WO2022085905A1 - Antibody binding specifically to sars-cov-2 spike protein and use thereof - Google Patents

Antibody binding specifically to sars-cov-2 spike protein and use thereof Download PDF

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WO2022085905A1
WO2022085905A1 PCT/KR2021/010133 KR2021010133W WO2022085905A1 WO 2022085905 A1 WO2022085905 A1 WO 2022085905A1 KR 2021010133 W KR2021010133 W KR 2021010133W WO 2022085905 A1 WO2022085905 A1 WO 2022085905A1
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seq
light chain
heavy chain
cov
sars
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Korean (ko)
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이상필
신지영
윤선하
최윤선
윤지혜
노한별
박재은
김수영
윤정호
이우정
구예림
박민영
양소영
김혜난
이동중
임주리
이재민
송영자
이한승
박범찬
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주식회사 와이바이오로직스
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6839—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses
    • A61K47/6841—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses the antibody targeting a RNA virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • 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 an antibody that specifically binds to the membrane protein spike of novel coronavirus SARS-CoV-2 and uses thereof.
  • the novel coronavirus infection (COVID-19) is a respiratory infection that occurred on a large scale in Wuhan, China in December 2019.
  • the fatality rate is about 5%, which is weaker than the existing SARS (about 9.6%) or MERS (about 34.4%), but it is highly contagious from person to person and spread rapidly worldwide, and in March 2020, it was declared a pandemic by the WHO.
  • SARS-CoV-2 (or 2019-nCoV), a virus that causes novel coronavirus infection, has low genomic homology with existing SARS (SARS-CoV) and MERS (MERS-CoV). There are currently no treatments available. Currently, few of the therapeutic antibodies targeting SARS-CoV-2 are in the clinical stage, and it is necessary to discover antibodies derived from infected patients or to secure various antibodies applicable to mutants. In addition, it is necessary to concurrently administer antibodies having different epitopes in consideration of viral variability.
  • Non-Patent Document 1 Haibo Zhang et.al., Intensive Care Med., 46(4):586-590, 2020
  • the present inventors developed antibodies that specifically bind to the membrane protein spike of SARS-CoV-2.
  • the present invention was completed by confirming that the antibodies can be used as a treatment for novel coronavirus infection by completely blocking the binding of the human cell receptor angiotensin converting enzyme-2 (ACE2) to the SARS-CoV-2 spike.
  • ACE2 human cell receptor angiotensin converting enzyme-2
  • one aspect of the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to the membrane protein spike of SARS-CoV-2 and exhibit a preventive or therapeutic effect on novel coronavirus infection.
  • an antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 spike protein comprising any one CDR (complementarity determining region) combination selected from the following group:
  • heavy chain CDR1 of SEQ ID NO: 1 heavy chain CDR2 of SEQ ID NO: 2
  • heavy chain CDR3 of SEQ ID NO: 3 light chain CDR1 of SEQ ID NO: 4
  • light chain CDR2 of SEQ ID NO: 5 light chain CDR3 of SEQ ID NO: 6;
  • heavy chain CDR1 of SEQ ID NO: 7 heavy chain CDR2 of SEQ ID NO: 8
  • heavy chain CDR3 of SEQ ID NO: 9 light chain CDR1 of SEQ ID NO: 10
  • light chain CDR2 of SEQ ID NO: 11 light chain CDR3 of SEQ ID NO: 12;
  • heavy chain CDR1 of SEQ ID NO: 13 heavy chain CDR2 of SEQ ID NO: 14
  • heavy chain CDR3 of SEQ ID NO: 15 light chain CDR1 of SEQ ID NO: 16
  • light chain CDR2 of SEQ ID NO: 17 light chain CDR3 of SEQ ID NO: 18;
  • heavy chain CDR1 of SEQ ID NO: 19 heavy chain CDR2 of SEQ ID NO: 20
  • heavy chain CDR3 of SEQ ID NO: 21 light chain CDR1 of SEQ ID NO: 22, light chain CDR2 of SEQ ID NO: 23, light chain CDR3 of SEQ ID NO: 24;
  • heavy chain CDR1 of SEQ ID NO: 25 heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 28, light chain CDR2 of SEQ ID NO: 29, light chain CDR3 of SEQ ID NO: 30;
  • heavy chain CDR1 of SEQ ID NO: 31 heavy chain CDR2 of SEQ ID NO: 32, heavy chain CDR3 of SEQ ID NO: 33, light chain CDR1 of SEQ ID NO: 34, light chain CDR2 of SEQ ID NO: 35, light chain CDR3 of SEQ ID NO: 36;
  • heavy chain CDR1 of SEQ ID NO: 37 heavy chain CDR2 of SEQ ID NO: 38, heavy chain CDR3 of SEQ ID NO: 39, light chain CDR1 of SEQ ID NO: 40, light chain CDR2 of SEQ ID NO: 41, light chain CDR3 of SEQ ID NO: 42;
  • heavy chain CDR1 of SEQ ID NO: 43 heavy chain CDR2 of SEQ ID NO: 44, heavy chain CDR3 of SEQ ID NO: 45, light chain CDR1 of SEQ ID NO: 46, light chain CDR2 of SEQ ID NO: 47, light chain CDR3 of SEQ ID NO: 48;
  • heavy chain CDR1 of SEQ ID NO: 49 heavy chain CDR2 of SEQ ID NO: 50, heavy chain CDR3 of SEQ ID NO: 51, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 53, light chain CDR3 of SEQ ID NO: 54;
  • heavy chain CDR1 of SEQ ID NO: 67 heavy chain CDR2 of SEQ ID NO: 68, heavy chain CDR3 of SEQ ID NO: 69, light chain CDR1 of SEQ ID NO: 70, light chain CDR2 of SEQ ID NO: 71, light chain CDR3 of SEQ ID NO: 72;
  • heavy chain CDR1 of SEQ ID NO: 73 heavy chain CDR2 of SEQ ID NO: 74, heavy chain CDR3 of SEQ ID NO: 75, light chain CDR1 of SEQ ID NO: 76, light chain CDR2 of SEQ ID NO: 77, light chain CDR3 of SEQ ID NO: 78;
  • heavy chain CDR1 of SEQ ID NO: 85 heavy chain CDR2 of SEQ ID NO: 86, heavy chain CDR3 of SEQ ID NO: 87, light chain CDR1 of SEQ ID NO: 88, light chain CDR2 of SEQ ID NO: 89, light chain CDR3 of SEQ ID NO: 90;
  • heavy chain CDR1 of SEQ ID NO: 1 heavy chain CDR2 of SEQ ID NO: 2
  • heavy chain CDR3 of SEQ ID NO: 3 light chain CDR1 of SEQ ID NO: 271, light chain CDR2 of SEQ ID NO: 272, light chain CDR3 of SEQ ID NO: 273;
  • heavy chain CDR1 of SEQ ID NO: 7 heavy chain CDR2 of SEQ ID NO: 8
  • heavy chain CDR3 of SEQ ID NO: 9 light chain CDR1 of SEQ ID NO: 274, light chain CDR2 of SEQ ID NO: 275, light chain CDR3 of SEQ ID NO: 276;
  • heavy chain CDR1 of SEQ ID NO: 13 heavy chain CDR2 of SEQ ID NO: 14
  • heavy chain CDR3 of SEQ ID NO: 15 light chain CDR1 of SEQ ID NO: 277, light chain CDR2 of SEQ ID NO: 278, light chain CDR3 of SEQ ID NO: 279;
  • heavy chain CDR1 of SEQ ID NO: 25 heavy chain CDR2 of SEQ ID NO: 26
  • heavy chain CDR3 of SEQ ID NO: 27 light chain CDR1 of SEQ ID NO: 280
  • light chain CDR2 of SEQ ID NO: 281 light chain CDR3 of SEQ ID NO: 282.
  • an antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 spike protein comprising a combination of any one variable region selected from the following group:
  • Another aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof and a recombinant expression vector comprising the same.
  • Another aspect of the present invention provides a cell transformed with the vector and a method for producing the antibody or antigen-binding fragment thereof using the same.
  • ADC antibody-drug conjugate
  • Another aspect of the present invention provides a multispecific antibody comprising the antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides a pharmaceutical composition for preventing or treating SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or multispecific antibody.
  • Another aspect of the present invention provides a composition for diagnosis of SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides a kit for detecting or quantifying a SARS-CoV-2 spike protein comprising the antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof according to the present invention specifically binds to the SARS-CoV-2 spike protein and completely blocks the binding of angiotensin converting enzyme-2 (ACE2), a receptor on the surface of human cells, to the SARS-CoV-2 spike protein. Because it can be blocked, it can be usefully used for prevention, treatment or diagnosis of novel coronavirus infection.
  • ACE2 angiotensin converting enzyme-2
  • FIG. 1 is a schematic diagram of a SARS-CoV-2 spike protein expression vector.
  • FIGS. 5A to 5D show anti-SARS derived from Ymax ® -ABL ( FIGS. 5A and 5B ) and a patient-immune library ( FIGS. 5C and 5D ) using HEK293E cell lines with different SARS-CoV-2 spike protein expression. -Shows the results of confirming the binding specificity of the CoV-2 spike antibody to the antigen by FACS.
  • FIG. 6A and 6B show anti-SARS-CoV-2 derived from Ymax ® -ABL (FIG. 6A) and patient-immune library (FIG. 6B) using the spike S1-His fusion protein of SARS-CoV or SARS-CoV-2. The results are shown by measuring the antigen-binding specificity of the spike antibody by ELISA.
  • FIG. 7A and 7B show the antigen-binding affinity of anti-SARS-CoV-2 spike antibodies derived from Ymax ® -ABL (FIG. 7A) and patient-immune library (FIG. 7B) using SARS-CoV-2 RBD-mFc fusion protein. The results measured by the Octet QKe analysis equipment are shown.
  • FIG. 8A and 8B show the HEK293E cell line (HEK293E/MocK) used as a control (FIG. 8A) and the HEK293E cell line (HEK293E/CoV-2 spike (chimeric)) with different expression of SARS-CoV-2 spike protein (FIG. 8B) Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variants using each of the antigen binding specificity confirmed by FACS is shown.
  • FIGS. 11A to 11C show SARS-CoV-2 RBD-His, a mutant in which the RBM of SARS-CoV-2 RBD is substituted with the RBM of SARS-CoV RBD (SARS-CoV-2_RBM_CoV-His) or vice versa Ymax ® -ABL and anti-SARS-CoV-2 spike antibody ( FIGS. 11A and 11B ) from the patient-immune library and Ymax using SARS-CoV_RBM_CoV-2-His, SARS-CoV RBD-His fusion proteins ® -This is the result of confirming the antigen-binding region of the ABL-derived antibody variant (FIG. 11c) through ELISA analysis.
  • FIG. 12A and 12B show recombinant mutant proteins (SARS-CoV-2 RBD-His) in which the amino acid sequence of the RBM region of SARS-CoV-2 RBD that binds ACE2 is substituted with other amino acids with reference to the RBM sequence of SARS-CoV RBD. mutants; M2 to M10, and M12) (FIG. 12A), the binding specificities of anti-SARS-CoV-2 spike antibodies and variants were measured by ELISA and grouped results (FIG. 12B) are shown.
  • FIGS. 13A-13D show that when RBD of SARS-CoV-2 ( FIGS. 13A and 13C ) or Spike S1 ( FIGS. 13B and 13D ) binds together with ACE2, Ymax ® -ABL ( FIGS. 13A and 13B ) and It is the result of measuring and confirming whether the anti-SARS-CoV-2 spike antibody derived from the patient-immune library ( FIGS. 13c and 13d ) competitively inhibits these binding in a concentration-dependent manner through ELISA.
  • 14A to 14C show Ymax ® -ABL and anti-SARS-CoV-2 spike antibody derived from a patient-immune library using HEK293E cells expressing recombinant SARS-CoV-2 RBD-mFc protein and ACE2 ( FIGS. 14A and 14A and 14C ).
  • 14b) and Ymax ® -ABL-derived antibody variant is the result of confirming the effect of inhibiting SARS-CoV-2 RBD / ACE2 binding in a concentration-dependent manner.
  • FIG. 15A and 15B show anti-SARS- derived from Ymax ® -ABL (FIG. 15A) and patient-immune library (FIG. 15B) using HEK293E cells expressing recombinant SARS-CoV-2 RBD-hFc protein and ACE2-GFP. This is the result of confirming the effect of the CoV-2 spike antibody concentration-dependently inhibiting the intracellular influx (internalization) of SARS-CoV-2 RBD through binding to cell surface ACE2.
  • FIG. 16A and 16B show whether anti-SARS-CoV-2 spike antibodies from Ymax ® -ABL (FIG. 16A) and patient-immune library (FIG. 16B) have neutralizing ability against Live SARS-CoV-2 virus in Vero cells. The confirmed results are shown.
  • FIG. 17A and 17B show that when RBD of SARS-CoV-2 (FIG. 17A) or Spike S1 (FIG. 17B) binds together with ACE2, Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variants dose-dependently It is the result of measuring and confirming whether these bindings are competitively inhibited by ELISA.
  • Figure 18 shows that Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variant SARS- through binding to cell surface ACE2 using HEK293E cells expressing recombinant SARS-CoV-2 RBD-hFc protein and ACE2-GFP. This is the result of confirming the effect of inhibiting the intracellular influx (internalization) of CoV-2 RBD in a concentration-dependent manner.
  • 19A and 19B show the results of confirming in Vero cells whether the Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variant has neutralizing ability against the Live SARS-CoV-2 virus.
  • Antibodies that specifically bind to the SARS-CoV-2 spike (S) protein specifically bind to the SARS-CoV-2 spike (S) protein
  • One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2; severe acute respiratory syndrome coronavirus 2
  • 2019-nCoV coronavirus infection
  • SARS-CoV-2 spike protein plays a key role in receptor recognition and cell membrane fusion process, and consists of two subunits S1 and S2 do.
  • the S1 subunit contains a receptor-binding domain (RBD) that recognizes and binds host angiotensin-converting enzyme 2 (ACE2), whereas the S2 subunit contains two heptad repeats. It mediates viral cell membrane fusion by forming a six-helical bundle through a two-heptad repeat domain.
  • RBD receptor-binding domain
  • ACE2 subunit contains two heptad repeats. It mediates viral cell membrane fusion by forming a six-helical bundle through a two-heptad repeat domain.
  • the SARS-CoV-2 spike (S) protein has a size of 180-200 kDa and is composed of an extracellular N-terminus, a transmembrane (TM) domain anchored to the viral membrane, and a short intracellular C-terminal segment.
  • TM transmembrane
  • C-terminal segment is composed Spikes generally exist in metastable and prefusion forms, and when the virus interacts with the host cell, extensive structural rearrangement of the S protein occurs, causing the virus to fuse with the host cell membrane.
  • the spikes can be coated with polysaccharide molecules to camouflage them and evade surveillance of the host immune system while entering.
  • SARS-CoV-2 S is 1,273 aa and consists of a signal peptide (amino acids 1-13) located at the N-terminus, S1 subunit (14-685 residues) and S2 subunit (686-1,273 residues).
  • the S1 subunit has an N-terminal domain (residues 14-305) and a receptor binding domain (RBD, residues 319-541), and the S2 subunit contains a fusion peptide (FP) (residues 788-806) and a heptapeptide.
  • FP fusion peptide
  • HR1 heptapeptide repeat sequence 1
  • HR2 residues 1,163-1,213
  • TM domain residues 1,213-1,237
  • cytoplasmic domain residues 1,237-1,273
  • the term "antibody that specifically binds to SARS-CoV-2 spike (S) protein” refers to SARS that binds to angiotensin converting enzyme-2 (ACE2), a receptor on the surface of human cells, and causes an infection mechanism.
  • S SARS-CoV-2 spike
  • ACE2 angiotensin converting enzyme-2
  • - refers to an antibody that targets the membrane protein spike of CoV-2.
  • the antibody exhibits neutralizing efficacy against SARS-CoV-2 by recognizing and binding the membrane protein spike of SARS-CoV-2, which is closely related to the mechanism of human infection of SARS-CoV-2, as an antigen. Meanwhile, in the present invention, the antibody is mixed with the anti-SARS-COV-2 spike antibody.
  • the antibody or antigen-binding fragment thereof according to the present invention may bind to the amino acid sequence of the membrane protein spike of SARS-CoV-2 or a part thereof.
  • the amino acid sequence of the SARS-CoV-2 spike protein is GenBank accession NO. It may be one described in QHD43416.1 (SEQ ID NO: 309).
  • the gene is GenBank accession No. It may be the nucleotide sequence described in MN908947.3 (SEQ ID NO: 310).
  • the antibody or antigen-binding fragment thereof may specifically bind to a site containing a SARS-CoV-2 receptor binding motif (RBM) binding to ACE2.
  • RBM SARS-CoV-2 receptor binding motif
  • the R319 to F541 amino acid sequence (SEQ ID NO: 311), L425 to V510 amino acid sequence (SEQ ID NO: 312), N437 to Y508 amino acid sequence (SEQ ID NO: 313) represented by SEQ ID NO: 309 is specific to the site may be combined with Preferably, it may be one that specifically binds to the sequence of SEQ ID NO: 313, but is not limited thereto.
  • the spike protein may be a polypeptide consisting of any sequence known in the art.
  • the polypeptide may be a variant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid substitutions in proteins or peptides that do not entirely alter the activity of the molecule are known in the art.
  • substitutions are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Substitutions between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.
  • the antibody or antigen-binding fragment thereof specifically binding to the SARS-CoV-2 spike protein according to the present invention is not limited thereto, but SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, a heavy chain CDR1 selected from the group consisting of 55, 61, 67, 73, 79 and 85; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80 and 86; a heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81 and 87; and SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 271, 274, 277 and 280 light chain ) CDR1; a light chain CDR2 selected from the group consist
  • the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein is not limited thereto, but may include any one CDR (complementarity determining region) combination selected from the following group. can:
  • heavy chain CDR1 of SEQ ID NO: 1 heavy chain CDR2 of SEQ ID NO: 2
  • heavy chain CDR3 of SEQ ID NO: 3 light chain CDR1 of SEQ ID NO: 4
  • light chain CDR2 of SEQ ID NO: 5 light chain CDR3 of SEQ ID NO: 6;
  • heavy chain CDR1 of SEQ ID NO: 7 heavy chain CDR2 of SEQ ID NO: 8
  • heavy chain CDR3 of SEQ ID NO: 9 light chain CDR1 of SEQ ID NO: 10
  • light chain CDR2 of SEQ ID NO: 11 light chain CDR3 of SEQ ID NO: 12;
  • heavy chain CDR1 of SEQ ID NO: 13 heavy chain CDR2 of SEQ ID NO: 14
  • heavy chain CDR3 of SEQ ID NO: 15 light chain CDR1 of SEQ ID NO: 16
  • light chain CDR2 of SEQ ID NO: 17 light chain CDR3 of SEQ ID NO: 18;
  • heavy chain CDR1 of SEQ ID NO: 19 heavy chain CDR2 of SEQ ID NO: 20
  • heavy chain CDR3 of SEQ ID NO: 21 light chain CDR1 of SEQ ID NO: 22, light chain CDR2 of SEQ ID NO: 23, light chain CDR3 of SEQ ID NO: 24;
  • heavy chain CDR1 of SEQ ID NO: 25 heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 28, light chain CDR2 of SEQ ID NO: 29, light chain CDR3 of SEQ ID NO: 30;
  • heavy chain CDR1 of SEQ ID NO: 31 heavy chain CDR2 of SEQ ID NO: 32, heavy chain CDR3 of SEQ ID NO: 33, light chain CDR1 of SEQ ID NO: 34, light chain CDR2 of SEQ ID NO: 35, light chain CDR3 of SEQ ID NO: 36;
  • heavy chain CDR1 of SEQ ID NO: 37 heavy chain CDR2 of SEQ ID NO: 38, heavy chain CDR3 of SEQ ID NO: 39, light chain CDR1 of SEQ ID NO: 40, light chain CDR2 of SEQ ID NO: 41, light chain CDR3 of SEQ ID NO: 42;
  • heavy chain CDR1 of SEQ ID NO: 43 heavy chain CDR2 of SEQ ID NO: 44, heavy chain CDR3 of SEQ ID NO: 45, light chain CDR1 of SEQ ID NO: 46, light chain CDR2 of SEQ ID NO: 47, light chain CDR3 of SEQ ID NO: 48;
  • heavy chain CDR1 of SEQ ID NO: 49 heavy chain CDR2 of SEQ ID NO: 50, heavy chain CDR3 of SEQ ID NO: 51, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 53, light chain CDR3 of SEQ ID NO: 54;
  • heavy chain CDR1 of SEQ ID NO: 67 heavy chain CDR2 of SEQ ID NO: 68, heavy chain CDR3 of SEQ ID NO: 69, light chain CDR1 of SEQ ID NO: 70, light chain CDR2 of SEQ ID NO: 71, light chain CDR3 of SEQ ID NO: 72;
  • heavy chain CDR1 of SEQ ID NO: 73 heavy chain CDR2 of SEQ ID NO: 74, heavy chain CDR3 of SEQ ID NO: 75, light chain CDR1 of SEQ ID NO: 76, light chain CDR2 of SEQ ID NO: 77, light chain CDR3 of SEQ ID NO: 78;
  • heavy chain CDR1 of SEQ ID NO: 85 heavy chain CDR2 of SEQ ID NO: 86, heavy chain CDR3 of SEQ ID NO: 87, light chain CDR1 of SEQ ID NO: 88, light chain CDR2 of SEQ ID NO: 89, light chain CDR3 of SEQ ID NO: 90;
  • heavy chain CDR1 of SEQ ID NO: 1 heavy chain CDR2 of SEQ ID NO: 2
  • heavy chain CDR3 of SEQ ID NO: 3 light chain CDR1 of SEQ ID NO: 271, light chain CDR2 of SEQ ID NO: 272, light chain CDR3 of SEQ ID NO: 273;
  • heavy chain CDR1 of SEQ ID NO: 7 heavy chain CDR2 of SEQ ID NO: 8
  • heavy chain CDR3 of SEQ ID NO: 9 light chain CDR1 of SEQ ID NO: 274, light chain CDR2 of SEQ ID NO: 275, light chain CDR3 of SEQ ID NO: 276;
  • heavy chain CDR1 of SEQ ID NO: 13 heavy chain CDR2 of SEQ ID NO: 14
  • heavy chain CDR3 of SEQ ID NO: 15 light chain CDR1 of SEQ ID NO: 277, light chain CDR2 of SEQ ID NO: 278, light chain CDR3 of SEQ ID NO: 279;
  • heavy chain CDR1 of SEQ ID NO: 25 heavy chain CDR2 of SEQ ID NO: 26
  • heavy chain CDR3 of SEQ ID NO: 27 light chain CDR1 of SEQ ID NO: 280
  • light chain CDR2 of SEQ ID NO: 281 light chain CDR3 of SEQ ID NO: 282.
  • the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein according to the present invention is not limited thereto, but SEQ ID NOs: 211, 213, 215, 217, 219, 221, 223, 225, 227, It may include any one heavy chain variable region selected from the group consisting of 229, 231, 233, 235, 237 and 239.
  • the antibody or antigen-binding fragment thereof is not limited thereto, but SEQ ID NOs: 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 299, It may include any one light chain variable region selected from the group consisting of 300, 301 and 302.
  • the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein is not limited thereto, but may include a combination of any one variable region selected from the following group. can:
  • the antibody or antigen-binding fragment thereof comprising the variable region combination is not limited thereto, but may have at least about 80% homology, at least about 90% homology, or at least about 95% homology with the variable region of each corresponding sequence. .
  • the term "antibody” refers to a protein that specifically binds to and neutralizes a specific antigen, such as a pathogenic bacteria or virus. It refers to an antibody that specifically binds.
  • the scope of the present invention includes not only complete antibody forms that specifically bind to the SARS-COV-2 spike, but also antigen-binding fragments of the antibody molecule.
  • a complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain connected to the heavy chain by a disulfide bond.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types, and subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha1 ( ⁇ 1) and alpha2 ( ⁇ 2).
  • the constant region of the light chain has kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • an "antigen-binding fragment” or “antibody fragment” of an antibody refers to a fragment having an antigen-binding function, and may be in the form of Fab, F(ab'), F(ab')2 and Fv.
  • Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1), and has one antigen-binding site.
  • Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • the F(ab')2 antibody is produced by forming a disulfide bond between two cysteine residues in the hinge region of Fab'.
  • Fv refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region.
  • a double-chain Fv two-chain Fv
  • the heavy chain variable region and the light chain variable region are connected by a non-covalent bond
  • single-chain Fv scFv
  • scFv single-chain Fv
  • scFv is generally a heavy chain variable region and light chain variable region through a peptide linker. Since the regions are covalently linked or linked directly at the C-terminus, a dimer-like structure can be formed like a double-stranded Fv.
  • Such antibody fragments can be obtained using proteolytic enzymes (for example, papain-restricted digestion of the whole antibody yields Fab, pepsin digestion yields F(ab')2 fragments), and gene It can also be produced through recombinant technology.
  • the antibody according to the invention is in the form of an Fv (eg scFv) or in the form of a complete antibody.
  • the heavy chain constant region may be any one isotype of gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) or epsilon ( ⁇ ).
  • the constant region is gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4).
  • the light chain constant region may be kappa or lambda type.
  • the antibodies of the invention consist of fully human antibody sequences.
  • the "human antibody” is a molecule derived from human immunoglobulin, and means that the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin. If necessary, the antibody of the present invention may be modified into various forms such as a humanized antibody, a chimeric antibody, and the like according to methods known in the art.
  • Antibody variable domain refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the Complementarity Determining Region (CDR) and Framework Region (FR).
  • CDR Complementarity Determining Region
  • FR Framework Region
  • VH refers to the variable domain of the heavy chain
  • VL refers to the variable domain of the light chain.
  • complementarity determining region As used herein, the term "complementarity determining region" (CDR; that is, CDR1, CDR2 and CDR3) is a ring-shaped region involved in antigen recognition, and as the sequence of this region changes, the specificity of the antibody for the antigen is decided
  • the complementarity determining region refers to an amino acid residue of an antibody variable domain as a region necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • the present invention provides an anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof comprising the CDR combinations as described above.
  • frame region refers to variable domain residues other than CDR residues, each variable domain having four FRs, typically identified as FR1, FR2, FR3 and FR4.
