) to glycine (G), has increased rapidly in Europe and the United States since March, and is now almost It appears in most areas. According to a recent report, more than 70 coronavirus mutations have been identified, 8 mutations with increased transmission power (D614G, etc.), 10 mutations that avoid neutralizing antibodies (A841V, etc.), mutations with low plasma treatment effect 17 (I472V, etc.) were identified.
일 구체예로, 본 발명의 결합 분자는 현재까지 단리된 사스-코로나바이러스-2 균주, 예를 들어 단리 일시와 장소를 알 수 없는 UNKNOWN-LR757996 균주(Strain), SARS-CoV-2/Hu/DP/Kng/19-027 균주; 2019년 12월 중국에서 단리된 Wuhan-Hu-1 균주; 2019년 12월 23일 최초로 중국에서 단리된 BetaCoV/Wuhan/IPBCAMS-WH-01/2019 균주; 2019년 12월 30일 중국에서 단리된 BetaCoV/Wuhan/IPBCAMS-WH-02/2019 균주, BetaCoV/Wuhan/IPBCAMS-WH-03/2019 균주, BetaCoV/Wuhan/IPBCAMS-WH-04/2019 균주, WIV02 균주, WIV04 균주, WIV05 균주, WIV06 균주, WIV07 균주; 2020년 1월 일본에서 단리된 2019-nCoV/Japan/TY/WK-521/2020 균주, 2019-nCoV/Japan/TY/WK-501/2020 균주, 2019-nCoV/Japan/TY/WK-012/2020 균주, 2019-nCoV/Japan/KY/V-029/2020 균주; 2020년 1월 대한민국에서 단리된 SNU01 균주; 대한민국에서 단리된 BetaCoV/Korea/KCDC03/2020 균주; 2020년 1월 1일 중국에서 단리된 BetaCoV/Wuhan/IPBCAMS-WH-05/2020 균주; 2020년 1월 2일 중국에서 단리된 2019-nCoV WHU02 균주, 2019-nCoV WHU01 균주; 2020년 1월 8일 중국에서 단리된 SARS-CoV-2/WH-09/human/2020/CHN 균주; 2020년 1월 10일 중국에서 단리된 2019-nCoV_HKU-SZ-002a_2020 균주; 2020년 1월 11일 중국에서 단리된 2019-nCoV_HKU-SZ-005b_2020 균주; 2020년 1월 17일 중국에서 단리된 SARS-CoV-2/Yunnan-01/human/2020/CHN 균주; 2020년 1월 19일 미국에서 단리된 2019-nCoV/USA-WA1/2020 균주; 2020년 1월 20일 중국에서 단리된 HZ-1 균주; 2020년 1월 21일 미국에서 단리된 2019-nCoV/USA-IL1/2020 균주; 2020년 1월 22일 미국에서 단리된 2019-nCoV/USA-CA2/2020 균주, 2019-nCoV/USA-AZ1/2020 균주; 2020년 1월 23일 미국에서 단리된 2019-nCoV/USA-CA1/2020 균주; 2020년 1월 25일 호주에서 단리된 Australia/VIC01/2020 균주; 2020년 1월 25일 미국에서 단리된 2019-nCoV/USA-WA1-F6/2020 균주, 2019-nCoV/USA-WA1-A12/2020 균주; 2020년 1월 27일 미국에서 단리된 2019-nCoV/USA-CA6/2020 균주; 2020년 1월 28일 미국에서 단리된 2019-nCoV/USA-IL2/2020 균주; 2020년 1월 29일 미국에서 단리된 2019-nCoV/USA-MA1/2020 균주, 2019-nCoV/USA-CA5/2020 균주, 2019-nCoV/USA-CA4/2020 균주, 2019-nCoV/USA-CA3/2020 균주; 2020년 1월 29일 핀란드에서 단리된 nCoV-FIN-29-Jan-2020 균주; 2020년 1월 29일 중국에서 단리된 SARS-CoV-2/IQTC02/human/2020/CHN 균주; 2020년 1월 31일 미국에서 단리된 2019-nCoV/USA-WI1/2020 균주; 2020년 1월 31일 타이완에서 단리된 SARS-CoV-2/NTU01/2020/TWN 균주; 2020년 2월 5일 타이완에서 단리된 SARS-CoV-2/NTU02/2020/TWN 균주; 2020년 2월 6일 미국에서 단리된 2019-nCoV/USA-CA7/2020 균주; 2020년 2월 7일 스웨덴에서 단리된 SARS-CoV-2/01/human/2020/SWE 균주; 2020년 2월 10일 미국에서 단리된 2019-nCoV/USA-CA8/2020 균주; 2020년 2월 11일 미국에서 단리된 2019-nCoV/USA-TX1/2020 균주; 2020년 2월 23일 미국에서 단리된 2019-nCoV/USA-CA9/2020 균주; 2020년 2월 28일 브라질에서 단리된 SARS-CoV-2/SP02/human/2020/BRA 균주; 단리 일시와 장소를 알 수 없는 UNKNOWN-LR757995, UNKNOWN-LR757997, UNKNOWN-LR757998, SARS-CoV-2/Hu/DP/Kng/19-020; 2020년 1월 일본에서 단리된 2019-nCoV/Japan/AI/I-004/2020; 2020년 1월 13일 네팔에서 단리된 SARS0CoV-2/61-TW/human/2020/ NPL; 2020년 2월 5일 중국에서 단리된 SARS-CoV-2/IQTC01/human/2020/CHN 균주; 대한민국에서 단리된 hCoV-19/South Korea/KUMC17/2020 균주와 향후 단리될 사스-코로나바이러스-2 균주에 대하여 결합 가능하나, 이들 균주에 한정되는 것은 아니다.In one embodiment, the binding molecule of the present invention is a SARS-coronavirus-2 strain isolated to date, for example, UNKNOWN-LR757996 strain (Strain), SARS-CoV-2/Hu/ DP/Kng/19-027 strain; Wuhan-Hu-1 strain isolated from China in December 2019; BetaCoV/Wuhan/IPBCAMS-WH-01/2019 strain first isolated in China on December 23, 2019; BetaCoV/Wuhan/IPBCAMS-WH-02/2019 strain, BetaCoV/Wuhan/IPBCAMS-WH-03/2019 strain, BetaCoV/Wuhan/IPBCAMS-WH-04/2019 strain, WIV02 isolated on December 30, 2019 in China strain, WIV04 strain, WIV05 strain, WIV06 strain, WIV07 strain; 2019-nCoV/Japan/TY/WK-521/2020 strain isolated from Japan in January 2020, 2019-nCoV/Japan/TY/WK-501/2020 strain, 2019-nCoV/Japan/TY/WK-012/ 2020 strain, 2019-nCoV/Japan/KY/V-029/2020 strain; SNU01 strain isolated from Korea in January 2020; BetaCoV/Korea/KCDC03/2020 strain isolated from Korea; BetaCoV/Wuhan/IPBCAMS-WH-05/2020 strain isolated from China on January 1, 2020; 2019-nCoV WHU02 strain, 2019-nCoV WHU01 strain isolated on January 2, 2020 in China; SARS-CoV-2/WH-09/human/2020/CHN strain isolated from China on January 8, 2020; 2019-nCoV_HKU-SZ-002a_2020 strain isolated from China on January 10, 2020; 2019-nCoV_HKU-SZ-005b_2020 strain isolated from China on January 11, 2020; SARS-CoV-2/Yunnan-01/human/2020/CHN strain isolated from China on January 17, 2020; 2019-nCoV/USA-WA1/2020 strain isolated in the United States on January 19, 2020; HZ-1 strain isolated in China on January 20, 2020; 2019-nCoV/USA-IL1/2020 strain isolated in the United States on January 21, 2020; 2019-nCoV/USA-CA2/2020 strain, 2019-nCoV/USA-AZ1/2020 strain isolated in the United States on January 22, 2020; 2019-nCoV/USA-CA1/2020 strain isolated in the United States on January 23, 2020; Australia/VIC01/2020 strain isolated in Australia on 25 January 2020; 2019-nCoV/USA-WA1-F6/2020 strain, 2019-nCoV/USA-WA1-A12/2020 strain isolated in the United States on January 25, 2020; 2019-nCoV/USA-CA6/2020 strain isolated in the United States on January 27, 2020; 2019-nCoV/USA-IL2/2020 strain isolated in the United States on January 28, 2020; 2019-nCoV/USA-MA1/2020 strain, 2019-nCoV/USA-CA5/2020 strain, 2019-nCoV/USA-CA4/2020 strain, 2019-nCoV/USA-CA3 isolated in USA on January 29, 2020 /2020 strain; nCoV-FIN-29-Jan-2020 strain isolated on January 29, 2020 in Finland; SARS-CoV-2/IQTC02/human/2020/CHN strain isolated from China on January 29, 2020; 2019-nCoV/USA-WI1/2020 strain isolated in the United States on January 31, 2020; SARS-CoV-2/NTU01/2020/TWN strain isolated in Taiwan on January 31, 2020; SARS-CoV-2/NTU02/2020/TWN strain isolated on February 5, 2020 in Taiwan; 2019-nCoV/USA-CA7/2020 strain isolated in the United States on February 6, 2020; SARS-CoV-2/01/human/2020/SWE strain isolated on February 7, 2020 in Sweden; 2019-nCoV/USA-CA8/2020 strain isolated in the United States on February 10, 2020; 2019-nCoV/USA-TX1/2020 strain isolated in the United States on February 11, 2020; 2019-nCoV/USA-CA9/2020 strain isolated in the United States on February 23, 2020; SARS-CoV-2/SP02/human/2020/BRA strain isolated on February 28, 2020 in Brazil; UNKNOWN-LR757995, UNKNOWN-LR757997, UNKNOWN-LR757998, SARS-CoV-2/Hu/DP/Kng/19-020 of unknown date and place of isolation; 2019-nCoV/Japan/AI/I-004/2020 isolated from Japan in January 2020; SARS0CoV-2/61-TW/human/2020/NPL isolated from Nepal on January 13, 2020; SARS-CoV-2/IQTC01/human/2020/CHN strain isolated from China on February 5, 2020; It is possible to bind to the hCoV-19/South Korea/KUMC17/2020 strain isolated in Korea and the SARS-coronavirus-2 strain to be isolated in the future, but is not limited to these strains.
본 발명의 일 구체예로서, 상기 결합 분자는 하기 특성 중 어느 하나 이상을 가질 수 있다: In one embodiment of the present invention, the binding molecule may have any one or more of the following properties:
a) 1 x 10-8 M 이하의 평형 해리 상수(KD)로 사스-코로나바이러스-2 표면의 스파이크 단백질에 결합함; a) binds to the spike protein on the surface of SARS-coronavirus-2 with an equilibrium dissociation constant (K D ) of 1 x 10 -8 M or less;
b) 1 x 104 1/Ms 이상의 결합 상수(Ka)로 사스-코로나바이러스-2 표면의 스파이크 단백질에 결합함; 또는b) binds to the spike protein on the surface of SARS-coronavirus-2 with a binding constant (Ka) greater than or equal to 1 x 10 4 1/Ms; or
c) 1 x 10-2 1/s 이하의 해리 상수(Kd)로 사스-코로나바이러스-2 표면의 스파이크 단백질에 결합함.c) Binding to the spike protein on the surface of SARS-coronavirus-2 with a dissociation constant (Kd) of 1 x 10 -2 1/s or less.
본 발명의 일 구체예로서, 본 발명의 결합분자는 사스-코로나바이러스-2 표면의 스파이크 단백질에 1.0×10-8 M 이하 또는 3.0×10-9M 이하의 결합친화도(KD)로 결합할 수 있다.In one embodiment of the present invention, the binding molecule of the present invention binds to the spike protein on the surface of SARS-coronavirus-2 with a binding affinity (K D ) of 1.0×10 -8 M or less or 3.0×10 -9 M or less can do.
본 발명의 일 구체예로서, 본 발명의 결합분자는 사스-코로나바이러스-2 표면의 스파이크 단백질에 1 x 104 1/Ms 이상 또는 1 x 105 1/Ms 이상의 결합 상수(Ka)로 결합할 수 있다.In one embodiment of the present invention, the binding molecule of the present invention binds to the spike protein on the surface of SARS-coronavirus-2 with a binding constant (Ka) of 1 x 10 4 1/Ms or more or 1 x 10 5 1/Ms or more. can
본 발명의 일 구체예로서, 본 발명의 결합분자는 사스-코로나바이러스-2 표면의 스파이크 단백질에 1 x 10-2 1/s 이하 또는 5 x 10-3 1/s 이하의 해리 상수(Kd)로 결합할 수 있다.In one embodiment of the present invention, the binding molecule of the present invention has a dissociation constant (Kd) of 1 x 10 -2 1/s or less or 5 x 10 -3 1/s or less to the spike protein on the surface of SARS-coronavirus-2 can be combined with
본 발명의 일 구체예로서, 상기 결합 분자는 하기 표 1의 결합 분자들로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있다. 하기 표 1에서 No.는 각 결합 분자의 번호를 의미한다.In one embodiment of the present invention, the binding molecule may be any one binding molecule selected from the group consisting of binding molecules shown in Table 1 below. In Table 1 below, No. means the number of each binding molecule.
본 발명에 있어서, 가변영역의 CDR은 Kabat 등에 의해 고안된 시스템에 따라 통상적인 방법으로 결정되었다(문헌[Kabat et al., Sequences of Proteins of Immunological Interest(5th), National Institutes of Health, Bethesda, MD. (1991)] 참조). 본 발명에 사용된 CDR 넘버링은 Kabat 방법을 사용했지만, 이외에 IMGT 방법, Chothia 방법, AbM 방법 등 다른 방법에 따라 결정된 CDR을 포함하는 결합 분자도 본 발명에 포함된다. In the present invention, the CDRs of the variable region 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)]). Although the Kabat method was used for CDR numbering used in the present invention, binding molecules comprising CDRs determined according to other methods such as the IMGT method, Chothia method, and AbM method are also included in the present invention.
본 발명의 일 구체예로서, 상기 결합 분자는 하기 표 2의 결합 분자들로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있다. 하기 표 2에서 No.는 각 결합 분자의 번호를 의미한다.In one embodiment of the present invention, the binding molecule may be any one binding molecule selected from the group consisting of binding molecules shown in Table 2 below. In Table 2 below, No. means the number of each binding molecule.
본 발명의 일 구체예에서, 상기 결합 분자는 scFv 절편, scFv-Fc 절편, Fab 절편, Fv 절편, 디아바디(diabody), 키메라 항체, 인간화 항체 또는 인간 항체일 수 있으나, 이에 한정되지 않는다. 본 발명의 일 실시예는 SARS-CoV-2 S 단백질에 결합하는 scFv-Fc 절편을 제공한다. 또한, 본 발명의 다른 일 실시예는 SARS-CoV-2 S 단백질에 결합하는 완전한 인간 항체(Full IgG)를 제공한다. In one embodiment of the present invention, the binding molecule may be a scFv fragment, an scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody, but is not limited thereto. One embodiment of the present invention provides a scFv-Fc fragment that binds to the SARS-CoV-2 S protein. In addition, another embodiment of the present invention provides a fully human antibody (Full IgG) that binds to the SARS-CoV-2 S protein.
본 명세서에서 '항체'는 최대한 넓은 의미로 사용되며, 구체적으로 무손상(intact) 단일클론 항체, 다클론 항체, 2종 이상의 무손상 항체로부터 형성된 다중특이성 항체(예를 들어, 이중특이성 항체), 및 목적하는 생물학적 활성을 나타내는 항체 단편을 포함한다. 항체는 특이적인 항원을 인식하고 결합할 수 있는 면역계에 의하여 생성되는 단백질이다. 그 구조적인 면에서, 항체는 통상적으로 4개의 아미노산 쇄(2개의 중쇄 및 2개의 경쇄)로 이루어진 Y-형상의 단백질을 가진다. 각각의 항체는 주로 가변 영역 및 불변 영역의 2개의 영역을 가진다. Y의 팔의 말단 부분에 위치한 가변 영역은 표적 항원에 결합하고 상호작용한다. 상기 가변 영역은 특정 항원 상의 특이적 결합 부위를 인식하고 결합하는 상보성 결정 영역(CDR)을 포함한다. Y의 꼬리 부분에 위치한 불변 영역은 면역계에 의하여 인식되고 상호작용한다. 표적 항원은 일반적으로 다수의 항체 상의 CDR에 의하여 인식되는, 에피토프라고 하는 다수의 결합 부위를 가지고 있다. 상이한 에피토프에 특이적으로 결합하는 각각의 항체는 상이한 구조를 가진다. 그러므로 한 항원은 하나 이상의 상응하는 항체를 가질 수 있다.As used herein, the term 'antibody' is used in the broadest sense, specifically, an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more intact antibodies (eg, a bispecific antibody); and antibody fragments exhibiting the desired biological activity. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. In terms of their structure, antibodies usually have a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody mainly has two regions: a variable region and a constant region. The variable region located in the distal portion of the arm of Y binds and interacts with the target antigen. The variable region comprises a complementarity determining region (CDR) that recognizes and binds a specific binding site on a specific antigen. The constant region located at the tail of Y is recognized and interacted with by the immune system. Target antigens have multiple binding sites, called epitopes, which are generally recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.