  • the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein of the present invention is not limited thereto, but SEQ ID NOs: 91, 99, 107, 115, 123, 131, 139, 147, any one heavy chain FR1 selected from the group consisting of 155, 163, 171, 179, 187, 195 and 203; any one heavy chain FR2 selected from the group consisting of SEQ ID NOs: 92, 100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196 and 204; any one heavy chain FR3 selected from the group consisting of SEQ ID NOs: 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, 189, 197 and 205; any one heavy chain FR4 selected from the group consisting of SEQ ID NOs: 94, 102, 110, 118
  • the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein of the present invention is not limited thereto, but may include any one FR combination selected from the following group:
  • heavy chain FR1 of SEQ ID NO: 107 heavy chain FR2 of SEQ ID NO: 108, heavy chain FR3 of SEQ ID NO: 109, heavy chain FR4 of SEQ ID NO: 110, light chain FR1 of SEQ ID NO: 111, light chain FR2 of SEQ ID NO: 112, light chain of SEQ ID NO: 113 FR3, light chain FR4 of SEQ ID NO: 114;
  • heavy chain FR1 of SEQ ID NO: 115 heavy chain FR2 of SEQ ID NO: 116, heavy chain FR3 of SEQ ID NO: 117, heavy chain FR4 of SEQ ID NO: 118, light chain FR1 of SEQ ID NO: 119, light chain FR2 of SEQ ID NO: 120, light chain of SEQ ID NO: 121 FR3, light chain FR4 of SEQ ID NO: 122;
  • heavy chain FR1 of SEQ ID NO: 139 heavy chain FR2 of SEQ ID NO: 140, heavy chain FR3 of SEQ ID NO: 141, heavy chain FR4 of SEQ ID NO: 142, light chain FR1 of SEQ ID NO: 143, light chain FR2 of SEQ ID NO: 144, light chain of SEQ ID NO: 145 FR3, light chain FR4 of SEQ ID NO: 146;
  • heavy chain FR1 of SEQ ID NO: 147 heavy chain FR2 of SEQ ID NO: 148, heavy chain FR3 of SEQ ID NO: 149, heavy chain FR4 of SEQ ID NO: 150, light chain FR1 of SEQ ID NO: 151, light chain FR2 of SEQ ID NO: 152, light chain of SEQ ID NO: 153 FR3, light chain FR4 of SEQ ID NO: 154;
  • heavy chain FR1 of SEQ ID NO: 155 heavy chain FR2 of SEQ ID NO: 156, heavy chain FR3 of SEQ ID NO: 157, heavy chain FR4 of SEQ ID NO: 158, light chain FR1 of SEQ ID NO: 159, light chain FR2 of SEQ ID NO: 160, light chain of SEQ ID NO: 161 FR3, light chain FR4 of SEQ ID NO: 162;
  • heavy chain FR1 of SEQ ID NO: 171 heavy chain FR2 of SEQ ID NO: 172, heavy chain FR3 of SEQ ID NO: 173, heavy chain FR4 of SEQ ID NO: 174, light chain FR1 of SEQ ID NO: 175, light chain FR2 of SEQ ID NO: 176, light chain of SEQ ID NO: 177 FR3, light chain FR4 of SEQ ID NO: 178;
  • heavy chain FR1 of SEQ ID NO: 203 heavy chain FR2 of SEQ ID NO: 204, heavy chain FR3 of SEQ ID NO: 205, heavy chain FR4 of SEQ ID NO: 206, light chain FR1 of SEQ ID NO: 207, light chain FR2 of SEQ ID NO: 208, light chain of SEQ ID NO: 209 FR3, light chain FR4 of SEQ ID NO: 210;
  • heavy chain FR1 of SEQ ID NO: 91 heavy chain FR2 of SEQ ID NO: 92, heavy chain FR3 of SEQ ID NO: 93, heavy chain FR4 of SEQ ID NO: 94, light chain FR1 of SEQ ID NO: 283, light chain FR2 of SEQ ID NO: 284, light chain of SEQ ID NO: 285 FR3, light chain FR4 of SEQ ID NO: 286;
  • heavy chain FR1 of SEQ ID NO: 99 heavy chain FR2 of SEQ ID NO: 100, heavy chain FR3 of SEQ ID NO: 101, heavy chain FR4 of SEQ ID NO: 102, light chain FR1 of SEQ ID NO: 287, light chain FR2 of SEQ ID NO: 288, light chain of SEQ ID NO: 289 FR3, light chain FR4 of SEQ ID NO: 290;
  • heavy chain FR1 of SEQ ID NO: 107 heavy chain FR2 of SEQ ID NO: 108, heavy chain FR3 of SEQ ID NO: 109, heavy chain FR4 of SEQ ID NO: 110, light chain FR1 of SEQ ID NO: 291, light chain FR2 of SEQ ID NO: 292, light chain of SEQ ID NO: 293 FR3, light chain FR4 of SEQ ID NO: 294;
  • An antibody or antigen-binding fragment thereof comprising the above framework region combination may have, but is not limited to, at least about 80% homology, at least about 90% homology, or at least about 95% homology to the framework region of each corresponding sequence. .
  • the present inventors have developed a naive human scFv library (referred to as Ymax ® -ABL; YBiologics Co., Ltd.) or an scFv immune library derived from a Covid-19 confirmed patient by a phage display method (Patient). -Immune Library) to prepare an antibody that specifically binds to the SARS-CoV-2 spike protein by biopanning.
  • Ymax ® -ABL YBiologics Co., Ltd.
  • scFv immune library derived from a Covid-19 confirmed patient by a phage display method (Patient).
  • -Immune Library to prepare an antibody that specifically binds to the SARS-CoV-2 spike protein by biopanning.
  • phage display is a technique for displaying a variant polypeptide as a fusion protein with at least a portion of an envelope protein on the surface of a phage, eg, a filamentous phage particle.
  • the usefulness of phage display resides in the fact that, by targeting a large library of randomized protein variants, it is possible to quickly and efficiently sort sequences that bind to a target antigen with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for polypeptides with specific binding properties.
  • the phage display technology has the advantage of being able to generate an antibody library having various sequences in a short time compared to conventional hybridoma and recombinant methods for producing an antibody having desired characteristics.
  • the phage antibody library can generate antibodies against antigens that are toxic or of low antigenicity.
  • Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.
  • a technology capable of identifying and isolating high-affinity antibodies from phage display libraries is important for isolating novel therapeutic antibodies. Isolation of high affinity antibodies from a library may depend on the size of the library, production efficiency in bacterial cells, and diversity of the library.
  • scFv single chain fragment variable
  • biopanning refers to a property of binding to a target molecule (antibody, enzyme, cell surface receptor, etc.) from a phage library that expresses a peptide on the outer wall of a phage. It refers to the process of selecting only phages expressing the peptides on the surface.
  • the antibody or antigen-binding fragment thereof is not particularly limited thereto, but may be glycosylated and/or PEGylated to improve residence time in the administered body.
  • the glycosylation and/or pegylation can be modified by various glycosylation and/or pegylation patterns by methods known in the art as long as the function of the antibody of the present invention is maintained, and the antibody of the present invention has various glycosylation and/or pegylation It includes all mutant monoclonal antibodies or antigen-binding fragments thereof in which the pegylation pattern is modified.
  • a total of 15 lead antibodies were obtained from 7 types of human antibody library Ymax ® -ABL and 8 types derived from the COVID-19 patient-immunity library, and by optimizing them, a final total of 19 types of SARS- Antibodies that specifically bind to the CoV-2 spike protein were obtained (Examples 2 and 3).
  • the antibody exhibits high antigen-binding affinity (K D ) at a sub-nanomolar level to SARS-CoV-2 S1 or RBD, and cell-based confirming neutralizing ability to inhibit the binding of SARS-CoV-2 RBD and ACE2
  • K D antigen-binding affinity
  • the IC 50 value was excellent at the nanomolar level, and it was confirmed that all of the neutralizing ability was superior to soluble ACE2.
  • the antibody effectively inhibits the internalization of SARS-CoV-2 RBD, binds specifically to SARS-CoV-2 receptor binding motif (RBM) binding to ACE2, and reacts directly with the virus. It was found that the antibody had excellent efficacy to neutralize infection (Examples 5 to 9).
  • the antibody of the present invention can completely block the binding of the membrane protein spike (S) of SARS-CoV-2 to ACE2, it can exhibit preventive and therapeutic effects on novel coronavirus infection.
  • the antibody according to the present invention is not limited thereto, but 1 ⁇ 10 -8 M to 1 ⁇ 10 -12 M, or 1 ⁇ 10 -9 M to 1 ⁇ 10 -11 M for SARS-CoV-2 spike protein binding affinity (K D ) within a range.
  • the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof of the present invention includes a portion of the amino acid sequence through conservative substitution in the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof according to the present invention.
  • a substituted antibody or antigen-binding fragment thereof may also be included.
  • the term “conservative substitution” refers to a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the polypeptide.
  • a “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains have been defined in the art and are well known.
  • amino acids with basic side chains eg lysine, arginine, histidine
  • amino acids with acidic side chains eg aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains eg glycine
  • amino acids with non-polar side chains eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains amino acids eg, threonine, valine, isoleucine
  • aromatic side chains eg, tyrosine, phenylalanine, tryptophan, histidine.
  • the antibody of the present invention may still retain activity even with such conservative amino acid substitutions.
  • Another aspect of the present invention provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
  • Nucleic acids as used herein may be present in cells, cell lysates, or may exist in partially purified or substantially pure form. Nucleic acids can be removed from other cellular components or other contaminants, e.g., by standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. "Isolated” or “substantially pure” when purified from the nucleic acid or protein of another cell.
  • the nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not contain an intron sequence.
  • the nucleotide which is the basic building block of nucleic acids, includes not only natural nucleotides, but also analogs in which sugar or base regions are modified.
  • sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
  • the nucleic acid encoding the anti-SARS-COV-2 spike antibody is not limited thereto, but may include a polynucleotide combination encoding any one variable region selected from the following group:
  • the antibody or the nucleic acid molecule encoding the same is not limited thereto, but it is construed to include a sequence exhibiting substantial identity to each corresponding sequence shown in SEQ ID NO.
  • the substantial identity is, when the sequence of the present invention and any other sequences are arranged to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art, homology of 90% or more, Preferably, it refers to a sequence that exhibits at least 95% homology, more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology.
  • sequence homology can be determined by sequence comparison and/or alignment by methods known in the art.
  • sequence homology of the nucleic acid or protein of the present invention may be determined using a sequence comparison algorithm (eg, NCBI Basic Local Alignment Search Tool; BLAST), manual alignment, visual inspection, and the like.
  • BLAST NCBI Basic Local Alignment Search Tool
  • Another aspect of the present invention provides a recombinant expression vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
  • DNA encoding partial or full-length light and heavy chains is prepared using standard molecular biology techniques (e.g., PCR amplification or the antibody of interest) cDNA cloning using hybridomas expressing
  • Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.
  • the term "vector” refers to a means for expressing a target gene in a host cell, including a plasmid vector; cozmid vector; viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, and the like.
  • the nucleic acid encoding the antibody or antigen-binding fragment thereof is operably linked to a promoter.
  • operably linked means that the gene encoding the antibody or antigen-binding fragment thereof is lysed into a vector such that transcriptional and translational control sequences in the vector serve the intended function of regulating the transcription and translation of the antibody gene. It means being ligated.
  • Expression vectors and expression control sequences are selected to be compatible with the cells for expression used.
  • the light chain gene and heavy chain gene of the antibody are inserted into separate vectors, or both genes are inserted into the same expression vector.
  • the antibody gene is inserted into the expression vector by standard methods (eg, ligation of complementary restriction enzyme sites on the antibody gene fragment and vector, or blunt end ligation if no restriction enzyme sites are present).
  • the recombinant expression vector may contain a sequence encoding a signal peptide that facilitates secretion of the antibody chain from the transformed cell.
  • the antibody chain gene and signal peptide-coding sequence can be cloned into a vector in frame so that the signal peptide is expressed by binding to the amino terminus of the antibody chain.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a protein other than immunoglobulin).
  • the recombinant expression vector may include a regulatory sequence for controlling the expression of the antibody chain gene in the transformed cell.
  • regulatory sequences may include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes.
  • expression control elements eg, polyadenylation signals
  • a person skilled in the art can recognize that the design of the expression vector may vary by selecting different control sequences depending on factors such as the selection of cells to be transformed, the level of protein expression, and the like.
  • the vectors of the present invention may also include other sequences to be fused to the antibody gene to facilitate purification of the antibody expressed from the vector.
  • This sequence may be, for example, a gene such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6 ⁇ His (hexahistidine; Quiagen, USA).
  • the vector contains an antibiotic resistance gene commonly used in the art as a selection marker, and such genes include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and There is a gene for resistance to tetracycline.
  • Another aspect of the present invention provides a cell transformed with the recombinant expression vector.
  • the host cell of the transformed cell may include, but is not limited to, a cell of prokaryotic, eukaryotic, mammalian, plant, insect, fungal or cellular origin.
  • a cell of prokaryotic, eukaryotic, mammalian, plant, insect, fungal or cellular origin As an example of the prokaryotic cell, E. coli may be used. In addition, yeast may be used as an example of eukaryotic cells.
  • CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells or HEK293T cells may be used as the mammalian cells. , but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art is available.
  • expression vectors suitable for eukaryotic cells include, but are not limited to, expression vectors derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. It is not Expression vectors usable for bacterial cells include E.
  • coli -derived bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC, col E1, pCR1, pBR322, pMB9 and derivatives thereof; plasmids with a wider host range, such as RP4; phage DNA, such as various phage lambda derivatives such as ⁇ gt10, ⁇ gt11, and NM989; and other DNA phages such as M13 and filamentous single-stranded DNA phages.
  • Useful expression vectors for yeast cells are YEp plasmids and derivatives thereof.
  • a useful vector for insect cells is pVL941.
  • the CaCl 2 precipitation method when introducing a recombinant expression vector into a host cell, the CaCl 2 precipitation method, the CaCl 2 precipitation method using a reducing material called DMSO (dimethyl sulfoxide), which increases the efficiency by using the Hanahan method, electroporation, calcium phosphate precipitation method, protoplast fusion method, agitation method using silicon carbide fiber, agrobacterium-mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/inhibition-mediated transformation method, etc. can be used. .
  • DMSO dimethyl sulfoxide
  • the vector introduced into the host cell can be expressed in the host cell, and in this case, a large amount of the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof of the present invention can be obtained.
  • Another aspect of the present invention (i) culturing the transformed cell; and (ii) recovering an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein from the obtained cell culture medium.
  • a method for producing an antigen-binding fragment thereof is provided.
  • the antibody or antigen-binding fragment thereof can be prepared by culturing the cells for a period sufficient to allow the antibody to be secreted into the culture medium in which the cells are cultured.
  • the cells can be cultured in various media, and commercially available media can be used without limitation as the culture media. All other essential supplements known to those skilled in the art may be included in appropriate concentrations. Suitable culture conditions, for example, temperature, pH, etc., have already been used for protein expression in the selected host cell, and will be apparent to those skilled in the art.
  • the expressed antibody can be separated from the cell culture medium and purified to uniformity. Isolation or purification of the antibody may be performed by a conventional protein separation and purification method, for example, chromatography.
  • the chromatography may include, for example, affinity chromatography using a protein A column or a protein G column, ion exchange chromatography, hydrophobic chromatography, or hydroxylapatite chromatography.
  • the antibody can be separated and purified by further combining filtration, ultrafiltration, salting out, dialysis, and the like.
  • ADC comprising antibody and drug that specifically binds to SARS-CoV-2 S protein
  • ADC antibody-drug conjugate
  • antibody-drug conjugate refers to a conjugate of the antibody or antigen-binding fragment thereof and a drug, and the drug is stable to the antibody until the drug is delivered to the target cell. It should be bound to the target, and after delivery to the target, the drug should be released from the antibody.
  • the antibody or antigen-binding fragment thereof and a drug are bound to each other (eg, covalent bond, peptide bond, etc.) to be used in the form of a conjugate or a fusion protein (when the drug is a protein).
  • the drug is an agent that exhibits a pharmacological effect, may be bound to the antibody or antigen-binding fragment thereof of the present invention, may be separated from the antibody or antigen-binding fragment thereof by acidic conditions, and exhibit a therapeutic effect on target cells means a compound.
  • the drug is not limited thereto, but may be an antiviral agent.
  • the antiviral agent is not limited thereto, but may be a conventional antiviral agent, such as an Ebola virus treatment agent, an HIV (human immunodeficiency virus) treatment agent, a hepatitis C treatment agent, a flu treatment agent, and the like.
  • Ebola virus treatment agent such as an Ebola virus treatment agent, an HIV (human immunodeficiency virus) treatment agent, a hepatitis C treatment agent, a flu treatment agent, and the like.
  • specific drugs include kaletra, remdesivir, placknil (hydroxychloroquine), resorcin (chloroquine), and the like.
  • the drug may use a substance being developed as a coronavirus treatment.
  • the applicable drugs include antiviral agents other than anti-diabetic agents (for example, dapagliflozin), rheumatoid arthritis agents (for example, anakinra, tocilizumab, sarirumab). (sarilumab)), a blood cancer treatment agent (eg, acalabrutinib), or a treatment agent for multiple myeloma (eg, selinexor), but is not limited thereto.
  • antiviral agents other than anti-diabetic agents for example, dapagliflozin
  • rheumatoid arthritis agents for example, anakinra, tocilizumab, sarirumab). (sarilumab)
  • a blood cancer treatment agent eg, acalabrutinib
  • a treatment agent for multiple myeloma eg, selinexor
  • the antibody-drug conjugate may be internalized into cells and may act by inhibiting the binding of SARS-CoV-2 to human cell surface ACE2 to block SARS-CoV-2 influx into cells.
  • the conjugate can be prepared by a known method by binding a drug to an antibody or antigen-binding fragment thereof.
  • Antibodies and drugs may be directly coupled through their own linker, etc., or may be coupled indirectly through a linker or other material.
  • the main mechanisms by which drugs are cleaved from antibodies include hydrolysis at acidic pH of lysosomes (hydrazone, acetal and cis-aconitate-like amides), peptide cleavage by lysosomal enzymes (cathepsin and other lysosomal enzymes), and disulfides includes the reduction of As a result of these various cleavage mechanisms, the mechanisms by which drugs are linked to the antibody are very diverse and any suitable linker can be used.
  • Suitable linking groups for binding an antibody to a drug include, for example, a disulfide group, a thioether group, an acid-cleavable group, a photo-degradable group, a peptidase-cleavable group, and an esterase-decomposable group.
  • the linking group may be, for example, a disulfide bond using an SH group or a bond mediated by maleimide.
  • a disulfide bond using an SH group for example, an intramolecular disulfide bond of the antibody Fc region and a drug disulfide bond are reduced, and both are connected by a disulfide bond.
  • Antibodies and drugs may be indirectly coupled through other substances (linkers).
  • the linker preferably has one or two or more functional groups that react with an antibody, drug, or both.
  • the functional group include an amino group, a carboxyl group, a mercapto group, a maleimide group, and a pyridinyl group.
  • Multispecific antibodies comprising antibodies that specifically bind to SARS-CoV-2 S protein
  • Another aspect of the present invention provides a multispecific antibody comprising an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
  • multispecific antibody refers to an antibody or antigen-binding fragment thereof targeting two or more antigens, including a bispecific antibody and a trispecific antibody.
  • a bispecific antibody comprises an antibody or antigen-binding fragment thereof to the SARS-CoV-2 spike protein according to the present invention, wherein one arm comprises an antibody or antigen-binding fragment thereof according to the present invention, among two arms of the antibody.
  • the arm refers to a form containing an antigen other than the SARS-CoV-2 spike protein.
  • the multispecific antibody is a form that can be prepared by genetic engineering or any method, and is a bi-specific antibody, a tri-specific antibody, or a tetra-specific antibody. may include.
  • the multispecific antibody may be in a form in which the anti-SARS-COV-2 spike antibody according to the present invention is combined with an antibody or a fragment thereof having binding ability to an immune effector cell-specific target molecule.
  • the immunopotentiator cell-specific target molecule is preferably selected from, but not limited to, TCR/CD3, CD16 (Fc ⁇ RIIIa), CD44, CD56, CD69, CD64 (Fc ⁇ RI), CD89 and CD11b/CD18 (CR3).
  • the multispecific antibody is preferably in a form in which the anti-SARS-COV-2 spike antibody according to the present invention is combined with an antibody or a fragment thereof having binding ability to a cytokine that stimulates or inhibits immunity.
  • the cytokine that stimulates or inhibits immunity is preferably selected from, for example, IL-2, IL-6, IL-7, IFN ⁇ , GM-CSF, IL-10, and TGF- ⁇ , but is limited thereto not.
  • the multispecific antibody is a target for which the anti-SARS-COV-2 spike antibody according to the present invention is being used in the treatment of a viral disease, such as influenza, herpes, hepatitis B infection, hepatitis C infection, AIDS, for example, CCR5 receptor, neuraminidase (neuraminidase), hemagglutinin (hemagglutinin), interferons (eg, interferon alpha) having a binding ability to the antibody or a fragment thereof having a binding form, but is not limited thereto. .
  • a viral disease such as influenza, herpes, hepatitis B infection, hepatitis C infection, AIDS, for example, CCR5 receptor, neuraminidase (neuraminidase), hemagglutinin (hemagglutinin), interferons (eg, interferon alpha) having a binding ability to the antibody or a fragment thereof having a binding form, but is not limited there
  • Antibodies belonging to multispecific antibodies may be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies.
  • scFv-based antibodies Fab-based antibodies
  • IgG-based antibodies IgG-based antibodies.
  • bispecific antibody since it can inhibit or amplify two signals at the same time, it can be more effective than the case of inhibiting / amplifying one signal. Low dose dosing is possible, and both signals can be suppressed/amplified in the same time and space.
  • bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy/light chain pairs under conditions in which the two heavy chains have different specificities.
  • a hybrid scFv in the form of a heterodimer by combining the VL and VH of different scFvs, respectively, to make a diabody, and different scFvs can be combined with each other.
  • a tandem ScFv can be prepared, and by expressing CH1 and CL of the Fab at the ends of each scFv, a heterodimeric miniantibody can be prepared, and the homodimeric domain of Fc
  • a heterodimeric scFv type minibody was prepared. can do.
  • Fab's directed against a specific antigen can be combined with each other using a disulfide bond or a mediator to form a heterodimeric Fab, and the ends of the heavy or light chains of the specific Fab It can be prepared to have two antigen valencies by expressing scFvs for different antigens in the .
  • scFvs for different antigens to the light and heavy chain ends of the Fab
  • a dual-target bibody with three antigen binding values and different scFvs to the light and heavy chain ends of the Fab are fused to the antigen, respectively. It can be prepared in the form of a triple-targeted bibody so as to have three binding valencies, and can also be obtained by chemically conjugating three different Fabs.
  • an IgG-based bispecific antibody a method for producing a bispecific antibody by re-crossing a mouse and a rat hybridoma to produce a hybrid hybridoma, aka quadromas, is known.
  • a bispecific antibody in the so-called 'Holes and Knob' form by modifying some amino acids of the CH3 homodimeric domain of Fc with respect to different heavy chains to form a heterodimer.
  • (scFv)4-IgG in a homodimeric form can also be prepared by fusion-expressing two different scFvs in constant domains instead of the variable domains of the light and heavy chains of IgG.
  • composition comprising an antibody that specifically binds to SARS-CoV-2 S protein
  • Another aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the above-described SARS-CoV-2 spike protein, an antibody-drug conjugate comprising the same, or SARS-CoV-2 comprising a multispecific antibody It provides a pharmaceutical composition for preventing or treating an infection.
  • the SARS-CoV-2 infection may be related to the expression or overexpression of the spike protein of SARS-COV-2, and the SARS-CoV-2 is as described above.
  • prevention refers to a novel coronavirus infection (COVID) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by administration of the composition according to the present invention. -19) means any action that inhibits or delays the progression, and “treatment” means the suppression, alleviation or elimination of novel coronavirus infection.
  • COVID coronavirus infection
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the pharmaceutical composition may include a therapeutically effective amount of an anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable additive.
  • a “pharmaceutically acceptable carrier” is a substance that can be added to an active ingredient to help formulate or stabilize a drug and does not cause significant toxic effects in the patient.
  • the additive refers to a carrier or diluent that does not irritate the patient and does not inhibit the biological activity and properties of the administered compound.
  • acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and A mixture thereof may be used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed.
  • diluents such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
  • compositions include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for extemporaneous administration.
  • the composition is preferably formulated for parenteral injection.
  • the compositions may be formulated as solutions, microemulsions, liposomes, or other customized formulations suitable for high drug concentrations.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof.
  • isotonic agents may be included in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride.
  • Each formulation can be prepared using methods well known in the pharmaceutical art.
  • the dosage of the pharmaceutical composition according to the present invention is not particularly limited, but may be changed according to various factors including the patient's health condition and weight, disease severity, drug type, administration route, and administration time.
  • the pharmaceutical composition according to the present invention can be administered in one or multiple doses per day through various routes of oral or parenteral routes typically accepted into mammals including humans, rats, mice, livestock, and the like. may be administered. Specifically, it may be administered in a conventional manner via oral, intrarectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalational, intraocular, intrapulmonary or intradermal routes, but is not limited thereto. not.
  • the pharmaceutical composition according to the present invention may be administered to a patient as a bolus or by continuous infusion, if desired.
  • bolus administration of the antigen-binding fragment of the spike antibody of anti-SARS-COV-2 of the present invention represented by the Fab fragment is 0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg /kg or 0.10 to 0.50 mg/kg.
  • the antigen-binding fragment of the spike antibody of anti-SARS-COV-2 of the present invention represented by the Fab fragment is 0.001 to 100 mg/kg body weight/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min or 0.10 to 0.50 mg/kg/min, 1 hour to 24 hours, 1 hour to 12 hours, 2 hours to 12 hours, 6 hours to 12 hours, 2 hours to 8 hours, or from 1 hour to 2 hours.
  • the dosage is about 1 to 10 mg/kg body weight, 2 to 8 mg/kg, or 5 to 6 mg/kg day can
  • the spike antibody of full length anti-SARS-COV-2 is typically administered via infusion lasting for a period of 30 to 35 minutes.
  • the frequency of administration depends on the severity of the condition. The frequency may range from 3 times per week to once every 1 or 2 weeks.
  • the present invention also provides a therapeutically effective amount of the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof or the multispecific antibody or antibody-drug conjugate to prevent or treat novel coronavirus infection (COVID-19). It relates to a method for preventing or treating novel coronavirus infection (COVID-19), comprising administering to a patient in need thereof.
  • the prevention or treatment method may further include the step of identifying a patient in need of the prevention or treatment of the disease before the administering step.
  • the antibody or antigen-binding fragment thereof may be a conventional antiviral agent, such as an Ebola virus treatment agent, an HIV (human immunodeficiency virus) treatment agent, a hepatitis C treatment agent, an influenza treatment agent, and the like.
  • an Ebola virus treatment agent such as an Ebola virus treatment agent, an HIV (human immunodeficiency virus) treatment agent, a hepatitis C treatment agent, an influenza treatment agent, and the like.
  • HIV human immunodeficiency virus
  • hepatitis C treatment agent such as a hepatitis C treatment agent
  • influenza treatment agent such as an influenza treatment agent, and the like.
  • Specific examples of the drug may be used together with kaletra, remdesivir, placknil (hydroxychloroquine), resorcin (chloroquine), and the like.
  • a treatment for diabetes eg, dapagliflozin
  • a treatment for rheumatoid arthritis eg, anakinra
  • tocilizumab e.g., tocilizumab
  • sarilumab e.g., hematologic cancer therapeutics
  • multiple myeloma therapeutics eg, selinexor
  • targets used in the treatment of influenza, herpes, hepatitis B, C infection, AIDS, etc. such as CCR5 receptor, neuraminidase, hemagglutinin ( hemagglutinin), interferons (eg, interferon alpha) may be used together with an antibody having a binding ability.