아울러 본 발명은 상기 결합 분자의 기능적 변이체를 포함한다. 결합 분자들은 변이체들이 SARS-CoV-2 또는 이것의 S 단백질에 특이적으로 결합하기 위해 본 발명의 결합 분자와 경쟁할 수 있고, SARS-CoV-2에 결합 능력을 보유한다면 본 발명의 결합 분자의 기능적 변이체로 간주된다. 기능적 변이체는 1차 구조적 서열이 실질적으로 유사한 유도체를 포함하지만, 이에 제한되는 것은 아니며, 예를 들면, 생체외(in vitro) 또는 생체내(in vivo) 변형, 화학약품 및/또는 생화학 약품을 포함하며, 이들은 본원 발명의 부모 단일클론 항체에서는 발견되지 않는다. 이와 같은 변형으로는 예를 들어 아세틸화, 아실화, 뉴클레오티드 또는 뉴클레오티드 유도체의 공유 결합, 지질 또는 지질 유도체의 공유 결합, 가교, 이황화 결합 형성, 글리코실화, 수산화, 메틸화, 산화, 페길화, 단백질 분해 및 인산화 등이 포함된다. 기능적 변이체는 선택적으로 부모 항체의 아미노산 서열과 비교하여 하나 이상의 아미노산의 치환, 삽입, 결실 또는 그들의 조합을 함유하는 아미노산 서열을 포함하는 항체일 수 있다. 더욱이 기능적 변이체는 아미노 말단 또는 카르복시 말단 중 하나 또는 모두에서 아미노산 서열의 절단체(truncated form)를 포함할 수 있다. 본 발명의 기능적 변이체는 본 발명의 부모 항체와 비교하여 동일하거나 다르거나, 더 높거나 낮은 결합 친화력을 가질 수 있지만, 여전히 SARS-CoV-2 또는 이것의 S 단백질에 결합할 수 있다. 일 예로, 골격구조, 초가변(Hypervariable) 영역, 특히 경쇄 또는 중쇄의 상보성 결정 영역(Complementarity-determining region, CDR)을 포함하나 이에 한정되지 않는 가변 영역의 아미노산 서열이 변형될 수 있다. 일반적으로 경쇄 또는 중쇄 영역은 3개의 CDR 영역을 포함하는, 3개의 초가변 영역 및 더욱 보존된 영역, 즉 골격 영역(FR)을 포함한다. 초가변 영역은 CDR로부터의 아미노산 잔기와 초가변 루프로부터의 아미노산 잔기를 포함한다. 본 발명의 범위에 속하는 기능적 변이체는 본 명세서의 부모 항체와 약 50%~99%, 약 60%~99%, 약 80%~99%, 약 90%~99%, 약 95%~99%, 또는 약 97%~99% 아미노산 서열 동질성을 가질 수 있다. 비교될 아미노산 서열을 최적으로 배열하고 유사하거나 또는 동일한 아미노산 잔기를 정의하기 위해 컴퓨터 알고리즘 중 당업자에게 알려진 Gap 또는 Bestfit를 사용할 수 있다. 기능적 변이체는 부모 항체 또는 그것의 일부를 PCR 방법, 올리고머 뉴클레오티드를 이용한 돌연변이 생성 및 부분 돌연변이 생성을 포함하는 공지의 일반 분자생물학적 방법에 의해 변화시키거나 유기합성 방법으로 얻을 수 있으나 이에 제한되는 것은 아니다. The present invention also includes functional variants of the binding molecule. Binding molecules are capable of competing with a binding molecule of the present invention for specific binding to SARS-CoV-2 or its S protein, and retain the ability to bind to SARS-CoV-2. It is considered a functional variant. Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, and include, for example, in vitro or in vivo modifications, chemicals and/or biochemicals. and they are not found in the parental monoclonal antibodies of the present invention. Such modifications include, for example, acetylation, acylation, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis. and phosphorylation. A functional variant may be an antibody comprising an amino acid sequence optionally containing one or more amino acid substitutions, insertions, deletions or combinations thereof compared to the amino acid sequence of the parent antibody. Furthermore, functional variants may include truncated forms of the amino acid sequence at either or both the amino terminus or the carboxy terminus. Functional variants of the invention may have the same, different, higher or lower binding affinity compared to the parent antibody of the invention, but still be capable of binding SARS-CoV-2 or its S protein. As an example, the amino acid sequence of a variable region including, but not limited to, a framework structure, a hypervariable region, in particular, a complementarity-determining region (CDR) of a light or heavy chain may be modified. Generally a light or heavy chain region comprises three hypervariable regions, comprising three CDR regions, and a more conserved region, namely a framework region (FR). A hypervariable region comprises amino acid residues from the CDRs and amino acid residues from the hypervariable loops. Functional variants within the scope of the present invention include about 50%-99%, about 60%-99%, about 80%-99%, about 90%-99%, about 95%-99%, or about 97%-99% amino acid sequence identity. To optimally align the amino acid sequences to be compared and to define similar or identical amino acid residues, the Gap or Bestfit of computer algorithms known to those skilled in the art can be used. A functional variant may be obtained by organic synthesis or by changing the parent antibody or a part thereof by known general molecular biological methods including PCR, mutagenesis using oligomeric nucleotides, and partial mutagenesis, but is not limited thereto.
또한, 본 발명은 상기 결합 분자에 추가적으로 하나 이상의 태그가 결합된 이뮤노컨쥬게이트를 제공한다. 일 구체예에서, 상기 결합 분자에 약물이 추가로 부착될 수 있다. 즉, 본 발명에 따른 결합 분자는 약제가 결합된 항체-약물 접합체(conjugate)의 형태로 사용될 수 있다. 약물을 국소 전달하기 위해 항체-약물 접합체(ADC), 즉 면역접합체를 사용하게 되면 상기 약물 모이어티를 감염된 세포에 표적화 전달할 수 있는데, 상기 약물 작용제를 접합시키지 않은 채로 투여하게 되면, 정상 세포에 대해서도 허용될 수 없는 수준의 독성이 야기될 수 있기 때문이다. 약물-연결성 및 약물-방출성 뿐만 아니라 폴리클로날 및 모노클로날 항체(mAb)의 선택성을 높임으로써 ADC의 최대 효능과 최소 독성을 개선할 수 있다.In addition, the present invention provides an immunoconjugate in which one or more tags are additionally bound to the binding molecule. In one embodiment, a drug may be further attached to the binding molecule. That is, the binding molecule according to the present invention may be used in the form of an antibody-drug conjugate to which a drug is bound. The use of antibody-drug conjugates (ADCs), i.e., immunoconjugates, for local delivery of drugs allows for targeted delivery of the drug moiety to infected cells, which when administered unconjugated to normal cells. This is because unacceptable levels of toxicity can result. Maximal efficacy and minimal toxicity of ADCs can be improved by increasing drug-connectivity and drug-releasing properties, as well as selectivity of polyclonal and monoclonal antibodies (mAbs).
약물 모이어티를 항체에 부착시키는, 즉 공유 결합을 통하여 연결시키는 통상적인 수단으로는, 일반적으로 약물 모이어티가 항체 상의 수 많은 부위에 부착되는 불균질한 분자 혼합물이 유발된다. 예를 들어, 세포독성 약물을 전형적으로, 종종 항체의 수 많은 리신 잔기를 통하여 항체와 접합시켜 불균질한 항체-약물 접합체 혼합물을 생성킬 수 있다. 반응 조건에 따라서, 이러한 불균질한 혼합물은 전형적으로, 약물 모이어티에 부착된 항체 분포도가 0 내지 약 8 이상이다. 또한, 특별한 정수 비의 약물 모이어티 대 항체를 수반한 접합체의 각 아군은, 약물 모이어티가 항체 상의 각종 부위에 부착되는 잠재적으로 불균질한 혼합물이다. 항체는 크고, 복잡하며 구조적으로 다양한 생체 분자이고, 종종 많은 반응성 관능기를 갖고 있다. 링커 시약 및 약물-링커 중간체와의 반응성은 pH, 농도, 염 농도 및 조용매와 같은 요인들에 의해 좌우된다.Conventional means of attaching drug moieties to antibodies, ie, linking them via covalent bonds, generally result in heterogeneous molecular mixtures in which drug moieties are attached to numerous sites on the antibody. For example, a cytotoxic drug can be conjugated with an antibody, typically through the numerous lysine residues of the antibody, resulting in a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, such heterogeneous mixtures typically have a distribution of 0 to about 8 or greater of antibody attached to the drug moiety. In addition, each subgroup of conjugates with a particular integer ratio of drug moieties to antibody is a potentially heterogeneous mixture in which drug moieties are attached to various sites on the antibody. Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Reactivity with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration and cosolvent.
또한, 본 발명의 다른 일 구체예는 상기 결합 분자를 암호화하는 핵산 분자를 제공한다. Further, another embodiment of the present invention provides a nucleic acid molecule encoding the binding molecule.
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본 발명의 핵산 분자는 본 발명에서 제공하는 항체의 아미노산 서열을 당업자에게 알려진 바와 같이 폴리뉴클레오티드 서열로 번역된 핵산 분자 모두를 포함한다. 그러므로 ORF(open reading frame)에 의한 다양한 폴리뉴클레오티드 서열이 제조될 수 있으며 이 또한 모두 본 발명의 핵산 분자에 포함된다.Nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of the antibody provided in the present invention is translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by an open reading frame (ORF), and all of these are also included in the nucleic acid molecule of the present invention.
또한, 본 발명의 다른 일 구체예는 상기 핵산 분자가 삽입된 발현 벡터를 제공한다.In addition, another embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.
상기 발현 벡터로는 셀트리온 고유의 발현 벡터인 MarEx 벡터(한국특허등록 제10-1076602호 참조) 및 상업적으로 널리 사용되는 pCDNA 벡터, F, R1, RP1, Col, pBR322, ToL, Ti 벡터; 코스미드; 람다, 람도이드(lambdoid), M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7 등의 파아지; 식물 바이러스로 이루어진 군으로부터 선택된 어느 하나에서 선택된 발현 벡터를 이용할 수 있으나 이에 한정되지 않으며, 당업자에게 발현 벡터로 알려진 모든 발현 벡터는 본 발명에 사용 가능하며, 발현 벡터를 선택할 때에는 목적으로 하는 숙주 세포의 성질에 따른다. 숙주세포로의 벡터 도입시 인산칼슘 트랜스펙션, 바이러스 감염, DEAE-덱스트란 조절 트랜스펙션, 리포펙타민 트랜스펙션 또는 전기천공법에 의해 수행될 수 있으나 이에 한정되지 않으며 당업자는 사용하는 발현 벡터 및 숙주 세포에 알맞은 도입 방법을 선택하여 이용할 수 있다. 예를 들어, 벡터는 하나 이상의 선별 마커를 함유하나 이에 한정되지 않으며, 선별 마커를 포함하지 않은 벡터도 이용하여 생산물 생산 여부에 따라 선별이 가능하다. 선별 마커의 선택은 목적하는 숙주 세포에 의해 선택되며, 이는 이미 당업자에게 알려진 방법을 이용하므로 본 발명은 이에 제한을 두지 않는다. Examples of the expression vector include the MarEx vector (refer to Korean Patent Registration No. 10-1076602), which is Celltrion's own expression vector, and the widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmid; phage such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7; An expression vector selected from any one selected from the group consisting of plant viruses may be used, but the present invention is not limited thereto, and any expression vector known to those skilled in the art can be used in the present invention. according to the nature When introducing a vector into a host cell, calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation may be performed, but not limited thereto, and expression used by those skilled in the art. An introduction method suitable for the vector and host cell can be selected and used. For example, the vector contains one or more selectable markers, but is not limited thereto, and a vector that does not contain a selectable marker may be used to select depending on whether a product is produced. The selection of the selection marker is selected by the host cell of interest, and the present invention is not limited thereto since it uses a method already known to those skilled in the art.