  • the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof according to the present invention may be administered simultaneously or sequentially with a conventional antiviral therapeutic agent.
  • Diagnostic composition and diagnostic method comprising an antibody that specifically binds to SARS-CoV-2 S protein
  • Another aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the above-described SARS-CoV-2 spike protein, an antibody-drug conjugate comprising the same, or SARS-CoV-2 comprising a multispecific antibody
  • a composition for diagnosing an infection is provided.
  • the diagnostic composition of the present invention refers to a main means used for diagnosing a target disease, and according to the purpose of the present invention, substances for diagnosing SARS-CoV-2 may be included.
  • the diagnostic method may comprise contacting the antibody or antibody fragment with a sample.
  • the sample is a tissue, cell taken from sputum, nostril, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin. , urine, whole blood, serum, plasma, feces, cell culture supernatant or ruptured eukaryotic cells.
  • the expression level can be measured according to a conventional immunoassay method, radioimmunoassay using an antibody against the SARS-COV-2 spike protein, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification, but not limited thereto.
  • a kit comprising an antibody that specifically binds to the SARS-CoV-2 S protein
  • kits for detecting or quantifying a SARS-CoV-2 spike protein comprising an antibody or antigen-binding fragment thereof that specifically binds to the above-described SARS-CoV-2 spike protein. That is, there is provided a diagnostic kit comprising a composition for diagnosis of SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof.
  • the kit according to the present invention may be prepared by a conventional manufacturing method known to those skilled in the art, and may further include a buffer, a stabilizer, an inactive protein, and the like.
  • the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof according to the present invention, or an antibody-drug conjugate or multispecific antibody comprising the same, and a label generating a detectable signal may be included. there is.
  • the label may include a chemical bound to the antibody (eg biotin), an enzyme (alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase, luciferase or cytochrome P450), a radioactive material (eg, C14, I125, P32, and S35), a fluorescent material (eg, fluorescein), a light emitting material, a chemiluminescent material, and a fluorescence resonance energy transfer (FRET), but are not limited thereto.
  • a chemical bound to the antibody eg biotin
  • an enzyme alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase, luciferase or cytochrome P450
  • a radioactive material eg, C14, I125, P32, and S35
  • a fluorescent material eg, fluorescein
  • a light emitting material e.g, chemilumin
  • the substrate for the enzyme is bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate when alkaline phosphatase is used as the enzyme.
  • BCIP bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • naphthol-AS-B1-phosphate naphthol-AS-B1-phosphate
  • a novel coronavirus infection can be diagnosed by analyzing the strength of the signal displayed by the reaction between the sample and the antibody. Measurement of the activity or signal of an enzyme used for diagnosis may be performed according to various methods known in the art, through which the spike protein expression of SARS-COV-2 may be qualitatively or quantitatively analyzed.
  • SARS-CoV-2 (2019-nCoV) spike S1 gene ORF cDNA (Sino biological, Cat: VG40591-CF) and extracellular domain only 5' and 3' A polymerase chain reaction (PCR) was performed using a primer pair (Table 1) for SARS-CoV-2-RBD containing restriction enzyme Sfi I/Nhe I sites.
  • Expression vectors each expressing a protein in which 6 ⁇ His or human Fc (hFc) was fused to the amino-terminus of SARS-CoV-2-RBD were prepared using the obtained PCR product and the N293F vector ( FIG. 1 ).
  • SARS-CoV-2-RBD cloning designation 5' ⁇ 3' sequence SEQ ID NO: SARS-CoV-2-RBD-F CGTGTTCAGCCTACCGAGAGC 307 SARS-CoV-2-RBD-R GAAGTTCACGCATTTGTTCTT 308
  • Example 1.2 Expression and purification of antigenic proteins
  • Transfection was performed using PEI (polyethylenimine; Aldrich, Cat. 408727) under optimized conditions.
  • Human HEK293F cells were adjusted to 5 ⁇ 10 5 per ml, inoculated into a medium (#Freestyle 293 AGT type; AG100009P1, Thermo.), and cultured until 1 ⁇ 10 6 cells/ml.
  • Each of the expression vectors obtained in Example 1.1 and PEI were mixed to form a polyplex, and then added to the cells for transfection and then 1 nM of valproic acid (VPA, valproate, Sigma-P4543) was added. After that, it was further cultured for 6 days.
  • VPA valproic acid
  • the expressed SARS-CoV-2-RBD was first purified using protein A agarose or Ni-NTA beads, and the first purified protein was purified using Superdex 200 (1.5 cm ⁇ 100 cm) gel filtration chromatography. . The purity of the purified protein was confirmed using SDS-PAGE and size exclusion chromatography (TSK-GEL G-3000 SWXL Size-exclusion chromatography (SEC) (Tosoh)), and the purity of the protein was 95% or more (Fig. 2). ). Whether or not SARS-CoV-2-RBD maintains function was confirmed by interaction with ACE2.
  • PBMCs were obtained with consent from 20 confirmed Covid-19 patients through the Department of Infectious Diseases, Chungnam National University.
  • Total RNA was isolated from PBMC, and cDNA was prepared by RT-PCR using this. The obtained cDNA was used to construct an immune library.
  • PCR was performed with the cDNA to amplify the heavy chain variable region and the light chain variable region. After confirming the diversity of the heavy chain variable region and the light chain variable region through nucleotide sequence analysis, PCR was performed again to make scFv (connection of the heavy chain variable region and the light chain variable region). Through sequencing, it was confirmed whether the heavy chain variable region and the light chain variable region were well connected.
  • the purified scFv was inserted into a phage vector (pYG100) and electroporated together with ElectroTen-Blue cells (Agilent, Cat #200159) for transformation at 2.50 kV, 1 pulse, followed by electric shock.
  • Cells were cultured on SOBCG agar medium (2% (w/v) bactotryptone, 1.0% (w/v) bacto-yeast, 0.05% (w/v) NaCl, 5 mM MgCl 2 , 10 mM glucose, 34 ⁇ g/ml chloramphenicol, 15 g bacto-agar) and incubated at 37° C. for 16 hours. The next day, the nucleotide sequence of the obtained library colony was confirmed to confirm the diversity of the immune library.
  • Example 2 50 ⁇ g of each of the SARS-CoV-2-RBD-hFc and SARS-CoV-2-RBD-His protein antigens prepared in Example 1 were coated on an Immunosorb tube, followed by blocking.
  • the library phages prepared as described above were put into the immunosorbent tube, and reacted for 2 hours at room temperature, washed with 1 ⁇ PBST (PBS + tween-20) and 1 ⁇ PBS, and then 100 mM TAE and Tris-HCl (pH7. 5) The solution was sequentially treated to elute only scFv-phages specifically binding to the antigen.
  • a pool of positive phages is obtained through the panning process in which the eluted phages are again infected with E. coli and amplified, and the number of times is increased only in the PBST washing step with the phages amplified in the first round of panning, and the rest are performed in the same manner as 2 Round and 3 rounds of pa
  • Monoclones were selected from the positive phage pool of 3 round panning confirmed to have high binding ability through poly phage ELISA, and they were grown in 96-deep well plates to infect and culture helper phages.
  • direct ELISA was performed by transferring the mono-scFv-phage present in the supernatant to an antigen-coated immune-plate.
  • monophage ELISA for SARS-CoV-2-RBD and ELISA for ITGA6-Fc protein, which is a non-specific antigen control were simultaneously performed to confirm whether the obtained positive phage clone was specific for SARS-CoV-2-RBD.
  • the phagemid DNA was isolated using a DNA purification kit (Qiagen, Germany) for 15 types of single clones finally selected based on binding ability, and the DNA sequence was analyzed.
  • the amino acid sequences of the heavy chain CDRs (Complementarity-determining regions) and the light chain CDRs, the amino acid sequences of the heavy chain FRs (Framework) and the light chain FRs are shown in Tables 3 and 4, respectively, and the amino acid sequences of the heavy and light chain variable regions and encoding them
  • the polynucleotide sequences are shown in Tables 5 and 6 below, respectively.
  • Monoclonal heavy and light chain variable region amino acid sequences antibody variable region amino acid sequence SEQ ID NO: SA3755 heavy chain QMQLVESGGGVVQPGGSLRLSCAAS GFTFDDHT MHWVRQAPGKGLEWVSL ISWDGGST YYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYC TRDASRRPGDGGYDFDV WGQGTQVTVSS 211 light chain QAVVTQEPSLTVSPGGTVTLTCGSS TGTVTRGHW PYWFQQKPGQAPRTLIY DTD NKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYC LLSYSDSRV FGTGTKVTVL 212 SA3779 heavy chain QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYA MHWVRQAPGKGLEWVAV ISYDGSNK YYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRGHYYDSSGYLY WGH
  • LC light chain shuffling (LS) library To construct an LC light chain shuffling (LS) library, the LC genes of the parent antibodies (SA3755, SA3779, SA3827, and SA3838) were digested with BstX I and then used as a vector, and Ymax ® -ABL ((main ) Y Biologics) was cut with BstX I and used as an insert. After ligation with ligase, transformation was performed using cells for electroporation transformation. As a result of preparing an antibody library by collecting transformed cells on a square plate, a library with a diversity of about 1 ⁇ 10 7 was obtained. As a result of nucleotide sequence analysis, all HC sequences are the same and LC sequences are different. confirmed that.
  • LS LC light chain shuffling
  • Example 1 After coating 50 ⁇ g of the SARS-CoV-2-RBD-hFc protein antigen prepared in Example 1 on an immunosorbent tube, blocking was performed. After the LS shuffling human scFv library having a diversity of 1 ⁇ 10 7 obtained in Example 3.1. was infected with E. coli, E. coli was cultured at 30° C. for 16 hours. The culture medium was centrifuged, and the supernatant was concentrated with PEG, and then dissolved in PBS buffer to prepare a human antibody library.
  • Single clones were selected from the positive phage pool of round 2 panning confirmed to have high binding capacity through poly phage ELISA, and direct ELISA was performed in the same manner as in Example 2.3. Specificity was confirmed. As a result, it was confirmed that the mono scFv-phage clones had strong binding ability only to SARS-CoV-2-RBD as shown in FIG. 4 .
  • phagemid DNA was isolated using a DNA purification kit (Qiagen, Germany), and DNA sequences were analyzed. Since antibody optimization by LC shuffling was performed, the sequence of the heavy chain is the same as that of the parent antibody. Accordingly, the amino acid sequence of the light chain CDR and the amino acid sequence of the light chain FR are shown in Tables 8 and 9, respectively, and the amino acid sequence of the light chain variable region and the polynucleotide sequence encoding it are shown in Tables 10 and 11, respectively.
  • the base sequence of the heavy chain variable region was cloned into pNATVH (YBiologics Co., Ltd.) using restriction enzymes SfiI/NheI site to N293F
  • An HC vector was prepared, and the nucleotide sequence of the light chain variable region was cloned into pNATVL (YBiologics Co., Ltd.) using restriction enzymes SfiI/BsiWI site to prepare an N293F LC vector.
  • N293F HC and N293F LC vectors were co-transfected into HEK293F cells, and the culture medium was collected on the 7th day of culture and centrifuged at 8,000 rpm for 30 minutes to remove cell debris. Thereafter, it was filtered using a bottle top filter (Steritop-GP Filter Unit. #SCGPS01RE, Millipore) having a pore size of 0.22 ⁇ m.
  • a bottle top filter Steitop-GP Filter Unit. #SCGPS01RE, Millipore having a pore size of 0.22 ⁇ m.
  • 4 ml of protein A sepharose resin slurry (KANEKA KanCapA TM , Cat. No.
  • KKC20170403_01 was put into an empty column (#BR731-1550, Bio-rad), and then 100 ml of DPBS (#LB001-02) ) to pack and wash the resin.
  • the filtered culture solution was loaded on the packed resin and flowed at a rate of 1 ml per minute (#EP-1 Econo pump, Bio-Rad).
  • After washing with 150 ml of DPBS it was eluted with 10 ml of 0.1 M glycine-HCl (pH 3.3). 10% of 1 M Tris-HCl (pH 9.0) was added to the eluate to neutralize the pH, and the buffer was changed to DPBS using Amicon Ultra-10 (#UFC901096, Millipore). After carrying out this process about 3 times, it was stopped when it was concentrated to about 1 ml, and the concentration was measured with a Nano-drop.
  • the antigen specificity of the SARS-CoV-2 spike human monoclonal antibody was confirmed by using a cell pool overexpressing the SARS-CoV-2 spike in HEK293E cells and analyzing the degree of binding of the antibody with a flow cytometer.
  • the pool of transgenic cells overexpressing the SARS-CoV-2 spike has the SARS-CoV-2 spike extracellular domain (amino acid sequence M1-P1213, UniProt#P0DTC2) and the transmembrane to intracellular domains of the epithelial cell adhesion molecule (EpCAM) (The pcDNA3.1 plasmid containing the chimeric sequence linked to the amino acid sequence A266-A314, UniProt#P16422) was transfected into HEK293E, and then 200 mg/ml of Zeocin (#R25001, thermo Fisher Scientific) was added. The selection process was performed in a selective culture medium.
  • SARS-CoV-2 spikes on the cell surface was detected by fluorescence activated cell sorting (FACS) using a SARS-CoV spike antibody (#40150-T62-CoV2, Sino Biological) that also recognizes SARS-CoV-2 spikes. ) was confirmed through analysis and used separately.
  • FACS fluorescence activated cell sorting
  • SARS-CoV-2 spike human monoclonal antibodies bound well to SARS-CoV-2 spike-overexpressing HEK293E cells (HEK293E/CoV-2 spike (chimeric)) (FIGS. 5b and 5d), but HEK293E cells (HEK293E) /Mock) did not bind ( FIGS. 5A and 5C ).
  • This result shows that Ymax ® -ABL-derived 7 types ( FIGS. 5a and 5b ) and patient-immune library-derived 8 types ( FIGS. 5c and 5d ) antibodies are non-specific to HEK293E, and expressed in HEK293E It means that these are antibodies that specifically bind to the SARS-CoV-2 spike protein.
  • SARS-CoV-2 spike human monoclonal antibody has binding specificity for SARS-CoV-2 spike S1 (amino acid sequence V16-R685, UniProt#P0DTC2) as well as SARS-CoV spike S1 (amino acid sequence S14-R667, UniProt#P59594)
  • SARS-CoV-2 spike S1 amino acid sequence V16-R685, UniProt#P0DTC2
  • SARS-CoV spike S1 amino acid sequence S14-R667, UniProt#P59594
  • SARS-CoV spike S1-His #40150-V08B1, Sino Biological
  • SARS-CoV-2 spike S1-His protein at a concentration of 200 ng per well was aliquoted into an immuno-96 microwell plate. After surface fixation at 4°C overnight, 200 ⁇ l of PBS-T containing 4% skim milk powder was put into all wells and reacted at 37°C for 1 hour to block non-specific protein binding.
  • SARS-CoV-2 spike human monoclonal antibodies of Ymax ® -ABL-derived 7 (FIG. 6a) and patient-immune library-derived 8 (FIG. 6B) did not bind to SARS-CoV spike S1, and specific was shown to bind to SARS-CoV-2 spike S1 ( FIGS. 6a and 6b ).
  • the binding affinity of the SARS-CoV-2 spike human monoclonal antibody to the antigen is determined by fusion of a mouse Fc (mFc)-tag to the carboxy-terminus of SARS-CoV-2 RBD (amino acid sequence R319-F541, UniProt#P0DTC2). Octet using the recombinant protein SARS-CoV-2 RBD-mFc antigen It was measured using QKe (Fortebio Inc., USA) analysis equipment.
  • mFc mouse Fc
  • Octet For affinity measurement between antigen-antibody using QKe, AHC (anti-human Fc capture: #18-5060, Fortebio Inc.) or AMC (anti-mouse Fc capture: #18-5088, Fortebio Inc.) biosensor buffer (#18-1042, Fortebio Inc.) After stabilization for 10 minutes, the antigen or antibody was fixed, and the non-immobilized antigen or antibody was washed with buffer for 5 minutes. Prepare the desired antibody or antigen for binding in a 96-well plate (#655209, Greiner Bio-One, USA) at each concentration (0.94 nM ⁇ 60 nM), perform an association reaction for 5 minutes, and then dissociate for 5 minutes (dissociation) reaction was performed.
  • AHC anti-human Fc capture: #18-5060, Fortebio Inc.
  • AMC anti-mouse Fc capture: #18-5088, Fortebio Inc.
  • biosensor buffer #18-1042, Fortebio Inc.
  • SARS-CoV-2 spike human monoclonal antibody, Ymax ® -ABL-derived 7 (Fig. 7a) and patient-immune library-derived 8 (Fig. 7b) antibodies had antigen affinity for SARS-CoV-2 RBD.
  • Figure (K D ) was excellent at 0.0284 nM ⁇ 1.06 nM, and the values of each antibody are shown in Table 12.
  • Table 12 summarizes the antigen affinity of the anti-SARS-CoV-2 spike monoclonal antibody by Octet analysis.
  • A affinity maturation
  • SARS-CoV-2 spiked human monoclonal antibody variants showed similar or superior binding compared to the parental antibody to SARS-CoV-2 spiked over-expressing HEK293E cells (HEK293E/CoV-2 spiked (chimeric)).
  • FIG. 8b did not bind to HEK293E cells (HEK293E/Mock) (FIG. 8a).
  • SA4079, SA4086, SA4114, SA4118 are non-specific to HEK293E, and antibodies that specifically bind to the SARS-CoV-2 spike protein expressed in HEK293E means to take
  • SARS-CoV-2 spike human monoclonal antibody mutant maintains binding specificity for SARS-CoV-2 spike S1 was confirmed by ELISA analysis as in Example 5.2 by comparison with SARS-CoV spike S1.
  • SARS-CoV-2 spike human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) did not bind to SARS-CoV spike S1 like the parent antibody, respectively, and specifically SARS-CoV-2 spike S1 was bound to (FIG. 9).
  • SARS-CoV-2 spike human monoclonal antibody variants is SARS-CoV-2 RBD-mFc, a recombinant protein in which a mouse Fc (mFc)-tag is fused to the carboxy-terminus as in Example 5.3.
  • SARS-CoV-2 spike human monoclonal antibody mutants SA4079, SA4086, SA4114, SA4118
  • K D antigen affinity for SARS-CoV-2 RBD from 0.0899 nM to 0.157 nM
  • Table 13 summarizes the antigen affinity of the anti-SARS-CoV-2 spike monoclonal antibody variants by Octet analysis.
  • Example 7 Determination of antigen binding site (epitope) of SARS-CoV-2 spike human monoclonal antibody and variants
  • the antibody binds to the RBM region of SARS-CoV-2 RBD that binds ACE2. Whether the antibody binds to the SARS-CoV-2 RBM region is determined by substituting the RBM amino acid region in the RBD of SARS-CoV-2 with the RBM of the SARS-CoV RBD (amino acid sequence R306-F527, UniProt#P59594) as shown in Table 14 and FIG. 12A.
  • Ymax ® -ABL-derived 7 types (FIG. 11a) and patient-immune library-derived 8 types (FIG. 11b) SARS-CoV-2 RBD human monoclonal antibodies and Ymax ® -ABL-derived SA4079 optimized for 4 types , SA4086, SA4114, SA4118 antibody variants ( FIG. 11C ) bound to the antigens SARS-CoV-2 RBD and SARS-CoV_RBM_CoV-2, but not SARS-CoV-2_RBM_CoV or SARS-CoV RBD.
  • This result means that the SARS-CoV-2 spike human monoclonal antibody and its variants specifically bind to the RBM region of SARS-CoV-2 RBD that binds to ACE2, indicating that antibodies having an epitope in this region. there is.
  • Table 14 shows the RBD mutant sequence of SARS-CoV-2 or SARS-CoV.
  • Example 7.2 Identification of antigen binding site (epitope) of SARS-CoV-2 spike human monoclonal antibody
  • SARS-CoV-2 spike antibodies did not bind to each recombinant mutant under conditions of sufficient binding to WT of SARS-CoV-2 RBD, or there were antibodies that did not bind or were very low.
  • gray box were grouped into 9 groups (G1 to G9) as shown in FIG. 12b.
  • the SARS-CoV-2 spike human monoclonal antibody and the mutants did not bind to the recombinant SARS-CoV-2 RBD mutants, or the region (dark gray box) was the epitope, the antigen-binding site of the actual antibodies.
  • points to The epitope sequence for each group of 15 SARS-CoV-2 spike human monoclonal antibodies and 4 antibody variants is the WT sequence of SARS-CoV-2 RBD corresponding to the mutant sequence of Table 14 and FIG. 12 .
  • antibodies SA4053, SA4055, SA4050, SA3779, SA4086 (SA3779AM), SA3902, SA3838, SA4118 (SA3838AM), SA3827, SA4114 (SA3827AM), SA4040, SA4043, SA4056, SA4057, SA3830, SA3856, SA4044) were antibodies that contained all or part of the M2, M8, M9, or M10 regions, or had additional epitopes in other regions externally.
  • SA4079 SA4079 (SA3755AM)
  • SA4053 had an epitope in M5
  • SA4053, SA3830, SA3856 was in M6
  • SA4044 was in M7
  • SA4053 was an antibody having or containing an epitope in the M12 region.
  • SARS-CoV-2 spike human monoclonal antibody can inhibit the formation of ACE2/SARS-CoV-2 RBD complex or ACE2/SARS-CoV-2 spike S1 complex
  • 1.94 nM SARS-CoV-2 RBD Antibodies to -mFc or 200 nM SARS-CoV-2 spike S1-mFc protein were serially diluted from 500 nM to 1/3 dilution fold. Thereafter, after pre-incubation at 37° C. for 30 minutes, the antigen-antibody mixture was prepared with 200 ng per well of ACE2-His, a recombinant protein in which His-tag was fused to the carboxy-terminus of ACE2 (UniProt#Q9BYF1). was added to an immune-96 microwell plate coated with , and reacted at 37° C. for 1 hour.
  • a rabbit anti-mouse IgG-HPR antibody (Cell signaling) bound to horseradish peroxidase (HRP) was diluted at 1:2,000 and added at 37°C. was reacted for 1 hour.
  • 100 ⁇ l of TMB buffer solution was added and reacted for 1 minute and 30 seconds in the absence of light, and then the reaction was stopped with 50 ⁇ l of 2.5 M sulfuric acid (H 2 SO 4 ).
  • absorbance was measured at 450 nm using a spectrophotometer (spectraMax M5 spectrophotometer, Molecular Devices, USA). Measurement results were analyzed using GraphPad Prism 8 (GraphPad Software, Inc., USA).
  • the ability of the SARS-CoV-2 spike human monoclonal antibody to inhibit ACE2/SARS-CoV-2 RBD or ACE2/SARS-CoV-2 spike S1 binding was related to the SARS-CoV-2 RBD-mFc or SARS-CoV-2 spike. It can be seen from the decrease in the binding force of S1-mFc, and the degree of inhibition can be expressed as the amount of sample (IC 50 ) required to reach 50% of the maximum inhibition by the antibody. From the experimental results, it was confirmed that the SARS-CoV-2 spike human monoclonal antibodies effectively inhibited ACE2 and SARS-CoV-2 RBD or SARS-CoV-2 spike S1 binding in a concentration-dependent manner ( FIGS. 13a to 13d ).
  • the IC 50 values for SARS-CoV-2 RBD of Ymax ® -ABL-derived 7 ( FIGS. 13a and 13b ) and 8 ( FIGS. 13c and 13d ) antibodies from the patient-immune library were 0.91 nM to 25.08 nM, respectively. 0.14 nM to 58.27 nM ( FIGS. 13A and 13C ), and 0.61 nM to 4.06 nM and 1.43 nM to 1.43 nM to 22 nM for SARS-CoV-2 spike S1, respectively ( FIGS. 13B and 13D ). showed superior characteristics.
  • the IC 50 values of each antibody are shown in Table 15.
  • Table 15 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody by the competitive enzyme immunoassay.
  • a pool of transformed cells overexpressing human ACE2 was selected in a selective culture medium containing 200 mg/ml Zeocin (#R25001, thermo Fisher Scientific) after transfection of the pcDNA3.1 plasmid containing human ACE2 into HEK293E. The process was carried out. After the selection process, the cell pool was isolated by confirming the expression status through FACS analysis using goat anti-human ACE2 antibody (#AF933, R&D system), and SARS-CoV-2 by SARS-CoV-2 spike human monoclonal antibody It was used to confirm the neutralizing ability of RBD and ACE2 binding inhibition.
  • the prepared human ACE2-overexpressing HEK293E cells (HEK293E/ACE2) were divided into 3 ⁇ 10 5 cells per sample, respectively.
  • the antigen-antibody mixture was reacted with the prepared cells at 4° C. for 30 minutes. Thereafter, the cells were washed 3 times with PBS containing 2% FBS, and an anti-mouse IgG antibody (#FI- 2000, Vectorlabs) after dark reaction at 4° C. for 30 minutes, followed by the same washing process. After suspension in PBS containing 0.2 ml of 2% FBS, analysis was performed using a flow cytometer, CytoFLEX (Beckman coulter, USA).
  • the neutralizing ability of the SARS-CoV-2 spike human monoclonal antibody to inhibit SARS-CoV-2 RBD and ACE2 binding can be seen from the decrease in the binding affinity of SARS-CoV-2 RBD-mFc binding to ACE2-expressing cells, and its inhibition
  • the degree can be expressed as the amount of sample (IC 50 ) required to reach 50% of the maximum inhibition by the antibody.
  • Table 16 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody by the cell-based competition assay.
  • SARS-CoV-2 RBD-hFc conjugated with Incucyte ® Human FabFluor-pH Red Antibody Labeling Reagent binds to cell surface ACE2 and enters the cell (internalization). It is possible to check the presence and extent of inflow.
  • Cells were imaged using the IncuCyte ZOOM HD/2CLR System (Essen Biosciences, USA) for 24 hours at intervals of 15 to 30 minutes, and all three channels of phase, green, and red were imaged, 'Total Red Object Area ( ⁇ m 2 /well)' was analyzed.
  • SARS-CoV-2 spike human monoclonal antibody inhibits the SARS-CoV-2 RBD influx through ACE2 can be known by reducing the red fluorescence of the SARS-CoV-2 RBD antigen in the cell, and the inhibition The degree can be expressed as the amount of sample required to reach 50% of the maximum inhibition by the antibody (IC 50 ), and was analyzed using GraphPad Prism 8 software.
  • FIGS. 15A and 15B The IC 50 values for SARS-CoV-2 RBD of Ymax ® -ABL-derived 7 ( FIG. 15A ) and 8 ( FIG. 15B ) antibodies from the patient-immune library were 11.92 nM to 40.02 nM and 12.78 nM to 62.72 nM, respectively. , IC 50 values of each antibody are shown in Table 17.
  • Table 17 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody by the intracellular influx inhibition assay.
  • Antibody neutralization ability against SARS-CoV-2 virus was confirmed by microneutralization assay together with Vero cells, an African green monkey kidney cell line.
  • the obtained image was obtained by obtaining the area intensity using the National Institute of Health (NIH) ImageJ program, and calculating the % viability/inhibition to SARS-CoV-2 virus through GrapPad Prism 5 software. Antibody neutralizing capacity IC 50 values were calculated.
  • NASH National Institute of Health
  • FIGS. 16A and 16B The IC 50 values of the Ymax ® -ABL-derived 7 ( FIG. 16A ) and 8 ( FIG. 16B ) antibodies from the patient-immune library against SARS-CoV-2 virus were 8.736 nM to 231 nM and 0.7102 nM to 346 nM, respectively. , IC 50 values of each antibody are shown in Table 18.
  • Table 18 summarizes the results of confirming the neutralization ability of the anti-SARS-CoV-2 spike monoclonal antibody by the microneutralization assay.
  • SARS-CoV-2 spike human monoclonal antibody variants can inhibit SARS-CoV-2 RBD or SARS-CoV-2 spike S1 binding binding to ACE2
  • competitive enzyme immunization was performed in the same manner as in Example 8.1. The assay was performed.
  • SARS-CoV-2 spike human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) inhibited ACE2 and SARS-CoV-2 RBD or SARS-CoV-2 spike S1 binding in a concentration-dependent manner. It was confirmed, and each parent antibody showed similar or superior neutralizing efficacy ( FIGS. 17A and 17B ).
  • the IC 50 values for SARS-CoV-2 RBD of SARS-CoV-2 spike human monoclonal antibody variants were between 0.7086 nM and 3.625 nM ( FIG. 17A ) and between 1.194 nM and 2.711 nM for SARS-CoV-2 spike S1.
  • FIG. 17b IC 50 values of the antibody variants compared with each parent antibody are shown in Table 19.
  • Table 19 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody mutant by the competitive enzyme immunoassay.
  • SARS-CoV-2 spike human monoclonal antibody variant can inhibit SARS-CoV-2 RBD and ACE2 binding, as in Example 8.2, human ACE2 overexpressing HEK293E cells (HEK293E/ACE2) and SARS-CoV -2 A cell-based competition assay using RBD-mFc protein was performed and analyzed.
  • SARS-CoV-2 spike human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) inhibited SARS-CoV-2 RBD and ACE2 binding in a concentration-dependent manner, and were similar or superior to the parent antibody, respectively. It showed neutralizing ability (Fig. 14c).
  • the IC 50 values for SARS-CoV-2 RBD of each antibody variant were all excellent at nanomolar levels between 2.346 nM and 1.705 nM, and it was confirmed that they were very superior compared to ACE2, the SARS-CoV-2 RBD receptor treated as a control. did Table 20 shows the IC 50 values of the antibody variants compared to each parent antibody.
  • Table 20 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody variants by the cell-based competition assay.
  • SARS-CoV-2 RBD When SARS-CoV-2 RBD binds to ACE2 present on the cell surface and enters the cell (internalization), the SARS-CoV-2 spike human monoclonal antibody variant can inhibit it like each parent antibody.
  • SARS-CoV-2 spike human monoclonal antibody variant can inhibit it like each parent antibody.
  • HEK293E cells overexpressing recombinant ACE2-GFP (HEK293E/ACE2-GFP) and Incucyte ® internalization assay using the SARS-CoV-2 RBD-hFc antigen was performed.
  • SARS-CoV-2 spiked human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) effectively inhibited the intracellular influx of SARS-CoV-2 RBD through binding to ACE2 like each parent antibody in a concentration-dependent manner. It was confirmed that inhibition (FIG. 18).
  • the IC 50 values for SARS-CoV-2 RBD of the antibody variants were between 24.06 nM and 39.97 nM, and the IC 50 values of the antibody variants compared to each parent antibody are shown in Table 21.
  • Table 21 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody variant by the intracellular influx inhibition assay.
  • Live SARS-CoV-2 virus was injected into Vero cells as in Example 8.4. After infection for a period of time, it was confirmed by performing a microneutralization assay.
  • the antibody variants (SA4079, SA4086, SA4114, SA4118) selected by optimizing the four SARS-CoV-2 spike human monoclonal antibodies (SA3755, SA3779, SA3827, SA3838) were concentration-dependent like each parent antibody. It was confirmed that it effectively neutralized the infection of SARS-CoV-2 virus ( FIGS. 19a and 19b ).
  • the IC 50 values of the antibody variants against SARS-CoV-2 virus were between 2.741 nM and 12.83 nM, and the IC 50 values of the antibody variants compared to each parent antibody are shown in Table 22.
  • Table 22 summarizes the results of confirming the neutralization ability of the anti-SARS-CoV-2 spike monoclonal antibody variant by the microneutralization assay.

Abstract

The present invention provides: an antibody binding specifically to membrane protein spikes of the new coronavirus SARS CoV-2, or an antigen-binding fragment thereof; a nucleic acid encoding same; a recombinant expression vector including the nucleic acid; a cell transformed with the vector; a method for preparing the antibody or the antigen-binding fragment thereof; an antibody-drug conjugate (ADC) or multispecific antibody including the antibody or the antigen-binding fragment thereof; a pharmaceutical composition for prevention or treatment of SARS-CoV-2 infection, a composition for diagnosis of SARS-CoV-2 infection, and a diagnostic kit, each comprising the antibody or the antigen-binding fragment thereof. The antibody or the antigen-binding fragment thereof according to the present invention binds specifically to SARS-CoV-2 spike protein and thus completely blocks the binding of the membrane protein spikes of SARS-CoV-2 to the receptor angiotensin converting enzyme-2 (ACE2) on human cell surfaces, finding advantageous applications in preventing, treating, or diagnosing new coronavirus infections.

Description

SARS-CｏV-2 스파이크 단백질에 특이적으로 결합하는 항체 및 이의 용도Antibodies specifically binding to SARS-CｏV-2 spike protein and uses thereof
본 발명은 신종 코로나바이러스 SARS-CoV-2의 막 단백질 스파이크(spike)에 특이적으로 결합하는 항체 및 이의 용도에 관한 것이다. The present invention relates to an antibody that specifically binds to the membrane protein spike of novel coronavirus SARS-CoV-2 and uses thereof.
신종 코로나바이러스 감염증(COVID-19)은 2019년 12월 중국 우한에서 대규모로 발병한 호흡기 감염질환이다. 치명률은 약 5%로서 기존 SARS(약 9.6%)나 MERS(약 34.4%)보다는 약하나, 사람간 전염성이 매우 강하여 세계적으로 빠르게 확산됨으로써 2020년 3월 WHO에 의해 팬더믹(pandemic)이 선언되었다. The novel coronavirus infection (COVID-19) is a respiratory infection that occurred on a large scale in Wuhan, China in December 2019. The fatality rate is about 5%, which is weaker than the existing SARS (about 9.6%) or MERS (about 34.4%), but it is highly contagious from person to person and spread rapidly worldwide, and in March 2020, it was declared a pandemic by the WHO.
한편, SARS-CoV-2의 막 단백질 스파이크(spike)가 인간 세포막에 있는 특정 수용체인 ACE2(angiotensin converting enzyme-2)에 결합한 후, 코로나바이러스의 유전적 정보를 인간 세포에 주입한 다음, 세포 내에서 복사 및 재조립을 계속한다. 결과적으로, 코로나바이러스는 세포를 파괴시킨 후, 세포 외로 방출되며, 짧은 시간내에 심각한 인체 감염을 일으킨다는 점이 보고된 바 있다(Haibo Zhang et.al., Intensive Care Med., 46(4):586-590, 2020).On the other hand, after the membrane protein spike of SARS-CoV-2 binds to ACE2 (angiotensin converting enzyme-2), a specific receptor in the human cell membrane, the genetic information of the coronavirus is injected into human cells, and then Continue copying and reassembling from As a result, it has been reported that coronaviruses are released extracellularly after destroying cells and cause serious human infection within a short time (Haibo Zhang et.al. , Intensive Care Med. , 46(4):586). -590, 2020).
신종 코로나바이러스 감염증을 일으키는 바이러스인 SARS-CoV-2(또는 2019-nCoV)는 기존 SARS(SARS-CoV) 및 MERS(MERS-CoV)와 게놈 유전체의 상동성이 낮아 COVID-19의 치료용으로 기존의 치료제들을 사용할 수 없는 실정이다. 현재 SARS-CoV-2를 타겟하는 치료용 항체 중 임상단계를 진행 중인 것은 소수이며, 감염환자 유래의 항체를 발굴하거나 변이체에 적용 가능한 다양한 항체를 확보하는 것이 필요하다. 또한, 바이러스 변이성을 고려하여 서로 다른 에피토프(epitope)를 갖는 항체들을 병용 투여하는 것이 필요하다.SARS-CoV-2 (or 2019-nCoV), a virus that causes novel coronavirus infection, has low genomic homology with existing SARS (SARS-CoV) and MERS (MERS-CoV). There are currently no treatments available. Currently, few of the therapeutic antibodies targeting SARS-CoV-2 are in the clinical stage, and it is necessary to discover antibodies derived from infected patients or to secure various antibodies applicable to mutants. In addition, it is necessary to concurrently administer antibodies having different epitopes in consideration of viral variability.
한편, COVID-19의 치료제로서 다른 용도로 개발된 기존 약물을 이용하는 약물 재창출의 경우, 에볼라바이러스의 치료제인 길리어드 사이언스사의 렘데시비르가 미국, 유럽, 일본에서 중증 환자 대상으로 승인되어 잠재적 치료제로 기대되나, 유효성 및 안전성 논란이 제기되고 있다. 또한, 에이즈(AIDS) 치료제인 로피나비르 및 리토나비르와 말라리아 치료제인 하이드록시클로로퀸 등은 효능이 거의 없는 것으로 보고되었다. 뿐만 아니라, 완치 환자의 혈장을 제제화하여 감염 환자에게 투입하는 혈장 치료제도 개발되고 있지만, 이는 생산 시 충분한 혈장 확보가 필요하므로 다수를 대상으로 이용하는 치료제로서는 적합하지 않다. 이러한 약물 재창출 치료제 및 혈장 치료제는 여러 가지 제한이 있고 효력 및 안전성의 확보가 미비하며 이들을 보완할 수 있는 항-SARS-CoV-2 항체 치료제의 개발이 요구되고 있다.On the other hand, in the case of drug re-creation using existing drugs developed for other purposes as a treatment for COVID-19, Gilead Sciences' remdesivir, a treatment for Ebola virus, has been approved for severe patients in the United States, Europe, and Japan and is a potential treatment. However, controversy over efficacy and safety is being raised. In addition, it has been reported that lopinavir and ritonavir, an AIDS treatment, and hydroxychloroquine, a treatment for malaria, have little effect. In addition, plasma therapeutics that are formulated from the plasma of cured patients and injected into infected patients are being developed, but this is not suitable as a therapeutic agent used for a large number of subjects because sufficient plasma must be secured during production. These drug re-creation therapeutics and plasma therapeutics have various limitations and lack of securing efficacy and safety, and the development of anti-SARS-CoV-2 antibody therapeutics that can supplement them is required.
[선행기술문헌][Prior art literature]
[비특허문헌][Non-patent literature]
(비특허문헌 1) Haibo Zhang et.al., Intensive Care Med., 46(4):586-590, 2020(Non-Patent Document 1) Haibo Zhang et.al., Intensive Care Med., 46(4):586-590, 2020
이에, 본 발명자들은 SARS-CoV-2를 직접 타겟하는 치료용 항체를 개발하기 위해 연구한 결과, SARS-CoV-2의 막 단백질 스파이크(spike)에 특이적으로 결합하는 항체들을 개발하였다. 또한, 상기 항체들이 인간 세포 수용체인 ACE2(angiotensin converting enzyme-2)와 SARS-CoV-2 스파이크의 결합을 완전히 차단함으로써 신종 코로나바이러스 감염증의 치료제로 이용될 수 있음을 확인하여 본 발명을 완성하였다. Accordingly, as a result of research to develop a therapeutic antibody that directly targets SARS-CoV-2, the present inventors developed antibodies that specifically bind to the membrane protein spike of SARS-CoV-2. In addition, the present invention was completed by confirming that the antibodies can be used as a treatment for novel coronavirus infection by completely blocking the binding of the human cell receptor angiotensin converting enzyme-2 (ACE2) to the SARS-CoV-2 spike.
상기 목적 달성을 위해, 본 발명의 일 측면은 SARS-CoV-2의 막 단백질 스파이크에 특이적으로 결합하여 신종 코로나바이러스 감염증에 대해 예방 또는 치료 효과를 나타낼 수 있는 항체 또는 이의 항원 결합 단편을 제공한다.In order to achieve the above object, one aspect of the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to the membrane protein spike of SARS-CoV-2 and exhibit a preventive or therapeutic effect on novel coronavirus infection. .
구체적으로, 하기 군에서 선택되는 어느 하나의 CDR(complementarity determining region) 조합을 포함하는 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 제공한다:Specifically, there is provided an antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 spike protein comprising any one CDR (complementarity determining region) combination selected from the following group:
(1) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 서열번호 3의 중쇄 CDR3, 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 서열번호 6의 경쇄 CDR3;(1) heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, light chain CDR3 of SEQ ID NO: 6;
(2) 서열번호 7의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 서열번호 9의 중쇄 CDR3, 서열번호 10의 경쇄 CDR1, 서열번호 11의 경쇄 CDR2, 서열번호 12의 경쇄 CDR3;(2) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 10, light chain CDR2 of SEQ ID NO: 11, light chain CDR3 of SEQ ID NO: 12;
(3) 서열번호 13의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 서열번호 15의 중쇄 CDR3, 서열번호 16의 경쇄 CDR1, 서열번호 17의 경쇄 CDR2, 서열번호 18의 경쇄 CDR3;(3) heavy chain CDR1 of SEQ ID NO: 13, heavy chain CDR2 of SEQ ID NO: 14, heavy chain CDR3 of SEQ ID NO: 15, light chain CDR1 of SEQ ID NO: 16, light chain CDR2 of SEQ ID NO: 17, light chain CDR3 of SEQ ID NO: 18;
(4) 서열번호 19의 중쇄 CDR1, 서열번호 20의 중쇄 CDR2, 서열번호 21의 중쇄 CDR3, 서열번호 22의 경쇄 CDR1, 서열번호 23의 경쇄 CDR2, 서열번호 24의 경쇄 CDR3;(4) heavy chain CDR1 of SEQ ID NO: 19, heavy chain CDR2 of SEQ ID NO: 20, heavy chain CDR3 of SEQ ID NO: 21, light chain CDR1 of SEQ ID NO: 22, light chain CDR2 of SEQ ID NO: 23, light chain CDR3 of SEQ ID NO: 24;
(5) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2, 서열번호 27의 중쇄 CDR3, 서열번호 28의 경쇄 CDR1, 서열번호 29의 경쇄 CDR2, 서열번호 30의 경쇄 CDR3;(5) heavy chain CDR1 of SEQ ID NO: 25, heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 28, light chain CDR2 of SEQ ID NO: 29, light chain CDR3 of SEQ ID NO: 30;
(6) 서열번호 31의 중쇄 CDR1, 서열번호 32의 중쇄 CDR2, 서열번호 33의 중쇄 CDR3, 서열번호 34의 경쇄 CDR1, 서열번호 35의 경쇄 CDR2, 서열번호 36의 경쇄 CDR3;(6) heavy chain CDR1 of SEQ ID NO: 31, heavy chain CDR2 of SEQ ID NO: 32, heavy chain CDR3 of SEQ ID NO: 33, light chain CDR1 of SEQ ID NO: 34, light chain CDR2 of SEQ ID NO: 35, light chain CDR3 of SEQ ID NO: 36;
(7) 서열번호 37의 중쇄 CDR1, 서열번호 38의 중쇄 CDR2, 서열번호 39의 중쇄 CDR3, 서열번호 40의 경쇄 CDR1, 서열번호 41의 경쇄 CDR2, 서열번호 42의 경쇄 CDR3;(7) heavy chain CDR1 of SEQ ID NO: 37, heavy chain CDR2 of SEQ ID NO: 38, heavy chain CDR3 of SEQ ID NO: 39, light chain CDR1 of SEQ ID NO: 40, light chain CDR2 of SEQ ID NO: 41, light chain CDR3 of SEQ ID NO: 42;
(8) 서열번호 43의 중쇄 CDR1, 서열번호 44의 중쇄 CDR2, 서열번호 45의 중쇄 CDR3, 서열번호 46의 경쇄 CDR1, 서열번호 47의 경쇄 CDR2, 서열번호 48의 경쇄 CDR3;(8) heavy chain CDR1 of SEQ ID NO: 43, heavy chain CDR2 of SEQ ID NO: 44, heavy chain CDR3 of SEQ ID NO: 45, light chain CDR1 of SEQ ID NO: 46, light chain CDR2 of SEQ ID NO: 47, light chain CDR3 of SEQ ID NO: 48;
(9) 서열번호 49의 중쇄 CDR1, 서열번호 50의 중쇄 CDR2, 서열번호 51의 중쇄 CDR3, 서열번호 52의 경쇄 CDR1, 서열번호 53의 경쇄 CDR2, 서열번호 54의 경쇄 CDR3;(9) heavy chain CDR1 of SEQ ID NO: 49, heavy chain CDR2 of SEQ ID NO: 50, heavy chain CDR3 of SEQ ID NO: 51, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 53, light chain CDR3 of SEQ ID NO: 54;
(10) 서열번호 55의 중쇄 CDR1, 서열번호 56의 중쇄 CDR2, 서열번호 57의 중쇄 CDR3, 서열번호 58의 경쇄 CDR1, 서열번호 59의 경쇄 CDR2, 서열번호 60의 경쇄 CDR3;(10) heavy chain CDR1 of SEQ ID NO: 55, heavy chain CDR2 of SEQ ID NO: 56, heavy chain CDR3 of SEQ ID NO: 57, light chain CDR1 of SEQ ID NO: 58, light chain CDR2 of SEQ ID NO: 59, light chain CDR3 of SEQ ID NO: 60;
(11) 서열번호 61의 중쇄 CDR1, 서열번호 62의 중쇄 CDR2, 서열번호 63의 중쇄 CDR3, 서열번호 64의 경쇄 CDR1, 서열번호 65의 경쇄 CDR2, 서열번호 66의 경쇄 CDR3;(11) heavy chain CDR1 of SEQ ID NO: 61, heavy chain CDR2 of SEQ ID NO: 62, heavy chain CDR3 of SEQ ID NO: 63, light chain CDR1 of SEQ ID NO: 64, light chain CDR2 of SEQ ID NO: 65, light chain CDR3 of SEQ ID NO: 66;
(12) 서열번호 67의 중쇄 CDR1, 서열번호 68의 중쇄 CDR2, 서열번호 69의 중쇄 CDR3, 서열번호 70의 경쇄 CDR1, 서열번호 71의 경쇄 CDR2, 서열번호 72의 경쇄 CDR3;(12) heavy chain CDR1 of SEQ ID NO: 67, heavy chain CDR2 of SEQ ID NO: 68, heavy chain CDR3 of SEQ ID NO: 69, light chain CDR1 of SEQ ID NO: 70, light chain CDR2 of SEQ ID NO: 71, light chain CDR3 of SEQ ID NO: 72;
(13) 서열번호 73의 중쇄 CDR1, 서열번호 74의 중쇄 CDR2, 서열번호 75의 중쇄 CDR3, 서열번호 76의 경쇄 CDR1, 서열번호 77의 경쇄 CDR2, 서열번호 78의 경쇄 CDR3;(13) heavy chain CDR1 of SEQ ID NO: 73, heavy chain CDR2 of SEQ ID NO: 74, heavy chain CDR3 of SEQ ID NO: 75, light chain CDR1 of SEQ ID NO: 76, light chain CDR2 of SEQ ID NO: 77, light chain CDR3 of SEQ ID NO: 78;
(14) 서열번호 79의 중쇄 CDR1, 서열번호 80의 중쇄 CDR2, 서열번호 81의 중쇄 CDR3, 서열번호 82의 경쇄 CDR1, 서열번호 83의 경쇄 CDR2, 서열번호 84의 경쇄 CDR3;(14) heavy chain CDR1 of SEQ ID NO: 79, heavy chain CDR2 of SEQ ID NO: 80, heavy chain CDR3 of SEQ ID NO: 81, light chain CDR1 of SEQ ID NO: 82, light chain CDR2 of SEQ ID NO: 83, light chain CDR3 of SEQ ID NO: 84;
(15) 서열번호 85의 중쇄 CDR1, 서열번호 86의 중쇄 CDR2, 서열번호 87의 중쇄 CDR3, 서열번호 88의 경쇄 CDR1, 서열번호 89의 경쇄 CDR2, 서열번호 90의 경쇄 CDR3;(15) heavy chain CDR1 of SEQ ID NO: 85, heavy chain CDR2 of SEQ ID NO: 86, heavy chain CDR3 of SEQ ID NO: 87, light chain CDR1 of SEQ ID NO: 88, light chain CDR2 of SEQ ID NO: 89, light chain CDR3 of SEQ ID NO: 90;
(16) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 서열번호 3의 중쇄 CDR3, 서열번호 271의 경쇄 CDR1, 서열번호 272의 경쇄 CDR2, 서열번호 273의 경쇄 CDR3;(16) heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 271, light chain CDR2 of SEQ ID NO: 272, light chain CDR3 of SEQ ID NO: 273;
(17) 서열번호 7의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 서열번호 9의 중쇄 CDR3, 서열번호 274의 경쇄 CDR1, 서열번호 275의 경쇄 CDR2, 서열번호 276의 경쇄 CDR3;(17) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 274, light chain CDR2 of SEQ ID NO: 275, light chain CDR3 of SEQ ID NO: 276;
(18) 서열번호 13의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 서열번호 15의 중쇄 CDR3, 서열번호 277의 경쇄 CDR1, 서열번호 278의 경쇄 CDR2, 서열번호 279의 경쇄 CDR3; 및 (18) heavy chain CDR1 of SEQ ID NO: 13, heavy chain CDR2 of SEQ ID NO: 14, heavy chain CDR3 of SEQ ID NO: 15, light chain CDR1 of SEQ ID NO: 277, light chain CDR2 of SEQ ID NO: 278, light chain CDR3 of SEQ ID NO: 279; and
(19) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2, 서열번호 27의 중쇄 CDR3, 서열번호 280의 경쇄 CDR1, 서열번호 281의 경쇄 CDR2, 서열번호 282의 경쇄 CDR3.(19) heavy chain CDR1 of SEQ ID NO: 25, heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 280, light chain CDR2 of SEQ ID NO: 281, light chain CDR3 of SEQ ID NO: 282.
또한, 하기 군에서 선택되는 어느 하나의 가변영역(variable region) 조합을 포함하는 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 제공한다:Also provided is an antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 spike protein comprising a combination of any one variable region selected from the following group:
(1) 서열번호 211의 중쇄 가변영역 및 서열번호 212의 경쇄 가변영역;(1) a heavy chain variable region of SEQ ID NO: 211 and a light chain variable region of SEQ ID NO: 212;
(2) 서열번호 213의 중쇄 가변영역 및 서열번호 214의 경쇄 가변영역;(2) the heavy chain variable region of SEQ ID NO: 213 and the light chain variable region of SEQ ID NO: 214;
(3) 서열번호 215의 중쇄 가변영역 및 서열번호 216의 경쇄 가변영역;(3) a heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 216;
(4) 서열번호 217의 중쇄 가변영역 및 서열번호 218의 경쇄 가변영역;(4) a heavy chain variable region of SEQ ID NO: 217 and a light chain variable region of SEQ ID NO: 218;
(5) 서열번호 219의 중쇄 가변영역 및 서열번호 220의 경쇄 가변영역;(5) a heavy chain variable region of SEQ ID NO: 219 and a light chain variable region of SEQ ID NO: 220;
(6) 서열번호 221의 중쇄 가변영역 및 서열번호 222의 경쇄 가변영역;(6) a heavy chain variable region of SEQ ID NO: 221 and a light chain variable region of SEQ ID NO: 222;
(7) 서열번호 223의 중쇄 가변영역 및 서열번호 224의 경쇄 가변영역;(7) a heavy chain variable region of SEQ ID NO: 223 and a light chain variable region of SEQ ID NO: 224;
(8) 서열번호 225의 중쇄 가변영역 및 서열번호 226의 경쇄 가변영역;(8) a heavy chain variable region of SEQ ID NO: 225 and a light chain variable region of SEQ ID NO: 226;
(9) 서열번호 227의 중쇄 가변영역 및 서열번호 228의 경쇄 가변영역;(9) a heavy chain variable region of SEQ ID NO: 227 and a light chain variable region of SEQ ID NO: 228;
(10) 서열번호 229의 중쇄 가변영역 및 서열번호 230의 경쇄 가변영역;(10) a heavy chain variable region of SEQ ID NO: 229 and a light chain variable region of SEQ ID NO: 230;
(11) 서열번호 231의 중쇄 가변영역 및 서열번호 232의 경쇄 가변영역;(11) a heavy chain variable region of SEQ ID NO: 231 and a light chain variable region of SEQ ID NO: 232;
(12) 서열번호 233의 중쇄 가변영역 및 서열번호 234의 경쇄 가변영역;(12) a heavy chain variable region of SEQ ID NO: 233 and a light chain variable region of SEQ ID NO: 234;
(13) 서열번호 235의 중쇄 가변영역 및 서열번호 236의 경쇄 가변영역;(13) a heavy chain variable region of SEQ ID NO: 235 and a light chain variable region of SEQ ID NO: 236;
(14) 서열번호 237의 중쇄 가변영역 및 서열번호 238의 경쇄 가변영역;(14) a heavy chain variable region of SEQ ID NO: 237 and a light chain variable region of SEQ ID NO: 238;
(15) 서열번호 239의 중쇄 가변영역 및 서열번호 240의 경쇄 가변영역;(15) a heavy chain variable region of SEQ ID NO: 239 and a light chain variable region of SEQ ID NO: 240;
(16) 서열번호 211의 중쇄 가변영역 및 서열번호 299의 경쇄 가변영역;(16) a heavy chain variable region of SEQ ID NO: 211 and a light chain variable region of SEQ ID NO: 299;
(17) 서열번호 213의 중쇄 가변영역 및 서열번호 300의 경쇄 가변영역;(17) a heavy chain variable region of SEQ ID NO: 213 and a light chain variable region of SEQ ID NO: 300;
(18) 서열번호 215의 중쇄 가변영역 및 서열번호 301의 경쇄 가변영역; 및 (18) a heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 301; and
(19) 서열번호 219의 중쇄 가변영역 및 서열번호 302의 경쇄 가변영역.(19) the heavy chain variable region of SEQ ID NO: 219 and the light chain variable region of SEQ ID NO: 302.
본 발명의 다른 측면은 상기 항체 또는 이의 항원 결합 단편을 코딩하는 핵산 및 이를 포함하는 재조합 발현 벡터를 제공한다.Another aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof and a recombinant expression vector comprising the same.
본 발명의 또 다른 측면은 상기 벡터로 형질전환된 세포 및 이를 이용한 상기 항체 또는 이의 항원 결합 단편의 제조 방법을 제공한다.Another aspect of the present invention provides a cell transformed with the vector and a method for producing the antibody or antigen-binding fragment thereof using the same.