본 발명의 결합 분자의 정제를 용이하게 하기 위하여 태그 서열을 발현 벡터 상에 삽입하여 융합시킬 수 있다. 상기 태그로는 헥사-히스티딘 태그, 헤마글루티닌 태그, myc 태그 또는 flag 태그를 포함하나 이에 한정되지 않으며 당업자에게 알려진 정제를 용이하게 하는 태그는 모두 본 발명에서 이용 가능하다. To facilitate purification of the binding molecule of the present invention, a tag sequence may be inserted and fused onto an expression vector. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
또한, 본 발명은 상기 발현 벡터로 형질전환된 세포주를 제공한다. 본 발명의 일 구체예로서, 상기 발현 벡터가 숙주 세포에 형질전환되어, SARS-CoV-2에 결합 능력을 가지는 결합 분자를 생산하는 세포주를 제공한다. In addition, the present invention provides a cell line transformed with the expression vector. In one embodiment of the present invention, the expression vector is transformed into a host cell to provide a cell line producing a binding molecule having the ability to bind to SARS-CoV-2.
본 발명에 있어서, 상기 세포주는 포유동물, 식물, 곤충, 균류 또는 세포성 기원의 세포를 포함할 수 있지만, 이에 한정되지 않는다. 상기 포유동물 세포로는 CHO 세포, F2N 세포, COS 세포, BHK 세포, 바우스(Bowes) 흑색종 세포, HeLa 세포, 911 세포, HT1080 세포, A549 세포, HEK 293 세포 및 HEK293T 세포로 이루어진 군에서 선택된 어느 하나를 사용할 수 있으나 이에 한정되지 않으며, 당업자에게 알려진 포유동물 숙주세포로 사용 가능한 세포는 모두 이용 가능하다.In the present invention, the cell line may include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin. The mammalian cells include any one selected from the group consisting of CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, A549 cells, HEK 293 cells and HEK293T cells. One can be used, but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art can be used.
또한, 본 발명은 상기 결합 분자를 포함하는 사스-코로나바이러스 감염증(COVID-19)의 진단용 조성물을 제공한다. 본 발명의 조성물은 상기 결합 분자 이외에 약제학적으로 허용 가능한 부형제를 포함할 수 있다. 약제학적으로 허용 가능한 부형제는 당업자에게 이미 잘 알려져 있다. 본 발명의 일 구체예로서, 상기 조성물은 멸균 주사용액, 동결건조(lyophilized) 제형, 사전 충전식 주사(pre-filled syringe) 용액제, 경구형 제형, 외용제 또는 좌제일 수 있으나, 이에 한정되는 것은 아니다.In addition, the present invention provides a composition for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule. The composition of the present invention may include a pharmaceutically acceptable excipient in addition to the binding molecule. Pharmaceutically acceptable excipients are well known to those skilled in the art. In one embodiment of the present invention, the composition may be a sterile injection solution, a lyophilized formulation, a pre-filled syringe solution, an oral formulation, an external formulation, or a suppository, but is not limited thereto. .
본 발명의 조성물은 추가로 적어도 하나의 다른 진단제를 포함할 수 있다. 예를 들어, 상기 결합 분자와 함께 사스-코로나바이러스-2(SARS-CoV-2) 표면의 N 단백질(Nucleocapsid protein, N protein)에 결합하는 결합 분자를 추가로 포함할 수 있다. The composition of the present invention may further comprise at least one other diagnostic agent. For example, it may further include a binding molecule that binds to a nucleocapsid protein (N protein) on the surface of SARS-CoV-2 together with the binding molecule.
또한, 본 발명의 다른 일 구체예로, 본 발명은 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 결합 분자를 포함하는 면역크로마토그래피 분석용 스트립을 제공한다. 상기 면역크로마토그래피 분석용 스트립은 코로나바이러스의 뉴클레오캡시드 단백질(Nucleocapsid protein, N protein)에 결합하는 결합 분자를 추가로 포함할 수 있다. 상기 코로나바이러스는 사스-코로나바이러스-2(SARS-CoV-2), 인간 코로나바이러스 229E (HCoV-229E), 인간 코로나바이러스 OC43 (HCoV-OC43), 중증급성호흡기증후군 코로나바이러스 (SARS-CoV), 인간 코로나바이러스 NL63 (HCoV-NL63), 인간 코로나바이러스 HKU1 및 중동호흡기증후군 코로나바이러스 (MERS-CoV)로 구성된 군으로부터 선택된 어느 하나일 수 있으나, 이에 한정되는 것은 아니다. In addition, in another embodiment of the present invention, the present invention provides an immunochromatographic analysis comprising a binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2) strips are provided. The immunochromatographic analysis strip may further include a binding molecule that binds to the nucleocapsid protein (N protein) of the coronavirus. Said coronavirus is SARS-coronavirus-2 (SARS-CoV-2), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), It may be any one selected from the group consisting of human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), but is not limited thereto.
상기 면역크로마토그래피 분석용 스트립은The strip for immunochromatographic analysis is
i) 지지체;i) a support;
ii) 분석하고자 하는 검체를 수용하고 버퍼 투입부 및 검체 투입부를 구비하는 검체 패드;ii) a sample pad for receiving a sample to be analyzed and having a buffer input unit and a sample input unit;
iii) 상기 검체 패드에서 유입된 검체에 함유되어 있는 코로나바이러스와 특이적으로 결합하는 결합 분자를 함유하는, 컨쥬게이트 패드;iii) a conjugate pad containing a binding molecule that specifically binds to the coronavirus contained in the sample introduced from the sample pad;
iv) 상기 검체에 코로나바이러스가 존재하는지 여부를 검출하는 신호검출부와 분석 물질의 존재 유무와 관계없이 검체가 흡수 패드로 이동하였는지 여부를 확인하는 대조부를 포함하는 신호 검출 패드; 및iv) a signal detection pad including a signal detection unit for detecting whether or not the coronavirus is present in the sample and a control unit for checking whether the sample has moved to the absorbent pad regardless of the presence or absence of an analyte; and
v) 신호 검출 반응이 종료된 검체를 흡수하는 흡수 패드v) Absorbent pad that absorbs the sample after the signal detection reaction has been completed
를 포함할 수 있다. may include.
상기 검체패드는 셀룰로오스, 아크릴 섬유, 레이온, 폴리에스터 섬유, 유리섬유, 울섬유, 실크섬유, 면사, 아마섬유 또는 펄프로 제조되거나, 한 종류 이상이 혼합되어 제조될 수 있다.The sample pad may be made of cellulose, acrylic fiber, rayon, polyester fiber, glass fiber, wool fiber, silk fiber, cotton yarn, flax fiber or pulp, or a mixture of one or more types.
상기 컨쥬게이트 패드는 유리섬유, 폴리에틸렌섬유 등으로 제조될 수 있다.The conjugate pad may be made of glass fiber, polyethylene fiber, or the like.
상기 신호검출패드는 다공성 멤브레인 패드로 구성되며, 니트로셀룰로오스, 셀룰로오즈, 폴리에틸렌, 폴리에테르설폰, 또는 나일론 등으로 제조될 수 있다. 신호 검출부는 1 개 이상 포함될 수 있다. The signal detection pad is composed of a porous membrane pad, and may be made of nitrocellulose, cellulose, polyethylene, polyethersulfone, nylon, or the like. One or more signal detectors may be included.
상기 흡수패드는 버퍼의 이동을 촉진시킬 수 있는 임의의 패드일 수 있으며, 예를 들어 펄프, 셀룰로즈, 또는 면섬유 등으로 제조될 수 있다.The absorbent pad may be any pad capable of accelerating the movement of the buffer, and may be made of, for example, pulp, cellulose, or cotton fiber.
상기 스트립은 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 결합 분자를 컨쥬게이트 패드 및 신호 검출 패드에 각각 포함할 수 있다. 상기 컨쥬게이트 패드와 신호 검출 패드에 포함된 SARS-CoV-2 S 단백질 결합 분자는 동일하거나, 다를 수 있다. 또한, 상기 컨쥬게이트 패드와 신호 검출 패드에 포함된 SARS-CoV-2 S 단백질 결합 분자는 앞서 상술한 서열을 포함하는 결합 분자일 수 있다. The strip may include, in a conjugate pad and a signal detection pad, a binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2, respectively. The SARS-CoV-2 S protein binding molecule included in the conjugate pad and the signal detection pad may be the same or different. In addition, the SARS-CoV-2 S protein binding molecule included in the conjugate pad and the signal detection pad may be a binding molecule including the above-described sequence.
상기 면역크로마토그래피 분석용 스트립에 있어서, 상기 컨쥬게이트 패드에 함유된 결합 분자는 금속 입자, 라텍스 입자, 형광물질 또는 효소로 라벨링될 수 있다. 일 예로서, 상기 금속 입자는 금 입자일 수 있다. In the strip for immunochromatographic analysis, the binding molecule contained in the conjugate pad may be labeled with a metal particle, a latex particle, a fluorescent substance, or an enzyme. As an example, the metal particles may be gold particles.
보다 자세히 설명하면, 상기 면역크로마토그래피 분석용 스트립의 컨쥬게이트 패드에 본 발명의 결합 분자는 검출 가능하게 표식될 수 있다. 생분자들을 표식시키는데 사용 가능한 다양한 방법들이 당업자에게 잘 알려져 있고, 본 발명의 범주 내에서 고려된다. 본 발명에서 사용될 수 있는 표식 종류의 예로는 효소, 방사성 동위원소, 콜로이드금속, 형광 화합물, 화학발광 화합물 및 생발광 화합물이 있다. 통상적으로 사용되는 표식들은 형광물질(가령, 플루레신, 로다민, 텍사스 레드 등), 효소(가령, 고추냉이 퍼옥시다아제, 베타-갈락토시다아제, 알칼리포스파타 아제), 방사성 동위원소(가령, 32P 또는 125I), 바이오틴, 디곡시게닌, 콜로이드 금속, 화학발광 또는 생발광 화합물(가령, 디옥세탄, 루미놀 또는 아크리디늄)을 포함한다. 효소 또는 바이오티닐기의 공유 결합법, 요오드화법, 인산화법, 바이오틴화법 등과 같은 표식 방법들이 당 분야에 잘 알려져 있다. 검출 방법들로는 오토라디오그래피, 형광 현미경, 직접 및 간접 효소반응 등이 있으며, 이에 제한되지는 않는다. 통상적으로 사용되는 검출 분석법으로는 방사성 동위원소 또는 비-방사성 동위원소 방법이 있다. 이들은 그 중에서도 웨스턴블롯팅, 오버레이-분석법, RIA(Radioimmuno Assay) 및 IRMA(ImmuneRadioimmunometric Assay), EIA(Enzyme Immuno Assay), ELISA(Enzyme Linked Immuno Sorbent Assay), FIA(Fluorescent Immuno Assay) 및 CLIA(Chemioluminescent Immune Assay)이 있다.More specifically, the binding molecule of the present invention may be detectably labeled on the conjugate pad of the strip for immunochromatographic analysis. The various methods available for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention. Examples of label types that can be used in the present invention include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds. Commonly used markers include fluorescent substances (eg, fluorescein, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, beta-galactosidase, alkaline phosphatase), radioactive isotopes (eg, 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (eg, dioxetane, luminol or acridinium). Labeling methods such as covalent bonding of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are well known in the art. Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, and the like. Commonly used detection assays include radioactive isotope or non-radioactive isotope methods. These include, among others, Western blotting, overlay-assay, Radioimmuno Assay (RIA) and ImmuneRadioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (FIA) and Chemioluminescent Immune (CLIA). Assay).