본 발명의 또 다른 측면은 상기 항체 또는 이의 항원 결합 단편 및 약물을 포함하는 항체-약물 접합체(antibody-drug conjugate, ADC)를 제공한다.Another aspect of the present invention provides an antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof and a drug.
본 발명의 또 다른 측면은 상기 항체 또는 이의 항원 결합 단편을 포함하는 다중특이적 항체(multispecific antibody)를 제공한다.Another aspect of the present invention provides a multispecific antibody comprising the antibody or antigen-binding fragment thereof.
본 발명의 또 다른 측면은 상기 항체 또는 이의 항원 결합 단편, 상기 항체-약물 접합체 또는 다중특이적 항체를 포함하는 SARS-CoV-2 감염증의 예방 또는 치료용 약학 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for preventing or treating SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or multispecific antibody.
본 발명의 또 다른 측면은 상기 항체 또는 이의 항원 결합 단편을 포함하는 SARS-CoV-2 감염증의 진단용 조성물을 제공한다.Another aspect of the present invention provides a composition for diagnosis of SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof.
본 발명의 또 다른 측면은 상기 항체 또는 이의 항원 결합 단편을 포함하는 SARS-CoV-2 스파이크 단백질의 검출 또는 정량용 키트를 제공한다.Another aspect of the present invention provides a kit for detecting or quantifying a SARS-CoV-2 spike protein comprising the antibody or antigen-binding fragment thereof.
본 발명에 따른 항체 또는 이의 항원 결합 단편은 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하여 인간세포 표면의 수용체인 ACE2(angiotensin converting enzyme-2)와 SARS-CoV-2 스파이크 단백질의 결합을 완전히 차단할 수 있으므로, 신종 코로나바이러스 감염증의 예방, 치료 또는 진단에 유용하게 사용될 수 있다.The antibody or antigen-binding fragment thereof according to the present invention specifically binds to the SARS-CoV-2 spike protein and completely blocks the binding of angiotensin converting enzyme-2 (ACE2), a receptor on the surface of human cells, to the SARS-CoV-2 spike protein. Because it can be blocked, it can be usefully used for prevention, treatment or diagnosis of novel coronavirus infection.
도 1은 SARS-CoV-2 스파이크 단백질 발현 벡터의 모식도이다.1 is a schematic diagram of a SARS-CoV-2 spike protein expression vector.
도 2는 정제된 SARS-CoV-2 스파이크 단백질들을 환원(reducing) 및 비환원(non-reducing) 조건에서 SDS-PAGE로 확인한 결과 및 SE-HPLC 수행 결과를 나타낸 것이다.2 shows the results of SDS-PAGE and SE-HPLC performance of purified SARS-CoV-2 spike proteins under reducing and non-reducing conditions.
도 3은 SARS-CoV-2 스파이크 항원에 대한 결합능이 강한 모노 파지를 선별하기 위한 ELISA 수행 결과를 나타낸 것이다.3 shows the results of ELISA to select monophages with strong binding ability to the SARS-CoV-2 spike antigen.
도 4는 SARS-CoV-2 스파이크 항원에 대한 최적화 항체의 결합능이 강한 모노 파지를 선별하기 위한 ELISA 수행 결과를 나타낸 것이다.4 shows the results of ELISA to select monophages with strong binding ability of the optimized antibody to the SARS-CoV-2 spike antigen.
도 5a 내지 도 5d는 SARS-CoV-2 스파이크 단백질의 발현이 서로 다른 HEK293E 세포주를 이용하여 Ymax®-ABL(도 5a 및 도 5b)과 환자-면역 라이브러리(도 5c 및 도 5d) 유래 항-SARS-CoV-2 스파이크 항체의 항원에 대한 결합 특이성을 FACS로 확인한 결과를 나타낸 것이다.5A to 5D show anti-SARS derived from Ymax ® -ABL ( FIGS. 5A and 5B ) and a patient-immune library ( FIGS. 5C and 5D ) using HEK293E cell lines with different SARS-CoV-2 spike protein expression. -Shows the results of confirming the binding specificity of the CoV-2 spike antibody to the antigen by FACS.
도 6a 및 도 6b는 SARS-CoV 또는 SARS-CoV-2의 스파이크 S1-His 융합 단백질을 이용하여 Ymax®-ABL(도 6a)과 환자-면역 라이브러리(도 6b) 유래 항-SARS-CoV-2 스파이크 항체의 항원 결합 특이성을 ELISA로 측정하여 나타낸 결과이다.6A and 6B show anti-SARS-CoV-2 derived from Ymax ® -ABL (FIG. 6A) and patient-immune library (FIG. 6B) using the spike S1-His fusion protein of SARS-CoV or SARS-CoV-2. The results are shown by measuring the antigen-binding specificity of the spike antibody by ELISA.
도 7a 및 도 7b는 SARS-CoV-2 RBD-mFc 융합 단백질을 이용하여 Ymax®-ABL(도 7a)과 환자-면역 라이브러리(도 7b) 유래 항-SARS-CoV-2 스파이크 항체의 항원 결합력을 Octet QKe 분석 장비로 측정한 결과를 나타낸 것이다.7A and 7B show the antigen-binding affinity of anti-SARS-CoV-2 spike antibodies derived from Ymax ® -ABL (FIG. 7A) and patient-immune library (FIG. 7B) using SARS-CoV-2 RBD-mFc fusion protein. The results measured by the Octet QKe analysis equipment are shown.
도 8a 및 도 8b는 대조군으로서 사용한 HEK293E 세포주(HEK293E/MocK) (도 8a) 및 SARS-CoV-2 스파이크 단백질의 발현이 서로 다른 HEK293E 세포주(HEK293E/CoV-2 스파이크(chimeric)) (도 8b)를 각각 이용하여 Ymax®-ABL 유래 항-SARS-CoV-2 스파이크 항체 변이체의 항원에 대한 결합 특이성을 FACS로 확인한 결과를 나타낸 것이다. 8A and 8B show the HEK293E cell line (HEK293E/MocK) used as a control (FIG. 8A) and the HEK293E cell line (HEK293E/CoV-2 spike (chimeric)) with different expression of SARS-CoV-2 spike protein (FIG. 8B) Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variants using each of the antigen binding specificity confirmed by FACS is shown.
도 9는 SARS-CoV 또는 SARS-CoV-2의 스파이크 S1-His 융합 단백질을 이용하여 Ymax®-ABL 유래 항-SARS-CoV-2 스파이크 항체 변이체의 항원 결합 특이성을 ELISA로 측정한 결과를 나타낸 것이다.9 shows the results of measuring the antigen-binding specificity of an anti-SARS-CoV-2 spike antibody variant derived from Ymax ® -ABL by ELISA using SARS-CoV or SARS-CoV-2 spike S1-His fusion protein. .
도 10은 SARS-CoV-2 RBD-mFc 융합 단백질을 이용하여 Ymax®-ABL 유래 항-SARS-CoV-2 스파이크 항체 변이체의 항원 결합력을 Octet QKe 분석 장비로 측정한 결과를 나타낸 것이다.10 shows the results of measuring the antigen-binding ability of the Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variant using the SARS-CoV-2 RBD-mFc fusion protein using Octet QKe analysis equipment.
도 11a 내지 도 11c는 SARS-CoV-2 RBD-His, SARS-CoV-2 RBD의 RBM을 SARS-CoV RBD의 RBM으로 치환한 돌연변이체(SARS-CoV-2_RBM_CoV-His) 또는 그 반대로 치환한 돌연변이체(SARS-CoV_RBM_CoV-2-His), SARS-CoV RBD-His 융합 단백질들을 이용하여 Ymax®-ABL과 환자-면역 라이브러리 유래 항-SARS-CoV-2 스파이크 항체(도 11a 및 도 11b)와 Ymax®-ABL 유래 항체 변이체(도 11c)의 항원 결합 영역(region)을 ELISA 분석을 통해 확인한 결과이다.11A to 11C show SARS-CoV-2 RBD-His, a mutant in which the RBM of SARS-CoV-2 RBD is substituted with the RBM of SARS-CoV RBD (SARS-CoV-2_RBM_CoV-His) or vice versa Ymax ® -ABL and anti-SARS-CoV-2 spike antibody ( FIGS. 11A and 11B ) from the patient-immune library and Ymax using SARS-CoV_RBM_CoV-2-His, SARS-CoV RBD-His fusion proteins ® -This is the result of confirming the antigen-binding region of the ABL-derived antibody variant (FIG. 11c) through ELISA analysis.
도 12a 및 도 12b는 ACE2와 결합하는 SARS-CoV-2 RBD의 RBM 영역 아미노산 서열을 SARS-CoV RBD의 RBM 서열을 참고하여 다른 아미노산으로 치환한 재조합 돌연변이 단백질들(SARS-CoV-2 RBD-His mutants; M2 내지 M10, 및 M12) (도 12a)을 사용하여, 항-SARS-CoV-2 스파이크 항체 및 변이체의 결합 특이성을 ELISA를 통해 측정하고 그룹화한 결과(도 12b)를 나타낸 것이다.12A and 12B show recombinant mutant proteins (SARS-CoV-2 RBD-His) in which the amino acid sequence of the RBM region of SARS-CoV-2 RBD that binds ACE2 is substituted with other amino acids with reference to the RBM sequence of SARS-CoV RBD. mutants; M2 to M10, and M12) (FIG. 12A), the binding specificities of anti-SARS-CoV-2 spike antibodies and variants were measured by ELISA and grouped results (FIG. 12B) are shown.
도 13a 내지 도 13d는 ACE2와 함께 SARS-CoV-2의 RBD(도 13a 및 도 13c) 또는 스파이크 S1(도 13b 및 도 13d)이 결합할 때, Ymax®-ABL(도 13a 및 도 13b)과 환자-면역 라이브러리(도 13c 및 도 13d) 유래 항-SARS-CoV-2 스파이크 항체가 농도 의존적으로 이들 결합을 경쟁적으로 저해하는지 ELISA를 통해 측정하고 확인한 결과이다.13A-13D show that when RBD of SARS-CoV-2 ( FIGS. 13A and 13C ) or Spike S1 ( FIGS. 13B and 13D ) binds together with ACE2, Ymax ® -ABL ( FIGS. 13A and 13B ) and It is the result of measuring and confirming whether the anti-SARS-CoV-2 spike antibody derived from the patient-immune library ( FIGS. 13c and 13d ) competitively inhibits these binding in a concentration-dependent manner through ELISA.
도 14a 내지 도 14c는 재조합 SARS-CoV-2 RBD-mFc 단백질과 ACE2를 발현하는 HEK293E 세포를 이용하여 Ymax®-ABL과 환자-면역 라이브러리 유래 항-SARS-CoV-2 스파이크 항체(도 14a 및 도 14b)와 Ymax®-ABL 유래 항체 변이체(도 14c)가 농도 의존적으로 SARS-CoV-2 RBD/ACE2 결합을 저해하는 효과를 확인한 결과이다.14A to 14C show Ymax ® -ABL and anti-SARS-CoV-2 spike antibody derived from a patient-immune library using HEK293E cells expressing recombinant SARS-CoV-2 RBD-mFc protein and ACE2 ( FIGS. 14A and 14A and 14C ). 14b) and Ymax ® -ABL-derived antibody variant (FIG. 14c) is the result of confirming the effect of inhibiting SARS-CoV-2 RBD / ACE2 binding in a concentration-dependent manner.
도 15a 및 도 15b는 재조합 SARS-CoV-2 RBD-hFc 단백질과 ACE2-GFP를 발현하는 HEK293E 세포를 이용하여 Ymax®-ABL(도 15a)과 환자-면역 라이브러리(도 15b) 유래 항-SARS-CoV-2 스파이크 항체가 세포 표면 ACE2와의 결합을 통한 SARS-CoV-2 RBD의 세포내 유입(internalization)을 농도 의존적으로 저해하는 효과를 확인한 결과이다.15A and 15B show anti-SARS- derived from Ymax ® -ABL (FIG. 15A) and patient-immune library (FIG. 15B) using HEK293E cells expressing recombinant SARS-CoV-2 RBD-hFc protein and ACE2-GFP. This is the result of confirming the effect of the CoV-2 spike antibody concentration-dependently inhibiting the intracellular influx (internalization) of SARS-CoV-2 RBD through binding to cell surface ACE2.
도 16a 및 도 16b는 Ymax®-ABL(도 16a)과 환자-면역 라이브러리(도 16b) 유래 항-SARS-CoV-2 스파이크 항체가 Live SARS-CoV-2 바이러스에 대해 중화능을 갖는지 Vero 세포에서 확인한 결과를 나타낸 것이다.16A and 16B show whether anti-SARS-CoV-2 spike antibodies from Ymax ® -ABL (FIG. 16A) and patient-immune library (FIG. 16B) have neutralizing ability against Live SARS-CoV-2 virus in Vero cells. The confirmed results are shown.
도 17a 및 도 17b는 ACE2와 함께 SARS-CoV-2의 RBD(도 17a) 또는 스파이크 S1(도 17b)이 결합할 때, Ymax®-ABL 유래 항-SARS-CoV-2 스파이크 항체 변이체가 농도 의존적으로 이들 결합을 경쟁적으로 저해하는지 ELISA를 통해 측정하고 확인한 결과이다.17A and 17B show that when RBD of SARS-CoV-2 (FIG. 17A) or Spike S1 (FIG. 17B) binds together with ACE2, Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variants dose-dependently It is the result of measuring and confirming whether these bindings are competitively inhibited by ELISA.
도 18은 재조합 SARS-CoV-2 RBD-hFc 단백질과 ACE2-GFP를 발현하는 HEK293E 세포를 이용하여 Ymax®-ABL 유래 항-SARS-CoV-2 스파이크 항체 변이체가 세포 표면 ACE2와의 결합을 통한 SARS-CoV-2 RBD의 세포내 유입(internalization)을 농도 의존적으로 저해하는 효과를 확인한 결과이다.Figure 18 shows that Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variant SARS- through binding to cell surface ACE2 using HEK293E cells expressing recombinant SARS-CoV-2 RBD-hFc protein and ACE2-GFP. This is the result of confirming the effect of inhibiting the intracellular influx (internalization) of CoV-2 RBD in a concentration-dependent manner.
도 19a 및 도 19b는 Ymax®-ABL 유래 항-SARS-CoV-2 스파이크 항체 변이체가 Live SARS-CoV-2 바이러스에 대해 중화능을 갖는지 Vero 세포에서 확인한 결과를 나타낸 것이다.19A and 19B show the results of confirming in Vero cells whether the Ymax ® -ABL-derived anti-SARS-CoV-2 spike antibody variant has neutralizing ability against the Live SARS-CoV-2 virus.
달리 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.
이하, 본 발명에 대하여 상세히 설명하도록 한다.Hereinafter, the present invention will be described in detail.
SARS-CoV-2 스파이크(S) 단백질에 특이적으로 결합하는 항체Antibodies that specifically bind to the SARS-CoV-2 spike (S) protein
본 발명의 일측면은, SARS-CoV-2 스파이크(spike) 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 제공한다.One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
본 명세서에 사용된 용어 "SARS-CoV-2(severe acute respiratory syndrome coronavirus 2; 중증급성호흡기증후군 코로나바이러스 2)"는 신종 코로나바이러스 감염증(COVID-19)을 일으키는 원인 바이러스로서 2019-nCoV라고도 지칭되며, 중국, 미국, 아시아, 유럽 등 각지에서 발견된 돌연변이 등 모든 임의의 변이체를 총칭하는 개념이다.As used herein, the term "SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2; severe acute respiratory syndrome coronavirus 2)" is a virus that causes novel coronavirus infection (COVID-19), also referred to as 2019-nCoV. , is a concept that collectively refers to all random variants such as mutations found in various places such as China, the United States, Asia, and Europe.
본 명세서에 사용된 용어 "SARS-CoV-2 스파이크(S) 단백질(SARS-CoV-2 spike protein)"은 수용체 인식 및 세포막 융합 과정에서 핵심적인 역할을 하며, S1과 S2 두 개의 서브유닛으로 구성된다. S1 서브유닛은 숙주 수용체 안지오텐신-전환효소 2(angiotensin-converting enzyme 2, ACE2)를 인식하고 결합하는 수용체 결합 도메인(receptor-binding domain, RBD)을 포함하는 반면, S2 서브유닛은 두 개의 헵타드 반복 도메인(two-heptad repeat domain)을 통해 6개의 나선 다발(six-helical bundle)을 형성하여 바이러스 세포막 융합을 매개한다.As used herein, the term "SARS-CoV-2 spike protein (SARS-CoV-2 spike protein)" plays a key role in receptor recognition and cell membrane fusion process, and consists of two subunits S1 and S2 do. The S1 subunit contains a receptor-binding domain (RBD) that recognizes and binds host angiotensin-converting enzyme 2 (ACE2), whereas the S2 subunit contains two heptad repeats. It mediates viral cell membrane fusion by forming a six-helical bundle through a two-heptad repeat domain.
구체적으로, SARS-CoV-2 스파이크(S) 단백질의 크기는 180~200 kDa이며, 세포외 N-말단, 바이러스 막에 고정된 막 횡단(transmembrane; TM) 도메인 및 짧은 세포내 C-말단 세그먼트로 구성된다. 스파이크는 일반적으로 준안정(metastable), 사전융합(prefusion) 형태로 존재하며, 바이러스가 숙주 세포와 상호작용하면 S 단백질의 광범위한 구조적 재배열이 발생하여 바이러스가 숙주 세포막과 융합하게 된다. 스파이크는 다당류 분자로 코팅되어 그들을 위장하여 진입하는 동안 숙주 면역 체계의 감시를 피할 수 있는 것으로 알려져 있다.Specifically, the SARS-CoV-2 spike (S) protein has a size of 180-200 kDa and is composed of an extracellular N-terminus, a transmembrane (TM) domain anchored to the viral membrane, and a short intracellular C-terminal segment. is composed Spikes generally exist in metastable and prefusion forms, and when the virus interacts with the host cell, extensive structural rearrangement of the S protein occurs, causing the virus to fuse with the host cell membrane. It is known that the spikes can be coated with polysaccharide molecules to camouflage them and evade surveillance of the host immune system while entering.
또한, SARS-CoV-2 S의 총 길이는 1,273 aa이고, N-말단에 위치한 신호 펩타이드(아미노산 1-13), S1 서브유닛(14-685 잔기) 및 S2 서브유닛(686-1,273 잔기)으로 구성된다. S1 서브유닛에는 N-말단 도메인(14-305 잔기)과 수용체 결합 도메인(RBD, 319-541 잔기)이 있으며, S2 서브유닛은 융합 펩타이드(fusion peptide; FP)(788-806 잔기), 헵타펩타이드 반복 서열 1(heptapeptide repeat sequence 1; HR1)(912-984 잔기), HR2(1,163-1,213 잔기), TM 도메인(1,213-1,237 잔기) 및 세포질 도메인(1,237-1,273 잔기)으로 구성된다.In addition, the total length of SARS-CoV-2 S is 1,273 aa and consists of a signal peptide (amino acids 1-13) located at the N-terminus, S1 subunit (14-685 residues) and S2 subunit (686-1,273 residues). is composed The S1 subunit has an N-terminal domain (residues 14-305) and a receptor binding domain (RBD, residues 319-541), and the S2 subunit contains a fusion peptide (FP) (residues 788-806) and a heptapeptide. It consists of a heptapeptide repeat sequence 1 (HR1) (residues 912-984), HR2 (residues 1,163-1,213), a TM domain (residues 1,213-1,237) and a cytoplasmic domain (residues 1,237-1,273).
본 명세서에 사용된 용어 "SARS-CoV-2 스파이크(spike; S) 단백질에 특이적으로 결합하는 항체"는 인간세포 표면의 수용체인 ACE2(angiotensin converting enzyme-2)와 결합하여 감염 기전을 일으키는 SARS-CoV-2의 막 단백질 스파이크를 타겟으로 하는 항체를 의미한다. 상기 항체는 SARS-CoV-2의 인간 감염 기전과 밀접한 관련이 있는 SARS-CoV-2의 막 단백질 스파이크를 항원으로 인식하여 결합함으로써 SARS-CoV-2에 대한 중화 효능을 나타낸다. 한편, 본 발명에 있어서, 상기 항체는 항-SARS-COV-2의 스파이크 항체와 혼용된다.As used herein, the term "antibody that specifically binds to SARS-CoV-2 spike (S) protein" refers to SARS that binds to angiotensin converting enzyme-2 (ACE2), a receptor on the surface of human cells, and causes an infection mechanism. - Refers to an antibody that targets the membrane protein spike of CoV-2. The antibody exhibits neutralizing efficacy against SARS-CoV-2 by recognizing and binding the membrane protein spike of SARS-CoV-2, which is closely related to the mechanism of human infection of SARS-CoV-2, as an antigen. Meanwhile, in the present invention, the antibody is mixed with the anti-SARS-COV-2 spike antibody.
상기 용어, "특이적으로 결합(specifically binding)" 또는 "특이적으로 인식(specifically recognizing)"은 당업자에게 통상적으로 알려진 의미와 동일한 것으로서, 항원 및 항체가 특이적으로 상호작용하여 항원-항체 복합체를 형성할 수 있으며, 또한 면역학적 반응을 하는 것을 의미할 수 있다.The term, "specifically binding" or "specifically recognizing" has the same meaning as commonly known to those skilled in the art, and an antigen and an antibody specifically interact to form an antigen-antibody complex. may form, and may also mean to undergo an immunological response.
본 발명에 따른 항체 또는 이의 항원 결합 단편은 SARS-CoV-2의 막 단백질 스파이크의 아미노산 서열 또는 그 일부에 결합하는 것일 수 있다. 구체적으로, 상기 SARS-CoV-2의 스파이크 단백질의 아미노산 서열은 GenBank accession NO. QHD43416.1(서열번호 309)에 기재된 것일 수 있다. 또한, 상기 SARS-CoV-2의 스파이크 단백질을 코딩하는 서열로서 해당 유전자는 GenBank accession No. MN908947.3에 기재된 뉴클레오타이드 서열(서열번호 310)일 수 있다. The antibody or antigen-binding fragment thereof according to the present invention may bind to the amino acid sequence of the membrane protein spike of SARS-CoV-2 or a part thereof. Specifically, the amino acid sequence of the SARS-CoV-2 spike protein is GenBank accession NO. It may be one described in QHD43416.1 (SEQ ID NO: 309). In addition, as a sequence encoding the SARS-CoV-2 spike protein, the gene is GenBank accession No. It may be the nucleotide sequence described in MN908947.3 (SEQ ID NO: 310).
구체적으로, 상기 항체 또는 이의 항원 결합 단편은 ACE2와 결합하는 SARS-CoV-2 RBM(receptor binding motif)을 포함하는 부위에 특이적으로 결합하는 것일 수 있다. 예를 들어, 상기 서열번호 309로 표시되는 R319 내지 F541번째 아미노산 서열(서열번호 311), L425 내지 V510번째 아미노산 서열(서열번호 312), N437 내지 Y508번째 아미노산 서열(서열번호 313) 부위에 특이적으로 결합하는 것일 수 있다. 바람직하게는 서열번호 313의 서열에 특이적으로 결합하는 것일 수 있지만, 이에 한정되는 것은 아니다.Specifically, the antibody or antigen-binding fragment thereof may specifically bind to a site containing a SARS-CoV-2 receptor binding motif (RBM) binding to ACE2. For example, the R319 to F541 amino acid sequence (SEQ ID NO: 311), L425 to V510 amino acid sequence (SEQ ID NO: 312), N437 to Y508 amino acid sequence (SEQ ID NO: 313) represented by SEQ ID NO: 309 is specific to the site may be combined with Preferably, it may be one that specifically binds to the sequence of SEQ ID NO: 313, but is not limited thereto.
한편, 상기 스파이크 단백질은 통상의 기술분야에 알려진 어떠한 서열로 구성되는 폴리펩티드일 수 있다. 상기 폴리펩티드는 단백질의 기능에 영향을 미치지 않는 범위 내에서, 아미노산 잔기의 결실, 삽입, 치환 또는 이들의 조합에 의해서 상이한 서열을 가지는 아미노산의 변이체 또는 단편일 수 있다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 또는 펩티드에서의 아미노산 치환은 당해 분야에 공지되어 있다. 가장 통상적으로 일어나는 치환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 치환이다. 경우에 따라서는 인산화(phosphorylation), 황화(sulfation), 아크릴화(acrylation), 당화(glycosylation), 메틸화(methylation), 파네실화(farnesylation) 등으로 변형(modification) 될 수 있다. On the other hand, the spike protein may be a polypeptide consisting of any sequence known in the art. The polypeptide may be a variant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid substitutions in proteins or peptides that do not entirely alter the activity of the molecule are known in the art. The most common substitutions are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Substitutions between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.
본 발명에 따른 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나, 서열번호 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79 및 85로 구성된 군에서 선택되는 중쇄(heavy chain) CDR1; 서열번호 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80 및 86으로 구성된 군에서 선택되는 중쇄 CDR2; 서열번호 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81 및 87로 구성된 군에서 선택되는 중쇄 CDR3를 포함하는 중쇄 가변영역; 및 서열번호 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 271, 274, 277 및 280으로 구성된 군에서 선택되는 경쇄(light chain) CDR1; 서열번호 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 272, 275, 278 및 281로 구성된 군에서 선택되는 경쇄 CDR2; 서열번호 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 273, 276, 279 및 282로 구성된 군에서 선택되는 경쇄 CDR3를 포함하는 경쇄 가변영역을 포함할 수 있다.The antibody or antigen-binding fragment thereof specifically binding to the SARS-CoV-2 spike protein according to the present invention is not limited thereto, but SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, a heavy chain CDR1 selected from the group consisting of 55, 61, 67, 73, 79 and 85; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80 and 86; a heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81 and 87; and SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 271, 274, 277 and 280 light chain ) CDR1; a light chain CDR2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 272, 275, 278 and 281; SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 273, 276, 279 and 282 comprising a light chain CDR3 selected from the group consisting of It may include a light chain variable region.
보다 구체적으로, 상기 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나, 하기 군에서 선택되는 어느 하나의 CDR(complementarity determining region) 조합을 포함하는 것일 수 있다:More specifically, the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein is not limited thereto, but may include any one CDR (complementarity determining region) combination selected from the following group. can:
(1) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 서열번호 3의 중쇄 CDR3, 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 서열번호 6의 경쇄 CDR3;(1) heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, light chain CDR3 of SEQ ID NO: 6;
(2) 서열번호 7의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 서열번호 9의 중쇄 CDR3, 서열번호 10의 경쇄 CDR1, 서열번호 11의 경쇄 CDR2, 서열번호 12의 경쇄 CDR3;(2) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 10, light chain CDR2 of SEQ ID NO: 11, light chain CDR3 of SEQ ID NO: 12;
(3) 서열번호 13의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 서열번호 15의 중쇄 CDR3, 서열번호 16의 경쇄 CDR1, 서열번호 17의 경쇄 CDR2, 서열번호 18의 경쇄 CDR3;(3) heavy chain CDR1 of SEQ ID NO: 13, heavy chain CDR2 of SEQ ID NO: 14, heavy chain CDR3 of SEQ ID NO: 15, light chain CDR1 of SEQ ID NO: 16, light chain CDR2 of SEQ ID NO: 17, light chain CDR3 of SEQ ID NO: 18;
(4) 서열번호 19의 중쇄 CDR1, 서열번호 20의 중쇄 CDR2, 서열번호 21의 중쇄 CDR3, 서열번호 22의 경쇄 CDR1, 서열번호 23의 경쇄 CDR2, 서열번호 24의 경쇄 CDR3;(4) heavy chain CDR1 of SEQ ID NO: 19, heavy chain CDR2 of SEQ ID NO: 20, heavy chain CDR3 of SEQ ID NO: 21, light chain CDR1 of SEQ ID NO: 22, light chain CDR2 of SEQ ID NO: 23, light chain CDR3 of SEQ ID NO: 24;
(5) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2, 서열번호 27의 중쇄 CDR3, 서열번호 28의 경쇄 CDR1, 서열번호 29의 경쇄 CDR2, 서열번호 30의 경쇄 CDR3;(5) heavy chain CDR1 of SEQ ID NO: 25, heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 28, light chain CDR2 of SEQ ID NO: 29, light chain CDR3 of SEQ ID NO: 30;
(6) 서열번호 31의 중쇄 CDR1, 서열번호 32의 중쇄 CDR2, 서열번호 33의 중쇄 CDR3, 서열번호 34의 경쇄 CDR1, 서열번호 35의 경쇄 CDR2, 서열번호 36의 경쇄 CDR3;(6) heavy chain CDR1 of SEQ ID NO: 31, heavy chain CDR2 of SEQ ID NO: 32, heavy chain CDR3 of SEQ ID NO: 33, light chain CDR1 of SEQ ID NO: 34, light chain CDR2 of SEQ ID NO: 35, light chain CDR3 of SEQ ID NO: 36;
(7) 서열번호 37의 중쇄 CDR1, 서열번호 38의 중쇄 CDR2, 서열번호 39의 중쇄 CDR3, 서열번호 40의 경쇄 CDR1, 서열번호 41의 경쇄 CDR2, 서열번호 42의 경쇄 CDR3;(7) heavy chain CDR1 of SEQ ID NO: 37, heavy chain CDR2 of SEQ ID NO: 38, heavy chain CDR3 of SEQ ID NO: 39, light chain CDR1 of SEQ ID NO: 40, light chain CDR2 of SEQ ID NO: 41, light chain CDR3 of SEQ ID NO: 42;
(8) 서열번호 43의 중쇄 CDR1, 서열번호 44의 중쇄 CDR2, 서열번호 45의 중쇄 CDR3, 서열번호 46의 경쇄 CDR1, 서열번호 47의 경쇄 CDR2, 서열번호 48의 경쇄 CDR3;(8) heavy chain CDR1 of SEQ ID NO: 43, heavy chain CDR2 of SEQ ID NO: 44, heavy chain CDR3 of SEQ ID NO: 45, light chain CDR1 of SEQ ID NO: 46, light chain CDR2 of SEQ ID NO: 47, light chain CDR3 of SEQ ID NO: 48;
(9) 서열번호 49의 중쇄 CDR1, 서열번호 50의 중쇄 CDR2, 서열번호 51의 중쇄 CDR3, 서열번호 52의 경쇄 CDR1, 서열번호 53의 경쇄 CDR2, 서열번호 54의 경쇄 CDR3;(9) heavy chain CDR1 of SEQ ID NO: 49, heavy chain CDR2 of SEQ ID NO: 50, heavy chain CDR3 of SEQ ID NO: 51, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 53, light chain CDR3 of SEQ ID NO: 54;
(10) 서열번호 55의 중쇄 CDR1, 서열번호 56의 중쇄 CDR2, 서열번호 57의 중쇄 CDR3, 서열번호 58의 경쇄 CDR1, 서열번호 59의 경쇄 CDR2, 서열번호 60의 경쇄 CDR3;(10) heavy chain CDR1 of SEQ ID NO: 55, heavy chain CDR2 of SEQ ID NO: 56, heavy chain CDR3 of SEQ ID NO: 57, light chain CDR1 of SEQ ID NO: 58, light chain CDR2 of SEQ ID NO: 59, light chain CDR3 of SEQ ID NO: 60;
(11) 서열번호 61의 중쇄 CDR1, 서열번호 62의 중쇄 CDR2, 서열번호 63의 중쇄 CDR3, 서열번호 64의 경쇄 CDR1, 서열번호 65의 경쇄 CDR2, 서열번호 66의 경쇄 CDR3;(11) heavy chain CDR1 of SEQ ID NO: 61, heavy chain CDR2 of SEQ ID NO: 62, heavy chain CDR3 of SEQ ID NO: 63, light chain CDR1 of SEQ ID NO: 64, light chain CDR2 of SEQ ID NO: 65, light chain CDR3 of SEQ ID NO: 66;
(12) 서열번호 67의 중쇄 CDR1, 서열번호 68의 중쇄 CDR2, 서열번호 69의 중쇄 CDR3, 서열번호 70의 경쇄 CDR1, 서열번호 71의 경쇄 CDR2, 서열번호 72의 경쇄 CDR3;(12) heavy chain CDR1 of SEQ ID NO: 67, heavy chain CDR2 of SEQ ID NO: 68, heavy chain CDR3 of SEQ ID NO: 69, light chain CDR1 of SEQ ID NO: 70, light chain CDR2 of SEQ ID NO: 71, light chain CDR3 of SEQ ID NO: 72;
(13) 서열번호 73의 중쇄 CDR1, 서열번호 74의 중쇄 CDR2, 서열번호 75의 중쇄 CDR3, 서열번호 76의 경쇄 CDR1, 서열번호 77의 경쇄 CDR2, 서열번호 78의 경쇄 CDR3;(13) heavy chain CDR1 of SEQ ID NO: 73, heavy chain CDR2 of SEQ ID NO: 74, heavy chain CDR3 of SEQ ID NO: 75, light chain CDR1 of SEQ ID NO: 76, light chain CDR2 of SEQ ID NO: 77, light chain CDR3 of SEQ ID NO: 78;
(14) 서열번호 79의 중쇄 CDR1, 서열번호 80의 중쇄 CDR2, 서열번호 81의 중쇄 CDR3, 서열번호 82의 경쇄 CDR1, 서열번호 83의 경쇄 CDR2, 서열번호 84의 경쇄 CDR3;(14) heavy chain CDR1 of SEQ ID NO: 79, heavy chain CDR2 of SEQ ID NO: 80, heavy chain CDR3 of SEQ ID NO: 81, light chain CDR1 of SEQ ID NO: 82, light chain CDR2 of SEQ ID NO: 83, light chain CDR3 of SEQ ID NO: 84;
(15) 서열번호 85의 중쇄 CDR1, 서열번호 86의 중쇄 CDR2, 서열번호 87의 중쇄 CDR3, 서열번호 88의 경쇄 CDR1, 서열번호 89의 경쇄 CDR2, 서열번호 90의 경쇄 CDR3;(15) heavy chain CDR1 of SEQ ID NO: 85, heavy chain CDR2 of SEQ ID NO: 86, heavy chain CDR3 of SEQ ID NO: 87, light chain CDR1 of SEQ ID NO: 88, light chain CDR2 of SEQ ID NO: 89, light chain CDR3 of SEQ ID NO: 90;
(16) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 서열번호 3의 중쇄 CDR3, 서열번호 271의 경쇄 CDR1, 서열번호 272의 경쇄 CDR2, 서열번호 273의 경쇄 CDR3;(16) heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 271, light chain CDR2 of SEQ ID NO: 272, light chain CDR3 of SEQ ID NO: 273;
(17) 서열번호 7의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 서열번호 9의 중쇄 CDR3, 서열번호 274의 경쇄 CDR1, 서열번호 275의 경쇄 CDR2, 서열번호 276의 경쇄 CDR3;(17) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 274, light chain CDR2 of SEQ ID NO: 275, light chain CDR3 of SEQ ID NO: 276;
(18) 서열번호 13의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 서열번호 15의 중쇄 CDR3, 서열번호 277의 경쇄 CDR1, 서열번호 278의 경쇄 CDR2, 서열번호 279의 경쇄 CDR3; 및 (18) heavy chain CDR1 of SEQ ID NO: 13, heavy chain CDR2 of SEQ ID NO: 14, heavy chain CDR3 of SEQ ID NO: 15, light chain CDR1 of SEQ ID NO: 277, light chain CDR2 of SEQ ID NO: 278, light chain CDR3 of SEQ ID NO: 279; and
(19) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2, 서열번호 27의 중쇄 CDR3, 서열번호 280의 경쇄 CDR1, 서열번호 281의 경쇄 CDR2, 서열번호 282의 경쇄 CDR3.(19) heavy chain CDR1 of SEQ ID NO: 25, heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 280, light chain CDR2 of SEQ ID NO: 281, light chain CDR3 of SEQ ID NO: 282.
본 발명에 따른 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나, 서열번호 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237 및 239로 이루어진 군에서 선택되는 어느 하나의 중쇄 가변영역을 포함하는 것일 수 있다.The antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein according to the present invention is not limited thereto, but SEQ ID NOs: 211, 213, 215, 217, 219, 221, 223, 225, 227, It may include any one heavy chain variable region selected from the group consisting of 229, 231, 233, 235, 237 and 239.
또한, 상기 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나, 서열번호 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 299, 300, 301 및 302로 이루어진 군에서 선택되는 어느 하나의 경쇄 가변영역을 포함하는 것일 수 있다.In addition, the antibody or antigen-binding fragment thereof is not limited thereto, but SEQ ID NOs: 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 299, It may include any one light chain variable region selected from the group consisting of 300, 301 and 302.
보다 구체적으로, 상기 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나, 하기 군에서 선택되는 어느 하나의 가변영역(variable region) 조합을 포함하는 것일 수 있다:More specifically, the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein is not limited thereto, but may include a combination of any one variable region selected from the following group. can:
(1) 서열번호 211의 중쇄 가변영역 및 서열번호 212의 경쇄 가변영역;(1) a heavy chain variable region of SEQ ID NO: 211 and a light chain variable region of SEQ ID NO: 212;
(2) 서열번호 213의 중쇄 가변영역 및 서열번호 214의 경쇄 가변영역;(2) the heavy chain variable region of SEQ ID NO: 213 and the light chain variable region of SEQ ID NO: 214;
(3) 서열번호 215의 중쇄 가변영역 및 서열번호 216의 경쇄 가변영역;(3) a heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 216;
(4) 서열번호 217의 중쇄 가변영역 및 서열번호 218의 경쇄 가변영역;(4) a heavy chain variable region of SEQ ID NO: 217 and a light chain variable region of SEQ ID NO: 218;
(5) 서열번호 219의 중쇄 가변영역 및 서열번호 220의 경쇄 가변영역;(5) a heavy chain variable region of SEQ ID NO: 219 and a light chain variable region of SEQ ID NO: 220;
(6) 서열번호 221의 중쇄 가변영역 및 서열번호 222의 경쇄 가변영역;(6) a heavy chain variable region of SEQ ID NO: 221 and a light chain variable region of SEQ ID NO: 222;
(7) 서열번호 223의 중쇄 가변영역 및 서열번호 224의 경쇄 가변영역;(7) a heavy chain variable region of SEQ ID NO: 223 and a light chain variable region of SEQ ID NO: 224;
(8) 서열번호 225의 중쇄 가변영역 및 서열번호 226의 경쇄 가변영역;(8) a heavy chain variable region of SEQ ID NO: 225 and a light chain variable region of SEQ ID NO: 226;
(9) 서열번호 227의 중쇄 가변영역 및 서열번호 228의 경쇄 가변영역;(9) a heavy chain variable region of SEQ ID NO: 227 and a light chain variable region of SEQ ID NO: 228;
(10) 서열번호 229의 중쇄 가변영역 및 서열번호 230의 경쇄 가변영역;(10) a heavy chain variable region of SEQ ID NO: 229 and a light chain variable region of SEQ ID NO: 230;
(11) 서열번호 231의 중쇄 가변영역 및 서열번호 232의 경쇄 가변영역;(11) a heavy chain variable region of SEQ ID NO: 231 and a light chain variable region of SEQ ID NO: 232;
(12) 서열번호 233의 중쇄 가변영역 및 서열번호 234의 경쇄 가변영역;(12) a heavy chain variable region of SEQ ID NO: 233 and a light chain variable region of SEQ ID NO: 234;
(13) 서열번호 235의 중쇄 가변영역 및 서열번호 236의 경쇄 가변영역;(13) a heavy chain variable region of SEQ ID NO: 235 and a light chain variable region of SEQ ID NO: 236;
(14) 서열번호 237의 중쇄 가변영역 및 서열번호 238의 경쇄 가변영역;(14) a heavy chain variable region of SEQ ID NO: 237 and a light chain variable region of SEQ ID NO: 238;
(15) 서열번호 239의 중쇄 가변영역 및 서열번호 240의 경쇄 가변영역;(15) a heavy chain variable region of SEQ ID NO: 239 and a light chain variable region of SEQ ID NO: 240;
(16) 서열번호 211의 중쇄 가변영역 및 서열번호 299의 경쇄 가변영역;(16) a heavy chain variable region of SEQ ID NO: 211 and a light chain variable region of SEQ ID NO: 299;
(17) 서열번호 213의 중쇄 가변영역 및 서열번호 300의 경쇄 가변영역;(17) a heavy chain variable region of SEQ ID NO: 213 and a light chain variable region of SEQ ID NO: 300;
(18) 서열번호 215의 중쇄 가변영역 및 서열번호 301의 경쇄 가변영역; 및 (18) a heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 301; and
(19) 서열번호 219의 중쇄 가변영역 및 서열번호 302의 경쇄 가변영역.(19) the heavy chain variable region of SEQ ID NO: 219 and the light chain variable region of SEQ ID NO: 302.
상기 가변영역 조합을 포함하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나 각 해당 서열의 가변영역과 적어도 약 80% 상동성, 적어도 약 90% 상동성, 적어도 약 95% 상동성을 가질 수 있다.The antibody or antigen-binding fragment thereof comprising the variable region combination is not limited thereto, but may have at least about 80% homology, at least about 90% homology, or at least about 95% homology with the variable region of each corresponding sequence. .
본 명세서에 사용된 용어, "항체(antibody)"는 특정 항원, 예컨대 병원성 박테리아나 바이러스와 특이적으로 결합하여 이들을 중화시키는 기능을 수행하는 단백질로, 본 발명에서는 SARS-CoV-2의 스파이크 단백질에 특이적으로 결합하는 항체를 의미한다. 본 발명의 범위에는 SARS-COV-2의 스파이크에 특이적으로 결합하는 완전한 항체 형태뿐만 아니라, 상기 항체 분자의 항원 결합 단편도 포함된다.As used herein, the term "antibody" refers to a protein that specifically binds to and neutralizes a specific antigen, such as a pathogenic bacteria or virus. It refers to an antibody that specifically binds. The scope of the present invention includes not only complete antibody forms that specifically bind to the SARS-COV-2 spike, but also antigen-binding fragments of the antibody molecule.
완전한 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며, 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고, 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다.A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 ( γ3), gamma 4 (γ4), alpha1 (α1) and alpha2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.
항체의 "항원 결합 단편" 또는 "항체 단편"이란 항원 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등의 형태일 수 있다. 항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변영역 및 중쇄의 첫 번째 불변영역(CH1)을 가지는 구조로서 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 두 개의 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변영역 및 경쇄 가변영역만을 가지고 있는 최소의 항체 조각을 의미한다. An "antigen-binding fragment" or "antibody fragment" of an antibody refers to a fragment having an antigen-binding function, and may be in the form of Fab, F(ab'), F(ab')2 and Fv. Among the antibody fragments, Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by forming a disulfide bond between two cysteine residues in the hinge region of Fab'. Fv refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region.
이중쇄 Fv(two-chain Fv)는 비공유 결합으로 중쇄 가변영역과 경쇄 가변영역이 연결되어 있고, 단쇄 Fv(single-chain Fv, scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변영역과 경쇄의 가변영역이 공유결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머(dimer)와 같은 구조를 이룰 수 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있음), 유전자 재조합 기술을 통하여 제작할 수도 있다.In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and single-chain Fv (scFv) is generally a heavy chain variable region and light chain variable region through a peptide linker. Since the regions are covalently linked or linked directly at the C-terminus, a dimer-like structure can be formed like a double-stranded Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, papain-restricted digestion of the whole antibody yields Fab, pepsin digestion yields F(ab')2 fragments), and gene It can also be produced through recombinant technology.
일 구현예에서, 본 발명에 따른 항체는 Fv 형태(예컨대, scFv)이거나, 완전한 항체 형태이다. 또한, 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 또는 엡실론(ε) 중 어느 하나의 이소타입일 수 있다. 예를 들어, 불변영역은 감마1(IgG1), 감마 3(IgG3) 또는 감마 4(IgG4)이다. 경쇄 불변영역은 카파 또는 람다 형일 수 있다.In one embodiment, the antibody according to the invention is in the form of an Fv (eg scFv) or in the form of a complete antibody. In addition, the heavy chain constant region may be any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region may be kappa or lambda type.
본 발명의 항체는 완전 인간 항체 서열로 이루어져 있다. 상기 "인간 항체"는 인간 면역글로불린으로부터 유래하는 분자로서 상보성 결정영역, 구조 영역을 포함한 항체를 구성하는 모든 아미노산 서열 전체가 인간의 면역글로불린으로 구성되어 있는 것을 의미한다. 필요에 따라, 본 발명의 항체는 당 분야에 알려진 방법에 따라 인간화 항체, 키메라 항체 등의 다양한 형태로 변형될 수 있다.The antibodies of the invention consist of fully human antibody sequences. The "human antibody" is a molecule derived from human immunoglobulin, and means that the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin. If necessary, the antibody of the present invention may be modified into various forms such as a humanized antibody, a chimeric antibody, and the like according to methods known in the art.
본 명세서에 사용된 바와 같은 "항체 가변 도메인"은 상보성 결정 영역(Complementarity Determining Region; CDR) 및 골격 영역(Framework Region; FR)의 아미노산 서열을 포함하는 항체 분자의 경쇄 및 중쇄 부분을 지칭한다. VH는 중쇄의 가변 도메인을 지칭하고, VL은 경쇄의 가변 도메인을 지칭한다."Antibody variable domain" as used herein refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the Complementarity Determining Region (CDR) and Framework Region (FR). VH refers to the variable domain of the heavy chain and VL refers to the variable domain of the light chain.
본 명세서에 사용된 용어, "상보성 결정 영역"(CDR; 즉, CDR1, CDR2 및 CDR3)은 항원의 인식에 관여하는 고리모양의 부위로서 이 부위의 서열이 변함에 따라 항체의 항원에 대한 특이성이 결정된다. 상기 상보성 결정 영역은 항원 결합을 위해 필요한 영역으로서 항체 가변 도메인의 아미노산 잔기를 지칭한다. 각 가변 도메인은 전형적으로, CDR1, CDR2 및 CDR3으로서 확인된 3개의 CDR 영역을 갖는다. As used herein, the term "complementarity determining region" (CDR; that is, CDR1, CDR2 and CDR3) is a ring-shaped region involved in antigen recognition, and as the sequence of this region changes, the specificity of the antibody for the antigen is decided The complementarity determining region refers to an amino acid residue of an antibody variable domain as a region necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
구체적으로, 본 발명은 전술한 바와 같은 CDR 조합을 포함하는 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편을 제공한다. Specifically, the present invention provides an anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof comprising the CDR combinations as described above.
본 명세서에 사용된 용어, "골격 영역(framework region; FR)"은 CDR 잔기 이외의 가변 도메인 잔기로, 각 가변 도메인은 전형적으로, FR1, FR2, FR3 및 FR4로서 확인된 4개의 FR들을 가진다.As used herein, the term "framework region (FR)" refers to variable domain residues other than CDR residues, each variable domain having four FRs, typically identified as FR1, FR2, FR3 and FR4.
구체적으로, 본 발명의 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나, 서열번호 91, 99, 107, 115, 123, 131, 139, 147, 155, 163, 171, 179, 187, 195 및 203으로 이루어진 군에서 선택되는 어느 하나의 중쇄 FR1; 서열번호 92, 100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196 및 204로 이루어진 군에서 선택되는 어느 하나의 중쇄 FR2; 서열번호 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, 189, 197 및 205로 이루어진 군에서 선택되는 어느 하나의 중쇄 FR3; 서열번호 94, 102, 110, 118, 126, 134, 142, 150, 158, 166, 174, 182, 190, 198 및 206으로 이루어진 군에서 선택되는 어느 하나의 중쇄 FR4; 서열번호 95, 103, 111, 119, 127, 135, 143, 151, 159, 167, 175, 183, 191, 199, 207, 283, 287, 291 및 295로 이루어진 군에서 선택되는 어느 하나의 경쇄 FR1; 서열번호 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 284, 288, 292 및 296으로 이루어진 군에서 선택되는 어느 하나의 경쇄 FR2; 서열번호 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, 193, 201, 209, 285, 289, 293 및 297로 이루어진 군에서 선택되는 어느 하나의 경쇄 FR3; 및 서열번호 98, 106, 114, 122, 130, 138, 146, 154, 162, 170, 178, 186, 194, 202, 210, 286, 290, 294 및 298로 이루어진 군에서 선택되는 어느 하나의 경쇄 FR4를 포함할 수 있다.Specifically, the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein of the present invention is not limited thereto, but SEQ ID NOs: 91, 99, 107, 115, 123, 131, 139, 147, any one heavy chain FR1 selected from the group consisting of 155, 163, 171, 179, 187, 195 and 203; any one heavy chain FR2 selected from the group consisting of SEQ ID NOs: 92, 100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196 and 204; any one heavy chain FR3 selected from the group consisting of SEQ ID NOs: 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, 189, 197 and 205; any one heavy chain FR4 selected from the group consisting of SEQ ID NOs: 94, 102, 110, 118, 126, 134, 142, 150, 158, 166, 174, 182, 190, 198 and 206; Any one light chain FR1 selected from the group consisting of SEQ ID NOs: 95, 103, 111, 119, 127, 135, 143, 151, 159, 167, 175, 183, 191, 199, 207, 283, 287, 291 and 295 ; Any one light chain FR2 selected from the group consisting of SEQ ID NOs: 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 284, 288, 292 and 296 ; Any one light chain FR3 selected from the group consisting of SEQ ID NOs: 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, 193, 201, 209, 285, 289, 293 and 297 ; And any one light chain selected from the group consisting of SEQ ID NOs: 98, 106, 114, 122, 130, 138, 146, 154, 162, 170, 178, 186, 194, 202, 210, 286, 290, 294 and 298 FR4 may be included.