상기 면역크로마토그래피 분석용 스트립에 있어서, 상기 검출은 육안, 광학, 전기화학, 또는 전기전도도에 의해 판독할 수 있으나, 이에 한정되는 것은 아니다. In the immunochromatographic analysis strip, the detection may be read by visual, optical, electrochemical, or electrical conductivity, but is not limited thereto.
또한, 본 발명의 다른 일 구체예로, 본 발명은 상기 면역크로마토그래피 분석용 스트립을 포함하는, 사스-코로나바이러스 감염증(COVID-19)의 진단용 키트를 제공한다. In another embodiment of the present invention, the present invention provides a kit for diagnosis of SARS-coronavirus infection (COVID-19), comprising the strip for immunochromatographic analysis.
또한, 본 발명의 다른 일 구체예로, 본 발명은 상기 결합 분자를 포함하는, 사스-코로나바이러스 감염증(COVID-19)의 진단용 키트를 제공한다. In another embodiment of the present invention, the present invention provides a kit for diagnosis of SARS-coronavirus infection (COVID-19), comprising the binding molecule.
본 발명의 다른 일 구체예로, 상기 진단용 키트는 코로나바이러스의 뉴클레오캡시드 단백질(Nucleocapsid protein, N protein)에 결합하는 결합 분자를 추가로 포함할 수 있다. 상기 코로나바이러스는 사스-코로나바이러스-2(SARS-CoV-2), 인간 코로나바이러스 229E (HCoV-229E), 인간 코로나바이러스 OC43 (HCoV-OC43), 중증급성호흡기증후군 코로나바이러스 (SARS-CoV), 인간 코로나바이러스 NL63 (HCoV-NL63), 인간 코로나바이러스 HKU1 및 중동호흡기증후군 코로나바이러스 (MERS-CoV)로 구성된 군으로부터 선택된 어느 하나일 수 있으나, 이에 한정되는 것은 아니다.In another embodiment of the present invention, the diagnostic kit may further include a binding molecule that binds to the nucleocapsid protein (N protein) of the coronavirus. Said coronavirus is SARS-coronavirus-2 (SARS-CoV-2), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), It may be any one selected from the group consisting of human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), but is not limited thereto.
본 발명의 진단용 키트는 샘플과 상기 결합 분자를 접촉시킨 후, 반응을 확인하여 SARS-CoV-2의 존재 여부를 검출하는 데 사용될 수 있다. 상기 샘플은 대상의 가래, 침, 혈액, 땀, 폐 세포, 폐 조직의 점액, 호흡기 조직 및 타액으로 이루어진 군으로부터 선택된 어느 하나일 수 있으나, 이에 한정되지 않으며 당업자에게 알려진 통상적인 방법으로 샘플 준비가 가능하다.The diagnostic kit of the present invention may be used to detect the presence or absence of SARS-CoV-2 by contacting a sample with the binding molecule and then confirming a reaction. The sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, lung tissue mucus, respiratory tissue, and saliva, but is not limited thereto, and the sample can be prepared by a conventional method known to those skilled in the art. possible.
또한, 본 발명의 다른 일 구체예는 In addition, another embodiment of the present invention is
a) 상기 결합 분자; 및a) said binding molecule; and
b) 용기b) container
를 포함하는 SARS-CoV-2로 인하여 발생하는 질환의 진단용 키트를 제공한다.It provides a kit for diagnosing diseases caused by SARS-CoV-2, comprising:
본 발명의 진단용 키트에 있어서, 키트 용기에는 고체 담체가 포함될 수 있다. 본 발명의 항체는 고체 담체에 부착될 수 있고, 이와 같은 고체 담체는 다공성 또는 비다공성, 평면 또는 비평면일 수 있다.In the diagnostic kit of the present invention, the kit container may contain a solid carrier. Antibodies of the invention may be attached to a solid carrier, which may be porous or non-porous, planar or non-planar.
또한, 본 발명의 다른 일 구체예로, 본 발명은 상기 진단용 키트를 이용하여 사스-코로나바이러스-2(SARS-CoV-2)를 검출하는 방법을 제공한다. In another embodiment of the present invention, the present invention provides a method for detecting SARS-coronavirus-2 (SARS-CoV-2) using the diagnostic kit.
또한, 본 발명의 다른 일 구체예로, 본 발명은 상기 진단용 키트를 이용하여 사스-코로나바이러스 감염증(COVID-19)을 진단하는 방법을 제공한다. In another embodiment of the present invention, the present invention provides a method for diagnosing SARS-coronavirus infection (COVID-19) using the diagnostic kit.
이하 본 발명에서 사용되는 용어를 다음과 같이 정의한다.Hereinafter, terms used in the present invention are defined as follows.
본 발명에서 사용되는 용어 "결합 분자"는 키메라, 인간화 또는 인간 단일클론 항체와 같은 단일클론 항체를 포함하는 온전한(intact)이뮤노글로블린(immunoglobulin), 또는 항원에 결합하는 이뮤노글로블린인 항원-결합 단편을 포함한다. 예를 들면 SARS-CoV-2의 스파이크 단백질(spike protein)과 결합에 있어서, 온전한(intact) 이뮤노글로블린과 경쟁하는 이뮤노글로블린 단편을 포함하는 가변성 도메인, 효소, 수용체, 단백질을 뜻한다. 구조와는 상관없이 항원-결합 단편은 온전한(intact) 이뮤노글로블린에 의해 인식된 동일한 항원과 결합된다. 항원-결합 단편은 항체의 아미노산 서열의 2개 이상의 연속기, 20개 이상의 연속 아미노산 잔기, 25개 이상의 연속 아미노산 잔기, 30개 이상의 연속 아미노산 잔기, 35개 이상의 연속 아미노산 잔기, 40개 이상의 연속 아미노산 잔기, 50개 이상의 연속 아미노산 잔기, 60개 이상의 연속 아미노산 잔기, 70개 이상의 연속 아미노산 잔기, 80개 이상의 연속 아미노산 잔기, 90개 이상의 연속 아미노산 잔기, 100개 이상의 연속 아미노산 잔기, 125개 이상의 연속 아미노산 잔기, 150개 이상의 연속 아미노산 잔기, 175개 이상 연속 아미노산 잔기, 200개 이상의 연속 아미노산 잔기, 또는 250개 이상의 연속 아미노산 잔기의 아미노산 서열을 포함하는 펩티드 또는 폴리펩티드를 포함할 수 있다. As used herein, the term "binding molecule" refers to an intact immunoglobulin, including a monoclonal antibody such as a chimeric, humanized or human monoclonal antibody, or an antigen-binding immunoglobulin that binds to an antigen. Includes fragments. For example, in binding to the spike protein of SARS-CoV-2, it refers to a variable domain, an enzyme, a receptor, or a protein comprising an immunoglobulin fragment that competes with an intact immunoglobulin. Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact immunoglobulin. An antigen-binding fragment comprises at least two contiguous groups of the amino acid sequence of the antibody, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, At least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, 150 a peptide or polypeptide comprising an amino acid sequence of at least two contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.
"항원-결합 단편"은 특히 Fab, F(ab'), F(ab')2, Fv, dAb, Fd, 상보성 결정 영역(CDR) 단편, 단일-쇄 항체(scFv-Fc), 2가(bivalent) 단일-쇄 항체, 단일-쇄 파지 항체, 디아바디(diabody), 트리아바디, 테트라바디, 폴리펩티드로의 특정 항원에 결합하기에 충분한 이뮤노글로블린의 하나 이상의 단편을 함유하는 폴리펩티드 등을 포함한다. 상기 단편은 합성으로 또는 완전한 이뮤노글로블린의 효소적 또는 화학적 분해에 의해 생성되거나, 재조합 DNA 기술에 의해 유전공학적으로 생성될 수 있다. 생성 방법은 당업계에 잘 알려져 있다."Antigen-binding fragments" are inter alia Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv-Fc), bivalent ( bivalent) single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind a particular antigen to the polypeptide, and the like. . The fragment may be produced synthetically or by enzymatic or chemical digestion of complete immunoglobulins, or may be genetically engineered by recombinant DNA technology. Methods of production are well known in the art.
본 발명에서 사용되는 "약제학적으로 허용 가능한 부형제"라는 용어는 용인 가능한 또는 편리한 투약 형태를 제조하기 위한 약물, 제제 또는 항체와 같은 활성 분자로 조합되는 불활성 물질을 의미한다. 약제학적으로 허용 가능한 부형제는 비독성이거나, 적어도 독성이 사용된 용량 및 농도에서 수용자에게 이의 의도된 용도를 위해 허용될 수 있는 부형제이고, 약물, 제제 또는 결합 분제를 포함하는 제형화의 다른 성분과 양립할 수 있다.As used herein, the term "pharmaceutically acceptable excipient" refers to an inert substance that is combined into an active molecule, such as a drug, agent, or antibody, to prepare an acceptable or convenient dosage form. Pharmaceutically acceptable excipients are excipients which are non-toxic, or at least toxic, acceptable for their intended use in recipients at the dosages and concentrations employed, and with the other ingredients of the formulation, including the drug, agent, or binding agent; compatible.
본원에 기재된 상기 각 특징들은 조합되어 사용될 수 있으며, 상기 각 특징들이 특허청구범위의 서로 다른 종속항에 기재된다는 사실은 이들이 조합되어 사용될 수 없음을 나타내는 것은 아니다. Each of the features described herein may be used in combination, and the fact that each of the features is recited in different dependent claims of the claims does not indicate that they cannot be used in combination.
본 발명의 결합 분자는 SARS-CoV-2의 S 단백질에 대한 우수한 결합 능력을 가짐으로써 우수한 진단 효과를 나타내므로, 사스-코로나바이러스 감염증(COVID-19)에 대한 신속한 진단에 매우 유용하다. Since the binding molecule of the present invention exhibits an excellent diagnostic effect by having an excellent binding ability to the S protein of SARS-CoV-2, it is very useful for rapid diagnosis of SARS-coronavirus infection (COVID-19).
도 1은 본 발명의 일 구체예에 따른 진단 키트의 면역크로마토그래피 분석용 스트립의 작동 원리를 도시한 모식도이다. 도 1은 SARS-CoV-2의 표면 단백질 S에 결합하는 항체 Anti-S 가 탑재된 면역크로마토그래피 분석용 스트립에 관한 설명이다. 1 is a schematic diagram illustrating the principle of operation of a strip for immunochromatographic analysis of a diagnostic kit according to an embodiment of the present invention. 1 is a description of a strip for immunochromatographic analysis loaded with the antibody Anti-S that binds to the surface protein S of SARS-CoV-2.
도 2는 SARS-CoV-2의 표면 단백질 S에 결합하는 항체 Anti-S 및 SARS-CoV-2 내에 존재하는 단백질 N에 결합하는 항체 Anti-N 두 항체가 탑재된 면역크로마토그래피 분석용 스트립에 관한 설명이다. 2 is an immunochromatographic analysis strip equipped with two antibodies, Anti-S, an antibody that binds to the surface protein S of SARS-CoV-2, and Anti-N, an antibody that binds to protein N present in SARS-CoV-2. explanation.
도 3a 내지 3r은 본 발명의 일 구체예에 따른 본 발명 항체 No. 1 내지 No. 18의 표면 플라스몬 공명(Surface Plasmon Resonance) 기술을 이용한 항원-항체 결합 친화도 측정 결과를 각각 나타낸 것이다.3a to 3r show the present invention antibody No. according to one embodiment of the present invention. 1 to No. 18 shows the results of antigen-antibody binding affinity measurement using Surface Plasmon Resonance technology, respectively.
이하 본 발명을 실시예를 통해 상세히 설명한다. 그러나 하기 실시예들은 본 발명의 내용을 예시하는 것일 뿐 발명의 범위가 실시예에 의해 한정되지 않는다. 본 발명에서 인용된 문헌은 본 발명의 명세서에 참조로서 통합된다.Hereinafter, the present invention will be described in detail through examples. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited by the examples. The documents cited herein are incorporated herein by reference.
실시예 1: SARS-CoV-2 회복 환자의 혈액으로부터 PBMC 분리Example 1: Isolation of PBMCs from the blood of patients recovering from SARS-CoV-2
혈액의 공여자는 2020년 SARS-CoV-2에 감염되었음을 확진 받고 치료를 통하여 더 이상 바이러스가 검출되지 않은 사람들을 대상으로 하였으며 공여자 선정과 채혈 과정은 임상시험심사 위원회(IRB)의 승인을 받고 이루어졌다. 공여자 선정 후 약 30㎖의 전혈을 채혈하여 Ficoll-PaqueTM PLUS(GE Healthcare) 방법을 사용하여 PBMC(peripheral blood mononuclear cell)를 분리하였다. 분리된 PBMC는 인산 완충용액으로 2회 세척한 후, 냉동 배지(RPMI:FBS:DMSO = 5:4:1)로 1x107cells/㎖ 농도로 맞추어 액체 질소 탱크(Liquid Nitrogen Tank)에 보관하였다.The donor of blood was people who were confirmed to have been infected with SARS-CoV-2 in 2020 and no longer had the virus detected through treatment. . After selecting a donor, about 30 ml of whole blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque TM PLUS (GE Healthcare) method. The separated PBMCs were washed twice with phosphate buffer, and then stored in a liquid nitrogen tank (Liquid Nitrogen Tank) at a concentration of 1x10 7 cells/ml with a freezing medium (RPMI:FBS:DMSO = 5:4:1).
실시예 2: 항체 디스플레이된 파아지 라이브러리(phage library) 제작Example 2: Preparation of antibody-displayed phage library
실시예 1 에서 분리한 PBMC에서 Trizol Reagent(Invitrogen)를 이용하여 전체 RNA를 추출한 후 the SuperScriptTM III First-Strand cDNA synthesis system(Invitrogen, USA)을 이용하여 cDNA를 합성하였다.Total RNA was extracted from the PBMC isolated in Example 1 using Trizol Reagent (Invitrogen), and then cDNA was synthesized using the SuperScript™ III First-Strand cDNA synthesis system (Invitrogen, USA).
합성된 cDNA로부터 항체 라이브러리의 제작은 선행문헌을 참고하였다(Barbas C. et. al. Phage display a laboratory manual. 2001. CSHL Press). 간단히 기술하면 합성된 cDNA로부터 High fidelity Taq polymerase(Roche)와 디제너레이티브 프라이머 세트(degenerative primer set)(IDT)을 이용하여 항체의 경쇄와 중쇄의 가변 영역을 PCR(polymerase chain reaction) 방법으로 증폭하였다. 분리된 경쇄와 중쇄의 가변영역 절편들이 무작위 조합으로 하나의 서열로서 연결되도록 overlap PCR 방법으로 scFv 형태의 유전자로 만들어 증폭한 후 제한 효소로 절단하고 1% 아가로스 겔 전기영동(agarose gel electrophoresis)과 gel extraction kit(Qiagen) 방법을 사용하여 scFv를 분리하였다. 파아지 벡터도 동일한 제한 효소로 절단하고 분리한 뒤 상기 scFv 유전자와 섞고 T4 DNA ligase(New England Biolab)를 넣은 후 16℃에서 12시간 이상 반응하였다. 반응액을 ER2738 수용성 세포(competent cell)과 섞고 전기천공(electroporation) 방법으로 형질 전환하였다. 형질 전환된 ER2738은 진탕 배양 후 VCSM13 helper phage(Agilent Technologies)를 넣고 12시간 이상 배양하였다.For the preparation of an antibody library from the synthesized cDNA, the literature was referred to (Barbas C. et. al. Phage display a laboratory manual. 2001. CSHL Press). In brief, the variable regions of the light and heavy chains of the antibody are amplified by PCR (polymerase chain reaction) method using high fidelity Taq polymerase (Roche) and degenerative primer set (IDT) from the synthesized cDNA. did So that the variable region fragments of the separated light and heavy chains are connected as one sequence in a random combination, the scFv-type gene is created and amplified by the overlap PCR method, cut with restriction enzymes, and 1% agarose gel electrophoresis and scFv was isolated using the gel extraction kit (Qiagen) method. The phage vector was also digested with the same restriction enzyme and separated, mixed with the scFv gene, added with T4 DNA ligase (New England Biolab), and reacted at 16° C. for more than 12 hours. The reaction solution was mixed with ER2738 competent cells and transformed by electroporation. Transformed ER2738 was cultured with shaking, followed by VCSM13 helper phage (Agilent Technologies) and incubated for more than 12 hours.
실시예 3: 파아지 효소 면역분석을 이용한 선별Example 3: Selection using phage enzyme immunoassay
실시예 2 에서 제작한 파아지 라이브러리 배양액을 원심 분리하여 숙주세포를 제거한 뒤 4% PEG와 0.5M NaCl을 넣고 원심 분리하여 파아지를 침강 시키고 상층액을 제거하였다. 침강된 파아지를 1% BSA/TBS 에 희석하여 파아지 라이브러리를 얻고, 이후 다양한 SARS-CoV-2 Spike 단백질(이하, S 단백질)들에 대한 결합 및 해리 반응으로 패닝(panning)을 독립적으로 진행하여 SARS-CoV-2 S 단백질에 대한 결합능력을 갖는 scFv-파아지를 분리하였다. 예를 들어, SARS-CoV-2 S 단백질의 한 부분인 RBD(Receptor binding domain) 영역(residues N331 to V524 on S1 glycoprotein)이 결합된 ELISA 플레이트(plate)에 파아지 라이브러리를 넣고 상온에서 2시간 동안 반응시켰다. 반응액을 제거한 뒤 ELISA 플레이트를 0.05% tween 20이 포함된 PBS로 세척한 뒤 60㎕ 의 0.1M glycine-HCl(pH 2.2)를 넣어 항원에 결합한 scFv-파아지를 떼어내고 2M Tris(pH 9.1)를 이용하여 중화 하였다. 중화된 scFv-파아지는 ER2738에 감염시킨 뒤 보조(helper) 파아지를 넣고 배양하여 다음 번 패닝에 사용하였다. 감염시킨 ER2738의 일부는 보조 파아지를 넣기 전에 LB 플레이트에 도말하여 다음날 콜로니(colony)를 얻었다.The phage library culture medium prepared in Example 2 was centrifuged to remove host cells, 4% PEG and 0.5 M NaCl were added thereto, centrifuged to settle the phage, and the supernatant was removed. The precipitated phage was diluted in 1% BSA/TBS to obtain a phage library, and then, the binding and dissociation reactions for various SARS-CoV-2 Spike proteins (hereinafter, S protein) were independently performed for panning to SARS. -ScFv-phage having binding ability to -CoV-2 S protein was isolated. For example, put the phage library on an ELISA plate to which the receptor binding domain (RBD) region (residues N331 to V524 on S1 glycoprotein), a part of the SARS-CoV-2 S protein, is bound, and react at room temperature for 2 hours. made it After removing the reaction solution, the ELISA plate was washed with PBS containing 0.05 % tween 20, and 60 μl of 0.1M glycine-HCl (pH 2.2) was added to remove the antigen-bound scFv-phage, and 2M Tris (pH 9.1) was added. was used for neutralization. After the neutralized scFv-phage was infected with ER2738, a helper phage was added and cultured to be used for the next panning. A portion of the infected ER2738 was plated on an LB plate before adding auxiliary phages, and colonies were obtained the next day.