보다 구체적으로, 본 발명의 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나, 하기 군에서 선택되는 어느 하나의 FR 조합을 포함하는 것일 수 있다: More specifically, the antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein of the present invention is not limited thereto, but may include any one FR combination selected from the following group:
(1) 서열번호 91의 중쇄 FR1, 서열번호 92의 중쇄 FR2, 서열번호 93의 중쇄 FR3, 서열번호 94의 중쇄 FR4, 서열번호 95의 경쇄 FR1, 서열번호 96의 경쇄 FR2, 서열번호 97의 경쇄 FR3, 서열번호 98의 경쇄 FR4;(1) heavy chain FR1 of SEQ ID NO: 91, heavy chain FR2 of SEQ ID NO: 92, heavy chain FR3 of SEQ ID NO: 93, heavy chain FR4 of SEQ ID NO: 94, light chain FR1 of SEQ ID NO: 95, light chain FR2 of SEQ ID NO: 96, light chain of SEQ ID NO: 97 FR3, light chain FR4 of SEQ ID NO: 98;
(2) 서열번호 99의 중쇄 FR1, 서열번호 100의 중쇄 FR2, 서열번호 101의 중쇄 FR3, 서열번호 102의 중쇄 FR4, 서열번호 103의 경쇄 FR1, 서열번호 104의 경쇄 FR2, 서열번호 105의 경쇄 FR3, 서열번호 106의 경쇄 FR4;(2) heavy chain FR1 of SEQ ID NO: 99, heavy chain FR2 of SEQ ID NO: 100, heavy chain FR3 of SEQ ID NO: 101, heavy chain FR4 of SEQ ID NO: 102, light chain FR1 of SEQ ID NO: 103, light chain FR2 of SEQ ID NO: 104, light chain of SEQ ID NO: 105 FR3, light chain FR4 of SEQ ID NO: 106;
(3) 서열번호 107의 중쇄 FR1, 서열번호 108의 중쇄 FR2, 서열번호 109의 중쇄 FR3, 서열번호 110의 중쇄 FR4, 서열번호 111의 경쇄 FR1, 서열번호 112의 경쇄 FR2, 서열번호 113의 경쇄 FR3, 서열번호 114의 경쇄 FR4;(3) heavy chain FR1 of SEQ ID NO: 107, heavy chain FR2 of SEQ ID NO: 108, heavy chain FR3 of SEQ ID NO: 109, heavy chain FR4 of SEQ ID NO: 110, light chain FR1 of SEQ ID NO: 111, light chain FR2 of SEQ ID NO: 112, light chain of SEQ ID NO: 113 FR3, light chain FR4 of SEQ ID NO: 114;
(4) 서열번호 115의 중쇄 FR1, 서열번호 116의 중쇄 FR2, 서열번호 117의 중쇄 FR3, 서열번호 118의 중쇄 FR4, 서열번호 119의 경쇄 FR1, 서열번호 120의 경쇄 FR2, 서열번호 121의 경쇄 FR3, 서열번호 122의 경쇄 FR4;(4) heavy chain FR1 of SEQ ID NO: 115, heavy chain FR2 of SEQ ID NO: 116, heavy chain FR3 of SEQ ID NO: 117, heavy chain FR4 of SEQ ID NO: 118, light chain FR1 of SEQ ID NO: 119, light chain FR2 of SEQ ID NO: 120, light chain of SEQ ID NO: 121 FR3, light chain FR4 of SEQ ID NO: 122;
(5) 서열번호 123의 중쇄 FR1, 서열번호 124의 중쇄 FR2, 서열번호 125의 중쇄 FR3, 서열번호 126의 중쇄 FR4, 서열번호 127의 경쇄 FR1, 서열번호 128의 경쇄 FR2, 서열번호 129의 경쇄 FR3, 서열번호 130의 경쇄 FR4;(5) heavy chain FR1 of SEQ ID NO: 123, heavy chain FR2 of SEQ ID NO: 124, heavy chain FR3 of SEQ ID NO: 125, heavy chain FR4 of SEQ ID NO: 126, light chain FR1 of SEQ ID NO: 127, light chain FR2 of SEQ ID NO: 128, light chain of SEQ ID NO: 129 FR3, light chain FR4 of SEQ ID NO: 130;
(6) 서열번호 131의 중쇄 FR1, 서열번호 132의 중쇄 FR2, 서열번호 133의 중쇄 FR3, 서열번호 134의 중쇄 FR4, 서열번호 135의 경쇄 FR1, 서열번호 136의 경쇄 FR2, 서열번호 137의 경쇄 FR3, 서열번호 138의 경쇄 FR4;(6) heavy chain FR1 of SEQ ID NO: 131, heavy chain FR2 of SEQ ID NO: 132, heavy chain FR3 of SEQ ID NO: 133, heavy chain FR4 of SEQ ID NO: 134, light chain FR1 of SEQ ID NO: 135, light chain FR2 of SEQ ID NO: 136, light chain of SEQ ID NO: 137 FR3, light chain FR4 of SEQ ID NO: 138;
(7) 서열번호 139의 중쇄 FR1, 서열번호 140의 중쇄 FR2, 서열번호 141의 중쇄 FR3, 서열번호 142의 중쇄 FR4, 서열번호 143의 경쇄 FR1, 서열번호 144의 경쇄 FR2, 서열번호 145의 경쇄 FR3, 서열번호 146의 경쇄 FR4;(7) heavy chain FR1 of SEQ ID NO: 139, heavy chain FR2 of SEQ ID NO: 140, heavy chain FR3 of SEQ ID NO: 141, heavy chain FR4 of SEQ ID NO: 142, light chain FR1 of SEQ ID NO: 143, light chain FR2 of SEQ ID NO: 144, light chain of SEQ ID NO: 145 FR3, light chain FR4 of SEQ ID NO: 146;
(8) 서열번호 147의 중쇄 FR1, 서열번호 148의 중쇄 FR2, 서열번호 149의 중쇄 FR3, 서열번호 150의 중쇄 FR4, 서열번호 151의 경쇄 FR1, 서열번호 152의 경쇄 FR2, 서열번호 153의 경쇄 FR3, 서열번호 154의 경쇄 FR4;(8) heavy chain FR1 of SEQ ID NO: 147, heavy chain FR2 of SEQ ID NO: 148, heavy chain FR3 of SEQ ID NO: 149, heavy chain FR4 of SEQ ID NO: 150, light chain FR1 of SEQ ID NO: 151, light chain FR2 of SEQ ID NO: 152, light chain of SEQ ID NO: 153 FR3, light chain FR4 of SEQ ID NO: 154;
(9) 서열번호 155의 중쇄 FR1, 서열번호 156의 중쇄 FR2, 서열번호 157의 중쇄 FR3, 서열번호 158의 중쇄 FR4, 서열번호 159의 경쇄 FR1, 서열번호 160의 경쇄 FR2, 서열번호 161의 경쇄 FR3, 서열번호 162의 경쇄 FR4;(9) heavy chain FR1 of SEQ ID NO: 155, heavy chain FR2 of SEQ ID NO: 156, heavy chain FR3 of SEQ ID NO: 157, heavy chain FR4 of SEQ ID NO: 158, light chain FR1 of SEQ ID NO: 159, light chain FR2 of SEQ ID NO: 160, light chain of SEQ ID NO: 161 FR3, light chain FR4 of SEQ ID NO: 162;
(10) 서열번호 163의 중쇄 FR1, 서열번호 164의 중쇄 FR2, 서열번호 165의 중쇄 FR3, 서열번호 166의 중쇄 FR4, 서열번호 167의 경쇄 FR1, 서열번호 168의 경쇄 FR2, 서열번호 169의 경쇄 FR3, 서열번호 170의 경쇄 FR4;(10) heavy chain FR1 of SEQ ID NO: 163, heavy chain FR2 of SEQ ID NO: 164, heavy chain FR3 of SEQ ID NO: 165, heavy chain FR4 of SEQ ID NO: 166, light chain FR1 of SEQ ID NO: 167, light chain FR2 of SEQ ID NO: 168, light chain of SEQ ID NO: 169 FR3, light chain FR4 of SEQ ID NO: 170;
(11) 서열번호 171의 중쇄 FR1, 서열번호 172의 중쇄 FR2, 서열번호 173의 중쇄 FR3, 서열번호 174의 중쇄 FR4, 서열번호 175의 경쇄 FR1, 서열번호 176의 경쇄 FR2, 서열번호 177의 경쇄 FR3, 서열번호 178의 경쇄 FR4;(11) heavy chain FR1 of SEQ ID NO: 171, heavy chain FR2 of SEQ ID NO: 172, heavy chain FR3 of SEQ ID NO: 173, heavy chain FR4 of SEQ ID NO: 174, light chain FR1 of SEQ ID NO: 175, light chain FR2 of SEQ ID NO: 176, light chain of SEQ ID NO: 177 FR3, light chain FR4 of SEQ ID NO: 178;
(12) 서열번호 179의 중쇄 FR1, 서열번호 180의 중쇄 FR2, 서열번호 181의 중쇄 FR3, 서열번호 182의 중쇄 FR4, 서열번호 183의 경쇄 FR1, 서열번호 184의 경쇄 FR2, 서열번호 185의 경쇄 FR3, 서열번호 186의 경쇄 FR4;(12) heavy chain FR1 of SEQ ID NO: 179, heavy chain FR2 of SEQ ID NO: 180, heavy chain FR3 of SEQ ID NO: 181, heavy chain FR4 of SEQ ID NO: 182, light chain FR1 of SEQ ID NO: 183, light chain FR2 of SEQ ID NO: 184, light chain of SEQ ID NO: 185 FR3, light chain FR4 of SEQ ID NO: 186;
(13) 서열번호 187의 중쇄 FR1, 서열번호 188의 중쇄 FR2, 서열번호 189의 중쇄 FR3, 서열번호 190의 중쇄 FR4, 서열번호 191의 경쇄 FR1, 서열번호 192의 경쇄 FR2, 서열번호 193의 경쇄 FR3, 서열번호 194의 경쇄 FR4;(13) heavy chain FR1 of SEQ ID NO: 187, heavy chain FR2 of SEQ ID NO: 188, heavy chain FR3 of SEQ ID NO: 189, heavy chain FR4 of SEQ ID NO: 190, light chain FR1 of SEQ ID NO: 191, light chain FR2 of SEQ ID NO: 192, light chain of SEQ ID NO: 193 FR3, light chain FR4 of SEQ ID NO: 194;
(14) 서열번호 195의 중쇄 FR1, 서열번호 196의 중쇄 FR2, 서열번호 197의 중쇄 FR3, 서열번호 198의 중쇄 FR4, 서열번호 199의 경쇄 FR1, 서열번호 200의 경쇄 FR2, 서열번호 201의 경쇄 FR3, 서열번호 202의 경쇄 FR4;(14) heavy chain FR1 of SEQ ID NO: 195, heavy chain FR2 of SEQ ID NO: 196, heavy chain FR3 of SEQ ID NO: 197, heavy chain FR4 of SEQ ID NO: 198, light chain FR1 of SEQ ID NO: 199, light chain FR2 of SEQ ID NO: 200, light chain of SEQ ID NO: 201 FR3, light chain FR4 of SEQ ID NO: 202;
(15) 서열번호 203의 중쇄 FR1, 서열번호 204의 중쇄 FR2, 서열번호 205의 중쇄 FR3, 서열번호 206의 중쇄 FR4, 서열번호 207의 경쇄 FR1, 서열번호 208의 경쇄 FR2, 서열번호 209의 경쇄 FR3, 서열번호 210의 경쇄 FR4;(15) heavy chain FR1 of SEQ ID NO: 203, heavy chain FR2 of SEQ ID NO: 204, heavy chain FR3 of SEQ ID NO: 205, heavy chain FR4 of SEQ ID NO: 206, light chain FR1 of SEQ ID NO: 207, light chain FR2 of SEQ ID NO: 208, light chain of SEQ ID NO: 209 FR3, light chain FR4 of SEQ ID NO: 210;
(16) 서열번호 91의 중쇄 FR1, 서열번호 92의 중쇄 FR2, 서열번호 93의 중쇄 FR3, 서열번호 94의 중쇄 FR4, 서열번호 283의 경쇄 FR1, 서열번호 284의 경쇄 FR2, 서열번호 285의 경쇄 FR3, 서열번호 286의 경쇄 FR4;(16) heavy chain FR1 of SEQ ID NO: 91, heavy chain FR2 of SEQ ID NO: 92, heavy chain FR3 of SEQ ID NO: 93, heavy chain FR4 of SEQ ID NO: 94, light chain FR1 of SEQ ID NO: 283, light chain FR2 of SEQ ID NO: 284, light chain of SEQ ID NO: 285 FR3, light chain FR4 of SEQ ID NO: 286;
(17) 서열번호 99의 중쇄 FR1, 서열번호 100의 중쇄 FR2, 서열번호 101의 중쇄 FR3, 서열번호 102의 중쇄 FR4, 서열번호 287의 경쇄 FR1, 서열번호 288의 경쇄 FR2, 서열번호 289의 경쇄 FR3, 서열번호 290의 경쇄 FR4;(17) heavy chain FR1 of SEQ ID NO: 99, heavy chain FR2 of SEQ ID NO: 100, heavy chain FR3 of SEQ ID NO: 101, heavy chain FR4 of SEQ ID NO: 102, light chain FR1 of SEQ ID NO: 287, light chain FR2 of SEQ ID NO: 288, light chain of SEQ ID NO: 289 FR3, light chain FR4 of SEQ ID NO: 290;
(18) 서열번호 107의 중쇄 FR1, 서열번호 108의 중쇄 FR2, 서열번호 109의 중쇄 FR3, 서열번호 110의 중쇄 FR4, 서열번호 291의 경쇄 FR1, 서열번호 292의 경쇄 FR2, 서열번호 293의 경쇄 FR3, 서열번호 294의 경쇄 FR4; 및 (18) heavy chain FR1 of SEQ ID NO: 107, heavy chain FR2 of SEQ ID NO: 108, heavy chain FR3 of SEQ ID NO: 109, heavy chain FR4 of SEQ ID NO: 110, light chain FR1 of SEQ ID NO: 291, light chain FR2 of SEQ ID NO: 292, light chain of SEQ ID NO: 293 FR3, light chain FR4 of SEQ ID NO: 294; and
(19) 서열번호 123의 중쇄 FR1, 서열번호 124의 중쇄 FR2, 서열번호 125의 중쇄 FR3, 서열번호 126의 중쇄 FR4, 서열번호 295의 경쇄 FR1, 서열번호 296의 경쇄 FR2, 서열번호 297의 경쇄 FR3, 서열번호 298의 경쇄 FR4.(19) heavy chain FR1 of SEQ ID NO: 123, heavy chain FR2 of SEQ ID NO: 124, heavy chain FR3 of SEQ ID NO: 125, heavy chain FR4 of SEQ ID NO: 126, light chain FR1 of SEQ ID NO: 295, light chain FR2 of SEQ ID NO: 296, light chain of SEQ ID NO: 297 FR3, light chain FR4 of SEQ ID NO: 298.
상기 골격 영역 조합을 포함하는 항체 또는 이의 항원 결합 단편은 이에 제한되지는 않으나 각 해당 서열의 골격 영역과 적어도 약 80% 상동성, 적어도 약 90% 상동성, 적어도 약 95% 상동성을 가질 수 있다.An antibody or antigen-binding fragment thereof comprising the above framework region combination may have, but is not limited to, at least about 80% homology, at least about 90% homology, or at least about 95% homology to the framework region of each corresponding sequence. .
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체의 특징Characteristics of antibodies that specifically bind to SARS-CoV-2 S protein
본 발명의 일 실시예에 의하면, 본 발명자들은 파지 디스플레이 방법으로 나이브(naive) 인간 scFv 라이브러리(Ymax®-ABL로 칭함; (주)와이바이오로직스) 또는 Covid-19 확진 환자 유래 scFv 면역 라이브러리(Patient-Immune Library)를 바이오패닝하여 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체를 제조하였다.According to an embodiment of the present invention, the present inventors have developed a naive human scFv library (referred to as Ymax ® -ABL; YBiologics Co., Ltd.) or an scFv immune library derived from a Covid-19 confirmed patient by a phage display method (Patient). -Immune Library) to prepare an antibody that specifically binds to the SARS-CoV-2 spike protein by biopanning.
본 명세서에 사용된 용어, "파지 디스플레이"는 변이체 폴리펩티드를 파지, 예를 들어 섬유상 파지 입자의 표면 상에 외피 단백질의 적어도 일부와의 융합 단백질로서 디스플레이하는 기술이다. 파지 디스플레이의 유용성은 무작위화 단백질 변이체의 큰 라이브러리를 대상으로 하여, 표적 항원과 고친화도로 결합하는 서열을 신속하고도 효율적으로 분류할 수 있다는 사실에 있다. 펩티드 및 단백질 라이브러리를 파지 상에 디스플레이하는 것은 특이적 결합 특성을 지닌 폴리펩티드를 알아보기 위해 수 백만개의 폴리펩티드를 스크리닝하는데 사용되어 왔다.As used herein, the term "phage display" is a technique for displaying a variant polypeptide as a fusion protein with at least a portion of an envelope protein on the surface of a phage, eg, a filamentous phage particle. The usefulness of phage display resides in the fact that, by targeting a large library of randomized protein variants, it is possible to quickly and efficiently sort sequences that bind to a target antigen with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for polypeptides with specific binding properties.
파지 디스플레이 기술은 목적하는 특징을 지닌 항체를 제조하기 위한 통상적인 하이브리도마 및 재조합 방법에 비해 짧은 시간에 다양한 서열을 지닌 항체 라이브러리를 생성시킬 수 있는 장점이 있다. 또한, 면역이 전혀 요구되지 않기 때문에, 파지 항체 라이브러리는 독성이거나 항원성이 낮은 항원에 대해서도 항체를 생성시킬 수 있다. 파지 항체 라이브러리를 사용하여 신규한 치료적 항체를 생성 및 확인할 수도 있다.The phage display technology has the advantage of being able to generate an antibody library having various sequences in a short time compared to conventional hybridoma and recombinant methods for producing an antibody having desired characteristics. In addition, since no immunization is required, the phage antibody library can generate antibodies against antigens that are toxic or of low antigenicity. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.
파지 디스플레이 라이브러리로부터 고친화성 항체를 확인 및 분리할 수 있는 기술은 치료용 신규 항체 분리에 중요하다. 라이브러리로부터 고친화성 항체를 분리하는 것은 라이브러리의 크기, 세균성 세포 중에서의 생산 효율 및 라이브러리의 다양성에 좌우될 수 있다. A technology capable of identifying and isolating high-affinity antibodies from phage display libraries is important for isolating novel therapeutic antibodies. Isolation of high affinity antibodies from a library may depend on the size of the library, production efficiency in bacterial cells, and diversity of the library.
본 명세서에 사용된 용어, "scFv(single chain fragment variable)"는 유전자 재조합을 통해 항체의 가변영역만을 발현시켜 만든 단쇄항체를 말하며, 항체의 VH 영역과 VL 영역을 짧은 펩타이드 사슬로 연결한 단쇄 형태의 항체를 말한다. As used herein, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and a single-chain form in which the VH region and the VL region of the antibody are connected by a short peptide chain. refers to the antibody of
본 명세서에 사용된 용어, "바이오패닝(biopanning)"은 파지의 외벽(coat)에 펩타이드를 발현(display)하는 파지 라이브러리로부터, 표적 분자(항체, 효소, 세포표면 수용체 등)과 결합하는 성질을 지닌 펩타이드를 표면에 발현하고 있는 파지만을 선택해 내는 과정을 일컫는다.As used herein, the term "biopanning" refers to a property of binding to a target molecule (antibody, enzyme, cell surface receptor, etc.) from a phage library that expresses a peptide on the outer wall of a phage. It refers to the process of selecting only phages expressing the peptides on the surface.
상기 항체 또는 이의 항원 결합 단편은 특별히 이에 제한되지 않으나, 투여된 생체 내에서의 체류시간을 증진시키기 위하여, 당화(glycosylation) 및/또는 페길화(PEGylation)될 수 있다.The antibody or antigen-binding fragment thereof is not particularly limited thereto, but may be glycosylated and/or PEGylated to improve residence time in the administered body.
상기 당화 및/또는 페길화는 본 발명의 항체의 기능을 유지하는 한 당업계의 공지된 방법에 의해 다양한 당화 및/또는 페길화 패턴이 변형될 수 있고, 본 발명의 항체는 다양한 당화 및/또는 페길화 패턴이 변형된 변이 단일클론 항체(monoclonal antibody) 또는 이의 항원 결합 단편을 모두 포함한다.The glycosylation and/or pegylation can be modified by various glycosylation and/or pegylation patterns by methods known in the art as long as the function of the antibody of the present invention is maintained, and the antibody of the present invention has various glycosylation and/or pegylation It includes all mutant monoclonal antibodies or antigen-binding fragments thereof in which the pegylation pattern is modified.
본 발명의 일 실시예에서, 인간항체 라이브러리 Ymax®-ABL 유래 7종과 COVID-19 환자-면역 라이브러리 유래 8종으로 총 15종의 선도항체를 수득하고, 이를 최적화하여 최종 총 19종의 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체를 확보하였다(실시예 2 및 실시예 3). In one embodiment of the present invention, a total of 15 lead antibodies were obtained from 7 types of human antibody library Ymax ® -ABL and 8 types derived from the COVID-19 patient-immunity library, and by optimizing them, a final total of 19 types of SARS- Antibodies that specifically bind to the CoV-2 spike protein were obtained (Examples 2 and 3).
상기 항체는 SARS-CoV-2 S1 또는 RBD에 나노몰(nanomolar) 이하의 수준으로 높은 항원 결합력(KD)을 보이고, SARS-CoV-2 RBD와 ACE2의 결합을 저해하는 중화능을 확인한 세포 기반 중화능 평가에서도 IC50 값이 나노몰 수준으로 우수하며, soluble ACE2에 대해서도 중화능이 모두 월등함을 확인하였다. 또한, 상기 항체는 SARS-CoV-2 RBD의 세포내 유입(internalization)을 효과적으로 억제하고, ACE2와 결합하는 SARS-CoV-2 RBM(receptor binding motif)에 특이적으로 결합하여 바이러스와 직접 반응함에 의해 감염을 중화할 수 있는 효능이 우수한 항체임을 알 수 있었다(실시예 5 내지 실시예 9).The antibody exhibits high antigen-binding affinity (K D ) at a sub-nanomolar level to SARS-CoV-2 S1 or RBD, and cell-based confirming neutralizing ability to inhibit the binding of SARS-CoV-2 RBD and ACE2 In the evaluation of neutralization ability, the IC 50 value was excellent at the nanomolar level, and it was confirmed that all of the neutralizing ability was superior to soluble ACE2. In addition, the antibody effectively inhibits the internalization of SARS-CoV-2 RBD, binds specifically to SARS-CoV-2 receptor binding motif (RBM) binding to ACE2, and reacts directly with the virus. It was found that the antibody had excellent efficacy to neutralize infection (Examples 5 to 9).
따라서, 본 발명의 항체는 SARS-CoV-2의 막 단백질 스파이크(S)와 ACE2의 결합을 완전히 차단할 수 있으므로, 신종 코로나바이러스 감염증에 대한 예방 및 치료 효과를 나타낼 수 있다.Therefore, since the antibody of the present invention can completely block the binding of the membrane protein spike (S) of SARS-CoV-2 to ACE2, it can exhibit preventive and therapeutic effects on novel coronavirus infection.
본 발명에 따른 상기 항체는 이에 제한되지는 않으나, SARS-CoV-2 스파이크 단백질에 대해 1×10-8 M 내지 1×10-12　M, 또는 1×10-9 M 내지 1×10-11　M 범위 내의 결합 친화도(KD)를 나타낼 수 있다. The antibody according to the present invention is not limited thereto, but 1×10 -8 M to 1×10 -12 M, or 1×10 -9 M to 1×10 -11 M for SARS-CoV-2 spike protein binding affinity (K D ) within a range.
또한, 본 발명의 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편에는 본 발명에 따른 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편에서 보존적 치환을 통해 아미노산 서열의 일부가 치환된 항체 또는 이의 항원 결합 단편도 포함될 수 있다.In addition, the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof of the present invention includes a portion of the amino acid sequence through conservative substitution in the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof according to the present invention. A substituted antibody or antigen-binding fragment thereof may also be included.
본 명세서에 사용된 용어, "보존적 치환"은 1개 이상의 아미노산을 해당 폴리펩티드의 생물학적 또는 생화학적 기능의 손실을 야기하지 않는 유사한 생화학적 특성을 갖는 아미노산으로 치환하는 것을 포함하는 폴리펩티드의 변형을 의미한다. "보존적 아미노산 치환"은 아미노산 잔기를 유사한 측쇄를 갖는 아미노산 잔기로 대체시키는 치환이다. 유사한 측쇄를 갖는 아미노산 잔기 부류는 해당 기술분야에 규정되어 있으며, 잘 알려져 있다. 이들 부류는 염기성 측쇄를 갖는 아미노산(예를 들어, 라이신, 아르기닌, 히스티딘), 산성 측쇄를 갖는 아미노산(예를 들어, 아스파르트산, 글루탐산), 대전되지 않은 극성 측쇄를 갖는 아미노산(예를 들어, 글리신, 아스파라진, 글루타민, 세린, 트레오닌, 티로신, 시스테인), 비-극성 측쇄를 갖는 아미노산(예를 들어, 알라닌, 발린, 류신, 이소류신, 프롤린, 페닐알라닌, 메티오닌, 트립토판), 베타-분지된 측쇄를 갖는 아미노산(예를 들어, 트레오닌, 발린, 이소류신) 및 방향족 측쇄를 갖는 아미노산(예를 들어, 티로신, 페닐알라닌, 트립토판, 히스티딘)이다. 본 발명의 항체는 상기와 같은 보존적 아미노산 치환을 가지더라도 여전히 활성을 보유할 수 있다.As used herein, the term "conservative substitution" refers to a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the polypeptide. . A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains have been defined in the art and are well known. These classes include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains amino acids (eg, threonine, valine, isoleucine) with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). The antibody of the present invention may still retain activity even with such conservative amino acid substitutions.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체를 코딩하는 핵산Nucleic acid encoding an antibody that specifically binds to the SARS-CoV-2 S protein
본 발명의 다른 측면은, 상기 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 코딩하는 핵산을 제공한다.Another aspect of the present invention provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
본 명세서에서 사용되는 핵산은 세포, 세포 용해물(lysate) 중에 존재하거나, 또는 부분적으로 정제된 형태 또는 실질적으로 순수한 형태로 존재할 수도 있다. 핵산은 알칼리/SDS 처리, CsCl 밴드화(banding), 컬럼 크로마토그래피, 아가로스 겔 전기 영동 및 해당 기술분야에 잘 알려진 기타의 것을 포함하는 표준 기술에 의해 다른 세포 성분 또는 기타 오염 물질, 예를 들어 다른 세포의 핵산 또는 단백질로부터 정제되어 나올 경우 "단리"되거나 "실질적으로 순수하게 된" 것이다. Nucleic acids as used herein may be present in cells, cell lysates, or may exist in partially purified or substantially pure form. Nucleic acids can be removed from other cellular components or other contaminants, e.g., by standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. "Isolated" or "substantially pure" when purified from the nucleic acid or protein of another cell.
본 발명의 핵산은 예를 들어 DNA 또는 RNA일 수 있으며, 인트론 서열을 포함하거나 포함하지 않을 수 있다. 핵산에서 기본 구성단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다. The nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not contain an intron sequence. The nucleotide, which is the basic building block of nucleic acids, includes not only natural nucleotides, but also analogs in which sugar or base regions are modified.
본 발명의 중쇄 및 경쇄 가변영역을 코딩하는 핵산의 서열은 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다.The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
본 발명에 있어서, 상기 항-SARS-COV-2의 스파이크 항체를 코딩하는 핵산은 이에 제한되지는 않으나, 하기 군에서 선택되는 어느 하나의 가변영역을 코딩하는 폴리뉴클레오티드 조합을 포함하는 것일 수 있다:In the present invention, the nucleic acid encoding the anti-SARS-COV-2 spike antibody is not limited thereto, but may include a polynucleotide combination encoding any one variable region selected from the following group:
(1) 서열번호 241의 중쇄 가변영역 및 서열번호 242의 경쇄 가변영역;(1) a heavy chain variable region of SEQ ID NO: 241 and a light chain variable region of SEQ ID NO: 242;
(2) 서열번호 243의 중쇄 가변영역 및 서열번호 244의 경쇄 가변영역;(2) a heavy chain variable region of SEQ ID NO: 243 and a light chain variable region of SEQ ID NO: 244;
(3) 서열번호 245의 중쇄 가변영역 및 서열번호 246의 경쇄 가변영역;(3) a heavy chain variable region of SEQ ID NO: 245 and a light chain variable region of SEQ ID NO: 246;
(4) 서열번호 247의 중쇄 가변영역 및 서열번호 248의 경쇄 가변영역;(4) the heavy chain variable region of SEQ ID NO: 247 and the light chain variable region of SEQ ID NO: 248;
(5) 서열번호 249의 중쇄 가변영역 및 서열번호 250의 경쇄 가변영역;(5) a heavy chain variable region of SEQ ID NO: 249 and a light chain variable region of SEQ ID NO: 250;
(6) 서열번호 251의 중쇄 가변영역 및 서열번호 252의 경쇄 가변영역;(6) a heavy chain variable region of SEQ ID NO: 251 and a light chain variable region of SEQ ID NO: 252;
(7) 서열번호 253의 중쇄 가변영역 및 서열번호 254의 경쇄 가변영역;(7) a heavy chain variable region of SEQ ID NO: 253 and a light chain variable region of SEQ ID NO: 254;
(8) 서열번호 255의 중쇄 가변영역 및 서열번호 256의 경쇄 가변영역;(8) a heavy chain variable region of SEQ ID NO: 255 and a light chain variable region of SEQ ID NO: 256;
(9) 서열번호 257의 중쇄 가변영역 및 서열번호 258의 경쇄 가변영역;(9) a heavy chain variable region of SEQ ID NO: 257 and a light chain variable region of SEQ ID NO: 258;
(10) 서열번호 259의 중쇄 가변영역 및 서열번호 260의 경쇄 가변영역;(10) a heavy chain variable region of SEQ ID NO: 259 and a light chain variable region of SEQ ID NO: 260;
(11) 서열번호 261의 중쇄 가변영역 및 서열번호 262의 경쇄 가변영역;(11) a heavy chain variable region of SEQ ID NO: 261 and a light chain variable region of SEQ ID NO: 262;
(12) 서열번호 263의 중쇄 가변영역 및 서열번호 264의 경쇄 가변영역;(12) a heavy chain variable region of SEQ ID NO: 263 and a light chain variable region of SEQ ID NO: 264;
(13) 서열번호 265의 중쇄 가변영역 및 서열번호 266의 경쇄 가변영역;(13) a heavy chain variable region of SEQ ID NO: 265 and a light chain variable region of SEQ ID NO: 266;
(14) 서열번호 267의 중쇄 가변영역 및 서열번호 268의 경쇄 가변영역;(14) a heavy chain variable region of SEQ ID NO: 267 and a light chain variable region of SEQ ID NO: 268;
(15) 서열번호 269의 중쇄 가변영역 및 서열번호 270의 경쇄 가변영역;(15) a heavy chain variable region of SEQ ID NO: 269 and a light chain variable region of SEQ ID NO: 270;
(16) 서열번호 241의 중쇄 가변영역 및 서열번호 303의 경쇄 가변영역;(16) a heavy chain variable region of SEQ ID NO: 241 and a light chain variable region of SEQ ID NO: 303;
(17) 서열번호 243의 중쇄 가변영역 및 서열번호 304의 경쇄 가변영역;(17) a heavy chain variable region of SEQ ID NO: 243 and a light chain variable region of SEQ ID NO: 304;
(18) 서열번호 245의 중쇄 가변영역 및 서열번호 305의 경쇄 가변영역; 및 (18) a heavy chain variable region of SEQ ID NO: 245 and a light chain variable region of SEQ ID NO: 305; and
(19) 서열번호 249의 중쇄 가변영역 및 서열번호 306의 경쇄 가변영역.(19) the heavy chain variable region of SEQ ID NO: 249 and the light chain variable region of SEQ ID NO: 306.