각 패닝 마다 형성된 콜로니는 96-well deep well plate(Axygen)에 담긴 배양액에 넣어 진탕 배양하고 OD600이 0.7 이상의 값이 되었을 때 보조 파아지를 넣은 후 37℃에서 12시간 이상 진탕 배양하였다. 배양액을 원심 분리하여 숙주세포를 제거하고 scFv-파아지를 포함하는 상등액을 준비하였다.Colonies formed for each panning were put into a culture solution contained in a 96-well deep well plate (Axygen) and cultured with shaking. The culture medium was centrifuged to remove host cells, and a supernatant containing scFv-phages was prepared.
준비된 scFv-파아지 상등액을 6% BSA/PBS 와 1:1 희석한 뒤 SARS-CoV-2 S 단백질들을 흡착 후 블로킹(blocking)한 96-웰 마이크로타이터 플레이트의 각 웰에 넣고 37℃에서 2시간 동안 정치하였다. 각 웰을 0.05% Tween 20가 포함된 PBS로 3회 세척한 뒤 HRP(겨자무과산화효소, horseradish peroxidase)가 표지 된 항 M13 항체를 넣고 37℃에서 1시간 동안 정치하였다. 각 웰을 0.05% Tween 20가 포함된 PBS로 3회 세척한 뒤 ABTS(2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt)를 넣고 405nm 에서의 흡광도를 측정하여 SARS-CoV-2 S 항원 단백질에 대한 결합력을 갖는 scFv-파아지를 선별하였다.The prepared scFv-phage supernatant was diluted 1:1 with 6% BSA/PBS, and then put into each well of a 96-well microtiter plate in which SARS-CoV-2 S proteins were adsorbed and blocked and placed at 37°C for 2 hours. been in politics for a while. Each well was washed three times with PBS containing 0.05% Tween 20, and then HRP (mustard peroxidase, horseradish peroxidase)-labeled anti-M13 antibody was added thereto and left at 37°C for 1 hour. After washing each well 3 times with PBS containing 0.05% Tween 20, add ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) and measure the absorbance at 405 nm to SARS- ScFv-phages having binding affinity to CoV-2 S antigen protein were selected.
실시예 4: scFv-Fc 항체 분절의 결합능 확인Example 4: Confirmation of binding ability of scFv-Fc antibody fragments
실시예 3에서 선별된 scFv-파아지는 이후 콜로니를 진탕 배양하여 DNA를 얻은 다음 항체 가변영역에 대한 서열을 분석하였다. 이 중에서 아미노산 서열로서 중복된 클론을 제외하고 선정된 scFv-파아지를 후보 항체 동물 세포 주에서의 발현능력을 평가하기 위하여 scFv 항체 분절(scFv-Fc) 형태로 벡터에 클로닝하였다. 형질도입 시약(Transfection reagent)을 이용하여 CHO 세포에 형질 감염시켜 발현시킨 후 그 배양액을 이용하여 scFv 항체 분절의 SARS-CoV-2의 S 단백질 2종에 대한 결합 능력을 ELISA로 확인하였다. 간단하게는, ELISA 플레이트에 SARS-CoV-2 S 단백질들을 붙이고 발현된 항체 분절을 넣어주었다. 결합하지 않은 항체를 0.05% Tween 20가 포함된 PBS로 씻어낸 후 HRP(겨자무과산화효소, horseradish peroxidase)가 결합된 항-인간 IgG 항체를 이용하여 항원과 결합한 항체 분절들을 선별 및 평가하였다. The scFv-phage selected in Example 3 was then cultured with shaking to obtain DNA and then sequenced for the antibody variable region was analyzed. Among them, the selected scFv-phages were cloned into a vector in the form of an scFv antibody fragment (scFv-Fc) in order to evaluate the expression ability in the candidate antibody animal cell line, except for duplicated clones as an amino acid sequence. CHO cells were transfected and expressed using a transfection reagent, and the ability of the scFv antibody fragment to bind to two S proteins of SARS-CoV-2 was confirmed by ELISA using the culture medium. Briefly, SARS-CoV-2 S proteins were attached to an ELISA plate and the expressed antibody fragment was added. After washing the unbound antibody with PBS containing 0.05% Tween 20, HRP (horseradish peroxidase)-conjugated anti-human IgG antibody was used to select and evaluate antigen-bound antibody fragments.
그 결과, [표 3]의 17종 항체 분절들은 다른 코로나바이러스들(메르스 코로나바이러스(MERS-CoV), 사스코로나바이러스(SARS-CoV), HCoV 코로나바이러스)에 대해서는 결합하지 않았으며 (데이터 표시하지 않음), SARS-CoV-2의 S 단백질들에 대해서는 강한 결합력을 가지는 것을 확인하였다. 양성 대조군 항체로 SARS-CoV-2 S 단백질에 강하게 결합한다고 알려진 항체를 이용하였으며, 결합력은 양성 대조군 항체 대비하여 상대적인 값으로 결합력을 표기하였다(Xiaolong Tian et al., Emerg Microbes Infect. 2020 Feb 17;9(1):382-385). 하기 표 3에서 No.는 표 1, 2에 기재된 각 결합 분자의 No.와 동일한 결합 분자를 지칭한다.As a result, 17 kinds of antibody fragments in [Table 3] did not bind to other coronaviruses (MERS-CoV, SARS-CoV, HCoV coronavirus) (data display) not), it was confirmed that the SARS-CoV-2 has a strong binding force to the S proteins. As a positive control antibody, an antibody known to strongly bind to SARS-CoV-2 S protein was used, and the binding strength was expressed as a relative value compared to the positive control antibody (Xiaolong Tian et al., Emerg Microbes Infect. 2020 Feb 17; 9(1):382-385). In Table 3 below, No. refers to the same binding molecule as No. of each binding molecule shown in Tables 1 and 2.
| No.No. | 결합력cohesion | No.No. | 결합력cohesion |
| 1One | 1.481.48 | 1111 | 2.092.09 |
| 22 | 1.431.43 | 1212 | 2.082.08 |
| 33 | 1.371.37 | 1313 | 2.512.51 |
| 44 | 1.471.47 | 1414 | 1.431.43 |
| 55 | 2.862.86 | 1515 | 1.71.7 |
| 66 | 2.292.29 | 1616 | 1.451.45 |
| 77 | 1.821.82 | 1717 | 1.461.46 |
| 88 | 1.41.4 | 1818 | 0.850.85 |
| 99 | 1.421.42 | ||
| 1010 | 1.471.47 | 양성 대조군 항체positive control antibody | 1.001.00 |
상기 scFv-Fc 항체 분절의 유전정보를 이용하여 추가로 완전 인간 항체(Full IgG)로 변환하였다.Using the genetic information of the scFv-Fc antibody fragment, it was further converted into a full human antibody (Full IgG).
실시예 5: 표면 플라스몬 공명 기술을 이용한 항원-항체 결합친화도 결정Example 5: Determination of antigen-antibody binding affinity using surface plasmon resonance technology
표면 플라스몬 공명(Surface Plasmon Resonance) 검정은 정반응 속도 상수 및 역반응 속도 상수의 역학적 측정에 의해 항체의 결합친화도를 결정한다.The Surface Plasmon Resonance assay determines the binding affinity of an antibody by kinetic measurements of forward and reverse rate constants.
실시예 4에서 scFv-Fc 항체 분절을 변환한 완전 인간 항체(Full IgG)의 SARS-CoV-2의 S RBD 단백질에 대한 결합친화도 값(KD)을 확인하기 위해 Biacore T200 장비로 SPR(표면 플라스몬 공명; Surface Plasmon Resonance) 분석을 진행하였다. 간단하게는 CM5 칩에 SARS-CoV-2 S 단백질들을 붙이고 HBS-EP 버퍼 (pH 7.4)를 사용하여 항체 분절을 연속적으로 희석하여 5개 농도를 만들었다. 각 농도를 SARS-CoV-2 S 단백질이 붙어있는 CM5 칩에 흘려준 다음, HBS-EP 버퍼를 주입하여 결합 및 해리 곡선을 생성해 이를 Biacore T200 콘트롤 소프트웨어, Biacore T200 이벨루에이션 소프트웨어를 통해 결합 친화도(KD)를 측정하였다(표 4 및 도 3a 내지 3r). 항체와 표적 항원 사이의 반응에 대한 평형 해리 상수 KD(M)는 반응 속도 상수로부터 다음의 등식에 의해 계산하였다: KD = Kd/Ka. 결합은 시간과 반응 속도 상수의 함수를 계산하여 기록한다. 하기 표 4에서 No.는 표 1, 2에 기재된 각 결합 분자의 No.와 동일한 결합 분자를 지칭한다.In Example 4, in order to confirm the binding affinity value (K D ) of SARS-CoV-2 for the S RBD protein of the full human antibody (Full IgG) transformed with the scFv-Fc antibody fragment, SPR (surface Plasmon resonance; Surface Plasmon Resonance) analysis was performed. Briefly, SARS-CoV-2 S proteins were attached to a CM5 chip and the antibody fragments were serially diluted using HBS-EP buffer (pH 7.4) to make 5 concentrations. Each concentration was flowed to the CM5 chip to which the SARS-CoV-2 S protein was attached, and then HBS-EP buffer was injected to generate association and dissociation curves. Figure (K D ) was measured (Table 4 and Figures 3A-3R). The equilibrium dissociation constant K D (M) for the reaction between the antibody and the target antigen was calculated from the reaction rate constant by the following equation: KD = Kd/Ka. Binding is recorded as a function of time and reaction rate constants. In Table 4 below, No. refers to the same binding molecule as the No. of each binding molecule shown in Tables 1 and 2.
표 4에 나타낸 바와 같이, No. 1 내지 No. 18 항체는 모두 SARS-CoV-2 S 항원에 대하여 높은 결합친화도를 나타내었다. As shown in Table 4, No. 1 to No. All 18 antibodies showed high binding affinity to the SARS-CoV-2 S antigen.
| No.No. | Ka (1/Ms)K a (1/Ms) | Kd (1/s)K d (1/s) | KD (M)K D (M) |
| 1One | 4.91.E+054.91.E+05 | 3.83.E-053.83.E-05 | 7.80.E-117.80.E-11 |
| 22 | 1.50.E+051.50.E+05 | 4.87.E-054.87.E-05 |
3.25.E-103.25. |
| 33 | 1.58.E+061.58.E+06 | 2.64.E-042.64.E-04 | 1.67.E-101.67.E-10 |
| 44 | 2.56.E+062.56.E+06 | 2.08.E-042.08.E-04 | 8.12.E-118.12.E-11 |
| 55 | 2.31.E+062.31.E+06 | 5.03.E-045.03.E-04 | 2.18.E-102.18.E-10 |
| 66 | 2.96.E+062.96.E+06 | 2.52.E-042.52.E-04 | 8.53.E-118.53.E-11 |
| 77 | 2.46.E+062.46.E+06 | 3.56.E-043.56.E-04 |
1.45.E-101.45. |
| 88 | 2.46.E+062.46.E+06 | 1.74.E-041.74.E-04 | 7.07.E-117.07.E-11 |
| 99 | 3.03.E+063.03.E+06 | 3.83.E-043.83.E-04 | 1.27.E-101.27.E-10 |
| 1010 | 2.20.E+062.20.E+06 | 1.67.E-041.67.E-04 | 7.57.E-117.57.E-11 |
| 1111 | 5.61.E+055.61.E+05 | 1.87.E-051.87.E-05 | 3.33.E-113.33.E-11 |
| 1212 | 9.83.E+059.83.E+05 | 2.98.E-052.98.E-05 | 3.04.E-113.04.E-11 |
| 1313 | 1.82.E+061.82.E+06 | 1.56.E-041.56.E-04 | 8.57.E-118.57.E-11 |
| 1414 | 3.07.E+063.07.E+06 | 4.80.E-034.80.E-03 | 1.56.E-091.56.E-09 |
| 1515 | 1.85.E+061.85.E+06 | 1.48.E-041.48.E-04 | 8.03.E-118.03.E-11 |
| 1616 | 2.89.E+062.89.E+06 | 1.14.E-031.14.E-03 | 4.34.E-104.34.E-10 |
| 1717 | 3.81.E+053.81.E+05 | 1.11.E-031.11.E-03 | 2.92.E-092.92.E-09 |
| 1818 | 1.65.E+061.65.E+06 | 3.30.E-033.30.E-03 | 2.00.E-092.00.E-09 |
Claims (18)
- 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 사스-코로나바이러스 감염증(COVID-19)의 진단용 결합 분자.A binding molecule for diagnosis of SARS-coronavirus infection (COVID-19) that binds to the spike protein (S protein) on the surface of SARS-CoV-2.
- 제1항에 있어서,According to claim 1,상기 결합 분자는 사스-코로나바이러스-2 표면의 스파이크 단백질의 RBD(Receptor binding domain) 영역에 결합하는, 결합 분자.Wherein the binding molecule binds to the receptor binding domain (RBD) region of the spike protein on the surface of the SARS-coronavirus-2 surface.
- 제1항에 있어서,The method of claim 1,상기 결합 분자는 하기 특성 중 어느 하나 이상을 갖는, 결합 분자: The binding molecule has any one or more of the following properties:a) 1 x 10-8 M 이하의 평형 해리 상수(KD)로 사스-코로나바이러스-2 표면의 스파이크 단백질에 결합함; a) binds to the spike protein on the surface of SARS-coronavirus-2 with an equilibrium dissociation constant (K D ) of 1 x 10 -8 M or less;b) 1 x 104 1/Ms 이상의 결합 상수(Ka)로 사스-코로나바이러스-2 표면의 스파이크 단백질에 결합함; 또는b) binds to the spike protein on the surface of SARS-coronavirus-2 with a binding constant (Ka) greater than or equal to 1 x 10 4 1/Ms; orc) 1 x 10-2 1/s 이하의 해리 상수(Kd)로 사스-코로나바이러스-2 표면의 스파이크 단백질에 결합함.c) Binding to the spike protein on the surface of SARS-coronavirus-2 with a dissociation constant (Kd) of 1 x 10 -2 1/s or less.
- 제1항에 있어서,According to claim 1,상기 결합 분자는 표 1의 결합 분자로 이루어진 군으로부터 선택되는 어느 하나인, 결합 분자. The binding molecule is any one selected from the group consisting of the binding molecules of Table 1.
- 제1항에 있어서,According to claim 1,상기 결합 분자는 표 2의 결합 분자로 이루어진 군으로부터 선택되는 어느 하나인, 결합 분자. The binding molecule is any one selected from the group consisting of the binding molecules of Table 2.
- 제1항 내지 제5항 중 어느 한 항에 있어서, 6. The method according to any one of claims 1 to 5,상기 결합 분자는 scFv 절편, scFv-Fc 절편, Fab 절편, Fv 절편, 디아바디(diabody), 키메라 항체, 인간화 항체 또는 인간 항체인, 결합 분자.The binding molecule is an scFv fragment, an scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody or a human antibody.
- 제1항 내지 제5항 중 어느 한 항의 결합 분자에 추가적으로 하나 이상의 태그가 결합된 이뮤노컨쥬게이트.The immunoconjugate to which one or more tags are additionally bound to the binding molecule of any one of claims 1 to 5.
- 제1항 내지 제5항 중 어느 한 항의 결합 분자를 암호화하는 핵산 분자.A nucleic acid molecule encoding the binding molecule of any one of claims 1-5.
- 제8항의 핵산 분자가 삽입된 발현 벡터. An expression vector into which the nucleic acid molecule of claim 8 is inserted.
- 제9항의 발현 벡터로 형질전환된 세포주.A cell line transformed with the expression vector of claim 9 .
- 제10항에 있어서, 11. The method of claim 10,상기 세포주는 CHO 세포, F2N 세포, COS 세포, BHK 세포, 바우스(Bowes) 흑색종 세포, HeLa 세포, 911 세포, HT1080 세포, A549 세포, HEK 293 세포 및 HEK293T 세포로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 세포주.The cell line is any one selected from the group consisting of CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, A549 cells, HEK 293 cells and HEK293T cells. Characterized cell lines.
- 제1항 내지 제5항 중 어느 한 항의 결합 분자를 포함하는 사스-코로나바이러스 감염증(COVID-19)의 진단용 조성물.A composition for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule of any one of claims 1 to 5.
- 제12항에 있어서,13. The method of claim 12,상기 조성물은 멸균 주사용액, 동결건조(lyophilized) 제형, 사전 충전식 주사(pre-filled syringe) 용액제, 경구형 제형, 외용제 또는 좌제임을 특징으로 하는 조성물.The composition is a sterile injection solution, a lyophilized formulation, a pre-filled syringe solution, an oral formulation, a composition for external use or a suppository.
- 제1항 내지 제5항 중 어느 한 항의 결합 분자를 포함하는 사스-코로나바이러스 감염증(COVID-19)의 진단용 키트.A kit for diagnosis of SARS-coronavirus infection (COVID-19) comprising the binding molecule of any one of claims 1 to 5.
- 제14항에 있어서, 15. The method of claim 14,코로나바이러스의 뉴클레오캡시드 단백질(Nucleocapsid protein, N protein)에 결합하는 결합 분자를 추가로 포함함을 특징으로 하는, 진단용 키트,Diagnostic kit, characterized in that it further comprises a binding molecule that binds to the nucleocapsid protein (N protein) of the coronavirus,
- 제15항에 있어서, 16. The method of claim 15,상기 코로나바이러스는 사스-코로나바이러스-2(SARS-CoV-2), 인간 코로나바이러스 229E (HCoV-229E), 인간 코로나바이러스 OC43 (HCoV-OC43), 중증급성호흡기증후군 코로나바이러스 (SARS-CoV), 인간 코로나바이러스 NL63 (HCoV-NL63), 인간 코로나바이러스 HKU1 및 중동호흡기증후군 코로나바이러스 (MERS-CoV)로 구성된 군으로부터 선택된 어느 하나인 것을 특징으로 하는, 진단용 키트.Said coronavirus is SARS-coronavirus-2 (SARS-CoV-2), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), Human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 and Middle East respiratory syndrome coronavirus (MERS-CoV), characterized in that any one selected from the group consisting of, a diagnostic kit.
- 제14항의 진단용 키트를 이용하여 사스-코로나바이러스-2(SARS-CoV-2)를 검출하는 방법. A method of detecting SARS-coronavirus-2 (SARS-CoV-2) using the diagnostic kit of claim 14 .
- 제14항의 진단용 키트를 이용하여 사스-코로나바이러스 감염증(COVID-19)을 진단하는 방법.A method of diagnosing SARS-coronavirus infection (COVID-19) using the diagnostic kit of claim 14.
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| KR102205028B1 (en) * | 2020-03-22 | 2021-01-20 | (주)셀트리온 | A binding molecules able to neutralize SARS-CoV-2 |
| SG11202103404PA (en) | 2020-04-02 | 2021-04-29 | Regeneron Pharma | Anti-sars-cov-2-spike glycoprotein antibodies and antigen-binding fragments |
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