상기 항체 또는 이를 코딩하는 핵산 분자는 이에 제한되지는 않으나 서열번호에 기재된 각 해당 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 배열하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 정렬된 서열을 분석한 경우에, 90% 이상의 상동성, 바람직하게는 95% 이상의 상동성, 더욱 바람직하게는 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 상동성을 나타내는 서열을 의미한다. The antibody or the nucleic acid molecule encoding the same is not limited thereto, but it is construed to include a sequence exhibiting substantial identity to each corresponding sequence shown in SEQ ID NO. The substantial identity is, when the sequence of the present invention and any other sequences are arranged to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art, homology of 90% or more, Preferably, it refers to a sequence that exhibits at least 95% homology, more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology.
이러한 상동성은 당업계에 공지된 방법에 의한 서열 비교 및/또는 정렬(alignment)에 의해 결정될 수 있다. 예를 들어, 서열 비교 알고리즘(예: NCBI Basic Local Alignment Search Tool; BLAST), 수동 정렬, 육안 검사 등을 이용하여 본 발명의 핵산 또는 단백질의 서열 상동성을 결정할 수 있다. Such homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, the sequence homology of the nucleic acid or protein of the present invention may be determined using a sequence comparison algorithm (eg, NCBI Basic Local Alignment Search Tool; BLAST), manual alignment, visual inspection, and the like.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체 제조용 벡터A vector for preparing an antibody that specifically binds to the SARS-CoV-2 S protein
본 발명의 다른 측면은, 상기 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 코딩하는 핵산을 포함하는 재조합 발현 벡터를 제공한다.Another aspect of the present invention provides a recombinant expression vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
본 발명에 따른 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편의 발현을 위하여, 부분적이거나 전장인 경쇄 및 중쇄를 코딩하는 DNA를 표준 분자 생물학 기술(예를 들어, PCR 증폭 또는 목적 항체를 발현하는 하이브리도마를 사용한 cDNA 클로닝)로 수득할 수 있으며, DNA가 전사 및 번역 제어 서열에 "작동되도록 연결"되어 발현 벡터 내로 삽입될 수 있다. 벡터 성분에는 일반적으로 다음 중의 하나 이상이 포함되지만, 그에 제한되지 않는다: 신호 서열, 복제 기점, 하나 이상의 마커 유전자, 증강인자 요소, 프로모터, 및 전사 종결 서열.For the expression of the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof according to the present invention, DNA encoding partial or full-length light and heavy chains is prepared using standard molecular biology techniques (e.g., PCR amplification or the antibody of interest) cDNA cloning using hybridomas expressing Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.
본 명세서에서 사용되는 용어, "벡터"는 숙주세포에서 목적 유전자를 발현시키기 위한 수단으로 플라스미드 벡터; 코즈미드 벡터; 박테리오파지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스벡터 같은 바이러스 벡터 등을 포함한다. 상기 벡터에서 항체 또는 이의 항원 결합 단편을 코딩하는 핵산은 프로모터에 작동적으로 연결되어 있다. As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, including a plasmid vector; cozmid vector; viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, and the like. In the vector, the nucleic acid encoding the antibody or antigen-binding fragment thereof is operably linked to a promoter.
본 명세서에 사용된 용어, "작동적으로 연결"은 벡터 내의 전사 및 번역 제어 서열이 항체 유전자의 전사 및 번역을 조절하는 의도된 기능을 하도록 항체 또는 이의 항원 결합 단편을 코딩하는 유전자가 벡터 내로 라이게이션(ligation)된다는 것을 의미한다. 발현 벡터 및 발현 제어 서열은 사용되는 발현용 세포와 상용성이 있도록 선택된다. 항체의 경쇄 유전자 및 중쇄 유전자는 별개의 벡터 내로 삽입되거나, 두 유전자 모두 동일한 발현 벡터 내로 삽입된다. 항체 유전자는 표준 방법(예를 들어 항체 유전자 단편 및 벡터 상의 상보성 제한 효소 부위의 라이게이션, 또는 제한 효소 부위가 전혀 존재하지 않을 경우 블런트(blunt) 말단 라이게이션)으로 발현 벡터 내로 삽입된다. As used herein, the term "operably linked" means that the gene encoding the antibody or antigen-binding fragment thereof is lysed into a vector such that transcriptional and translational control sequences in the vector serve the intended function of regulating the transcription and translation of the antibody gene. It means being ligated. Expression vectors and expression control sequences are selected to be compatible with the cells for expression used. The light chain gene and heavy chain gene of the antibody are inserted into separate vectors, or both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (eg, ligation of complementary restriction enzyme sites on the antibody gene fragment and vector, or blunt end ligation if no restriction enzyme sites are present).
경우에 따라서, 상기 재조합 발현 벡터는 형질전환된 세포로부터 항체 사슬의 분비를 용이하게 하는 신호 펩티드를 코딩하는 서열을 포함할 수 있다. 상기 항체 사슬 유전자 및 신호 펩티드-코딩 서열은 신호 펩티드가 항체 사슬의 아미노 말단에 결합되어 발현되도록 프레임에 맞게 벡터 내로 클로닝될 수 있다. 신호 펩티드는 면역글로불린 신호 펩티드 또는 이종성 신호 펩티드(즉, 면역글로불린 외 단백질 유래의 신호 펩티드)일 수 있다. 또한, 상기 재조합 발현 벡터는 형질전환된 세포에서 항체 사슬 유전자의 발현을 제어하는 조절서열을 포함할 수 있다. "조절서열"은 항체 사슬 유전자의 전사 또는 번역을 제어하는 프로모터, 인핸서 및 기타 발현 제어 요소(예를 들어, 폴리아데닐화 신호)를 포함할 수 있다. 통상의 기술자는 형질전환시킬 세포의 선택, 단백질의 발현 수준 등과 같은 인자에 따라 조절 서열을 달리 선택하여, 발현 벡터의 디자인이 달라질 수 있음을 인식할 수 있다.Optionally, the recombinant expression vector may contain a sequence encoding a signal peptide that facilitates secretion of the antibody chain from the transformed cell. The antibody chain gene and signal peptide-coding sequence can be cloned into a vector in frame so that the signal peptide is expressed by binding to the amino terminus of the antibody chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a protein other than immunoglobulin). In addition, the recombinant expression vector may include a regulatory sequence for controlling the expression of the antibody chain gene in the transformed cell. “Regulatory sequences” may include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. A person skilled in the art can recognize that the design of the expression vector may vary by selecting different control sequences depending on factors such as the selection of cells to be transformed, the level of protein expression, and the like.
본 발명의 벡터는 또한 벡터로부터 발현되는 항체의 정제를 용이하게 하기 위하여 항체 유전자에 융합시킬 다른 서열을 포함할 수 있다. 이 서열은 예를 들어, 글루타티온 S-트랜스퍼라제(Pharmacia, USA), 말토스 결합 단백질(NEB, USA), FLAG(IBI, USA), 6× His(hexahistidine; Quiagen, USA) 등의 유전자일 수 있다. 상기 벡터는 선택 표지로서 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 이러한 유전자로는 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.The vectors of the present invention may also include other sequences to be fused to the antibody gene to facilitate purification of the antibody expressed from the vector. This sequence may be, for example, a gene such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6×His (hexahistidine; Quiagen, USA). there is. The vector contains an antibiotic resistance gene commonly used in the art as a selection marker, and such genes include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and There is a gene for resistance to tetracycline.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체 제조용 벡터 도입 세포Cells introduced with a vector for preparing an antibody that specifically binds to the SARS-CoV-2 S protein
본 발명의 또 다른 측면은, 상기 재조합 발현 벡터로 형질전환된 세포를 제공한다.Another aspect of the present invention provides a cell transformed with the recombinant expression vector.
상기 형질전환 세포의 숙주세포로서, 원핵세포, 진핵세포, 포유동물, 식물, 곤충, 균류 또는 세포성 기원의 세포를 포함할 수 있지만 이에 한정되지 않는다. 상기 원핵세포의 일 예로는 대장균을 사용할 수 있다. 또한, 진핵세포의 일 예로는 효모를 사용할 수 있다. 또한, 상기 포유동물 세포로 CHO 세포, F2N 세포, CSO 세포, BHK 세포, 바우스(Bowes) 흑색종 세포, HeLa 세포, 911 세포, AT1080 세포, A549 세포, HEK 293 세포 또는 HEK293T 세포 등을 사용할 수 있으나, 이에 한정되지 않으며, 당업자에게 알려진 포유동물 숙주세포로 사용 가능한 세포는 모두 이용 가능하다.The host cell of the transformed cell may include, but is not limited to, a cell of prokaryotic, eukaryotic, mammalian, plant, insect, fungal or cellular origin. As an example of the prokaryotic cell, E. coli may be used. In addition, yeast may be used as an example of eukaryotic cells. In addition, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells or HEK293T cells may be used as the mammalian cells. , but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art is available.
본 발명에 따른 항-SARS-COV-2의 스파이크 항체를 발현시키기 위해 다양한 세포/벡터 조합이 이용될 수 있다. 구체적으로, 진핵 세포에 적합한 발현 벡터로는 SV40, 소 유두종바이러스, 아데노바이러스, 아데노-연관 바이러스(adeno-associated virus), 시토메갈로바이러스 및 레트로바이러스로부터 유래된 발현 벡터가 포함되나, 이들로 한정되는 것은 아니다. 세균 세포에 사용할 수 있는 발현 벡터로는 pET, pRSET, pBluescript, pGEX2T, pUC, col E1, pCR1, pBR322, pMB9 및 이들의 유도체와 같은 대장균(E. coli)-유래 세균성 플라스미드; RP4와 같이 보다 넓은 숙주 범위를 갖는 플라스미드; λgt10, λgt11, 및 NM989와 같은 다양한 파지 람다(phage lambda) 유도체 등의 파지 DNA; 및 M13과 필라멘트성 단일가닥 DNA 파지와 같은 기타 DNA 파지가 포함된다. 효모 세포에 유용한 발현 벡터는 YEp 플라스미드 및 그의 유도체이다. 곤충 세포에 유용한 벡터는 pVL941이다.Various cell/vector combinations can be used to express the spike antibody of anti-SARS-COV-2 according to the present invention. Specifically, expression vectors suitable for eukaryotic cells include, but are not limited to, expression vectors derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. it is not Expression vectors usable for bacterial cells include E. coli -derived bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC, col E1, pCR1, pBR322, pMB9 and derivatives thereof; plasmids with a wider host range, such as RP4; phage DNA, such as various phage lambda derivatives such as λgt10, λgt11, and NM989; and other DNA phages such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells are YEp plasmids and derivatives thereof. A useful vector for insect cells is pVL941.
또한, 숙주세포로 재조합 발현 벡터를 도입하는 경우, CaCl2 침전법, CaCl2 침전법에 DMSO(dimethyl sulfoxide)라는 환원물질을 사용함으로써 효율을 높인 Hanahan 방법, 전기천공법(electroporation), 인산칼슘 침전법, 원형질 융합법, 실리콘 카바이드 섬유를 이용한 교반법, 아그로박테리아 매개된 형질전환법, PEG를 이용한 형질전환법, 덱스트란 설페이트, 리포펙타민 및 건조/억제 매개된 형질전환 방법 등이 사용될 수 있다.In addition, when introducing a recombinant expression vector into a host cell, the CaCl 2 precipitation method, the CaCl 2 precipitation method using a reducing material called DMSO (dimethyl sulfoxide), which increases the efficiency by using the Hanahan method, electroporation, calcium phosphate precipitation method, protoplast fusion method, agitation method using silicon carbide fiber, agrobacterium-mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/inhibition-mediated transformation method, etc. can be used. .
숙주세포 내로 도입된 벡터는 숙주세포 내에서 발현될 수 있으며, 이러한 경우에는 다량의 본 발명의 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편을 수득할 수 있다.The vector introduced into the host cell can be expressed in the host cell, and in this case, a large amount of the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof of the present invention can be obtained.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체 제조 방법Method for producing an antibody that specifically binds to SARS-CoV-2 S protein
본 발명의 또 다른 측면은, (i) 상기 형질전환된 세포를 배양하는 단계; 및 (ii) 얻어진 세포 배양액으로부터 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 회수하는 단계를 포함하는, SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편의 제조 방법을 제공한다.Another aspect of the present invention, (i) culturing the transformed cell; and (ii) recovering an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein from the obtained cell culture medium. A method for producing an antigen-binding fragment thereof is provided.
상기 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 발현할 수 있는 재조합 발현 벡터가 포유류 세포 내로 도입될 경우, 세포에서 항체가 발현되게 하기에 충분한 기간 동안, 또는 더 바람직하게는 세포가 배양되는 배양 배지 내로 항체가 분비되게 하기에 충분한 기간 동안 세포를 배양함으로써 상기 항체 또는 이의 항원 결합 단편을 제조할 수 있다.When a recombinant expression vector capable of expressing an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein is introduced into a mammalian cell, for a period sufficient to allow the antibody to be expressed in the cell, or for more Preferably, the antibody or antigen-binding fragment thereof can be prepared by culturing the cells for a period sufficient to allow the antibody to be secreted into the culture medium in which the cells are cultured.
상기 세포는 각종 배지에서 배양할 수 있으며, 배양 배지로는 시판용 배지를 제한 없이 사용할 수 있다. 당업자에게 공지되어 있는 기타 모든 필수 보충물이 적당한 농도로 포함될 수도 있다. 선별된 숙주세포에서 단백질 발현을 위해 적합한 배양 조건, 예를 들어 온도, pH 등이 이미 사용되고 있고, 이는 당업자에게 명백할 것이다.The cells can be cultured in various media, and commercially available media can be used without limitation as the culture media. All other essential supplements known to those skilled in the art may be included in appropriate concentrations. Suitable culture conditions, for example, temperature, pH, etc., have already been used for protein expression in the selected host cell, and will be apparent to those skilled in the art.
경우에 따라서, 발현된 항체를 세포 배양액으로부터 분리하여 균일하게 정제할 수 있다. 상기 항체의 분리 또는 정제는 통상의 단백질 분리 및 정제 방법, 예를 들어 크로마토그래피에 의해 수행될 수 있다. 상기 크로마토그래피는 예를 들어, 프로틴 A 컬럼 또는 프로틴 G 컬럼을 사용하는 친화성 크로마토그래피, 이온 교환 크로마토그래피, 소수성 크로마토그래피, 또는 히드록실아파타이트 크로마토그래피를 포함할 수 있다. 상기 크로마토그래피 이외에, 추가로 여과, 초여과, 염석, 투석 등을 조합함으로써 항체를 분리, 정제할 수 있다.In some cases, the expressed antibody can be separated from the cell culture medium and purified to uniformity. Isolation or purification of the antibody may be performed by a conventional protein separation and purification method, for example, chromatography. The chromatography may include, for example, affinity chromatography using a protein A column or a protein G column, ion exchange chromatography, hydrophobic chromatography, or hydroxylapatite chromatography. In addition to the above chromatography, the antibody can be separated and purified by further combining filtration, ultrafiltration, salting out, dialysis, and the like.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체 및 약물을 포함하는 ADCADC comprising antibody and drug that specifically binds to SARS-CoV-2 S protein
본 발명의 또 다른 측면은, 상기 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편 및 약물을 포함하는 항체-약물 접합체(antibody-drug conjugate, ADC)를 제공한다.Another aspect of the present invention provides an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein and a drug.
본 명세서에 사용된 용어, "항체-약물 접합체(Antibody-drug conjugate, ADC)"는 상기 항체 또는 이의 항원 결합 단편과 약물의 결합체를 의미하며, 타겟 세포로 약물을 전달하기 전까지 약물이 항체에 안정적으로 결합되어 있어야 하고, 타겟으로 전달된 후 약물은 항체로부터 유리되어야 한다. 본 발명에 있어서, 상기 항체 또는 이의 항원 결합 단편과 약물(항암제 등)은 서로 결합(예컨대, 공유결합, 펩타이드 결합 등)되어 접합체(conjugate) 또는 융합 단백질(약물이 단백질인 경우)의 형태로 사용될 수 있다.As used herein, the term "antibody-drug conjugate (ADC)" refers to a conjugate of the antibody or antigen-binding fragment thereof and a drug, and the drug is stable to the antibody until the drug is delivered to the target cell. It should be bound to the target, and after delivery to the target, the drug should be released from the antibody. In the present invention, the antibody or antigen-binding fragment thereof and a drug (anticancer agent, etc.) are bound to each other (eg, covalent bond, peptide bond, etc.) to be used in the form of a conjugate or a fusion protein (when the drug is a protein). can
상기 약물은 약리학적 효과를 나타내는 제제로 본 발명의 항체 또는 이의 항원 결합 단편에 결합될 수 있고, 산성조건에 의하여 상기 항체 또는 이의 항원 결합 단편과 분리될 수 있으며, 표적 세포에 대한 치료 효과를 나타내는 화합물을 의미한다. 상기 약물은 이에 제한되지는 않으나 항바이러스제일 수 있다. The drug is an agent that exhibits a pharmacological effect, may be bound to the antibody or antigen-binding fragment thereof of the present invention, may be separated from the antibody or antigen-binding fragment thereof by acidic conditions, and exhibit a therapeutic effect on target cells means a compound. The drug is not limited thereto, but may be an antiviral agent.
상기 항바이러스제는 이에 제한되지는 않으나, 기존의 항바이러스제, 예컨대 에볼라바이러스치료제, HIV(인간면역결핍바이러스)치료제, C형간염치료제, 독감치료제 등일 수 있다. 구체적 약물의 일예로는 칼레트라(kaletra), 렘데시비르(remdesivir), 플라크닐(하이드록시클로로퀸), 레소친(클로로퀸) 등이 있다.The antiviral agent is not limited thereto, but may be a conventional antiviral agent, such as an Ebola virus treatment agent, an HIV (human immunodeficiency virus) treatment agent, a hepatitis C treatment agent, a flu treatment agent, and the like. Examples of specific drugs include kaletra, remdesivir, placknil (hydroxychloroquine), resorcin (chloroquine), and the like.
또한, 상기 약물은 코로나바이러스 치료제로 개발되고 있는 물질을 이용할 수 있다. 구체적으로, 상기 적용될 수 있는 약물로는 항바이러스제 이외의 당뇨병 치료제(예컨대, 다파글리플로진(dapagliflozin)), 류마티스 관절염 치료제(예컨대, 아나킨라(anakinra), 토실리주맙(tocilizumab), 사리루맵(sarilumab)), 혈액암 치료제(예컨대, 아칼라브루티닙(acalabrutinib), 다발골수종 치료제(예컨대, 셀리넥서(selinexor)) 등일 수 있으나, 이에 제한되지는 않는다.In addition, the drug may use a substance being developed as a coronavirus treatment. Specifically, the applicable drugs include antiviral agents other than anti-diabetic agents (for example, dapagliflozin), rheumatoid arthritis agents (for example, anakinra, tocilizumab, sarirumab). (sarilumab)), a blood cancer treatment agent (eg, acalabrutinib), or a treatment agent for multiple myeloma (eg, selinexor), but is not limited thereto.
상기 항체-약물 접합체는 세포로 내재화될 수 있으며, SARS-CoV-2와 인간세포 표면 ACE2와의 결합을 저해시켜 SARS-CoV-2의 세포내 유입을 차단함에 의해 작용할 수 있다.The antibody-drug conjugate may be internalized into cells and may act by inhibiting the binding of SARS-CoV-2 to human cell surface ACE2 to block SARS-CoV-2 influx into cells.
상기 접합체는 약물을 항체 또는 이의 항원 결합 단편과 결합시켜 공지된 방법으로 제작할 수 있다. 항체와 약물은 그들 자신이 가진 연결기 등을 통해 직접 결합할 수도 있고, 링커나 다른 물질을 통해 간접적으로 결합할 수도 있다. 약물이 항체로부터 절단되도록 하는 주요 기전에는 리소좀(히드라존, 아세탈 및 시스-아코니테이트-유사 아미드)의 산성 pH에서의 가수분해, 리소좀 효소(카텝신 및 기타 리소좀 효소)에 의한 펩타이드 절단 및 디설파이드의 환원이 포함된다. 이러한 다양한 절단 기전의 결과로서, 약물을 항체에 연결시키는 기전은 매우 다양하며 어떠한 적합한 링커라도 사용될 수 있다.The conjugate can be prepared by a known method by binding a drug to an antibody or antigen-binding fragment thereof. Antibodies and drugs may be directly coupled through their own linker, etc., or may be coupled indirectly through a linker or other material. The main mechanisms by which drugs are cleaved from antibodies include hydrolysis at acidic pH of lysosomes (hydrazone, acetal and cis-aconitate-like amides), peptide cleavage by lysosomal enzymes (cathepsin and other lysosomal enzymes), and disulfides includes the reduction of As a result of these various cleavage mechanisms, the mechanisms by which drugs are linked to the antibody are very diverse and any suitable linker can be used.
항체와 약물이 결합하기 위한 적절한 연결기는 당해 분야에 잘 알려져 있으며, 예를 들어 이황화기, 티오에테르기, 산분해성기, 광분해성기, 펩티다제 분해성기 및 에스테라아제 분해성기를 포함한다.Suitable linking groups for binding an antibody to a drug are well known in the art and include, for example, a disulfide group, a thioether group, an acid-cleavable group, a photo-degradable group, a peptidase-cleavable group, and an esterase-decomposable group.
약물이 직접 결합될 경우 연결기(linking group)는 예로서는, SH기를 이용한 이황화결합이나 말레이미드를 매개로 하는 결합을 들 수 있다. 예를 들면, 항체 Fc영역의 분자 내 이황화결합과 약물의 이황화결합을 환원하여, 양자를 이황화결합으로 연결한다. 또한, 말레이미드를 통하는 방법 및 항체 내에 시스테인을 유전공학적으로 도입하는 방법도 있다.When the drug is directly bound, the linking group may be, for example, a disulfide bond using an SH group or a bond mediated by maleimide. For example, an intramolecular disulfide bond of the antibody Fc region and a drug disulfide bond are reduced, and both are connected by a disulfide bond. In addition, there is a method through maleimide and a method for genetically introducing cysteine into the antibody.
항체와 약물을 다른 물질(링커)을 통해 간접적으로 결합시킬 수도 있다. 링커로는 항체, 약물 또는 모두와 반응하는 작용기를 1 또는 2종류 이상 가지는 것이 바람직하다. 작용기의 예로는 아미노기, 카르복실기, 머캡토기, 말레이미드기, 피리디닐기 등을 들 수 있다.Antibodies and drugs may be indirectly coupled through other substances (linkers). The linker preferably has one or two or more functional groups that react with an antibody, drug, or both. Examples of the functional group include an amino group, a carboxyl group, a mercapto group, a maleimide group, and a pyridinyl group.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체를 포함하는 다중특이적 항체Multispecific antibodies comprising antibodies that specifically bind to SARS-CoV-2 S protein
본 발명의 또 다른 측면은, 상기 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 포함하는 다중특이적 항체(multispecific antibody)를 제공한다.Another aspect of the present invention provides a multispecific antibody comprising an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein.
본 명세서에 사용된 용어, "다중특이적 항체(multispecific antibody)"는 이중특이 항체(bispecific antibody) 및 삼중특이 항체(trispecific antibody)를 포함하는 2개 이상의 항원을 표적으로 하는 항체 또는 이의 항원 결합 단편을 의미한다. 예를 들어, 이중특이 항체는 항체의 2개의 암(arm) 중에서, 하나의 암(arm)은 본 발명에 따른 SARS-CoV-2 스파이크 단백질에 대한 항체 또는 이의 항원 결합 단편을 포함하고, 나머지 다른 암(arm)은 SARS-CoV-2 스파이크 단백질 이외의 다른 항원을 포함하는 형태를 의미한다.As used herein, the term "multispecific antibody" refers to an antibody or antigen-binding fragment thereof targeting two or more antigens, including a bispecific antibody and a trispecific antibody. means For example, a bispecific antibody comprises an antibody or antigen-binding fragment thereof to the SARS-CoV-2 spike protein according to the present invention, wherein one arm comprises an antibody or antigen-binding fragment thereof according to the present invention, among two arms of the antibody. The arm refers to a form containing an antigen other than the SARS-CoV-2 spike protein.
상기 다중특이적 항체는 유전공학 또는 임의의 방법에 의해 제조될 수 있는 형태이며, 이중특이적(Bi-specific) 항체, 삼중특이적(Tri-specific) 항체 또는 사중특이적(Tetra-specific) 항체를 포함할 수 있다. The multispecific antibody is a form that can be prepared by genetic engineering or any method, and is a bi-specific antibody, a tri-specific antibody, or a tetra-specific antibody. may include.
다중특이적 항체는 본 발명에 따른 항-SARS-COV-2의 스파이크 항체가 면역효능세포-특이적 표적분자에 대한 결합능을 가지는 항체 또는 그 단편과 결합된 형태일 수 있다. 면역효능세포-특이적 표적 분자는 TCR/CD3, CD16(FcγRIIIa), CD44, CD56, CD69, CD64(FcγRI), CD89 및 CD11b/CD18(CR3)에서 선택되는 것이 바람직하지만, 이에 한정되는 것은 아니다.The multispecific antibody may be in a form in which the anti-SARS-COV-2 spike antibody according to the present invention is combined with an antibody or a fragment thereof having binding ability to an immune effector cell-specific target molecule. The immunopotentiator cell-specific target molecule is preferably selected from, but not limited to, TCR/CD3, CD16 (FcγRIIIa), CD44, CD56, CD69, CD64 (FcγRI), CD89 and CD11b/CD18 (CR3).
다중특이적 항체는 본 발명에 따른 항-SARS-COV-2의 스파이크 항체가 면역을 자극하거나 저해시키는 사이토카인에 대한 결합능을 가지는 항체 또는 그 단편과 결합된 형태인 것이 바람직하다. 면역을 자극하거나 저해시키는 사이토카인은 예를 들면, IL-2, IL-6, IL-7, IFNα, GM-CSF, IL-10, 및 TGF-β에서 선택되는 것이 바람직하지만, 이에 한정되는 것은 아니다.The multispecific antibody is preferably in a form in which the anti-SARS-COV-2 spike antibody according to the present invention is combined with an antibody or a fragment thereof having binding ability to a cytokine that stimulates or inhibits immunity. The cytokine that stimulates or inhibits immunity is preferably selected from, for example, IL-2, IL-6, IL-7, IFNα, GM-CSF, IL-10, and TGF-β, but is limited thereto not.
다중특이적 항체는 본 발명에 따른 항-SARS-COV-2의 스파이크 항체가 바이러스성 질환, 예컨대 독감, 헤르페스, B형 감염, C형 감염, 에이즈 등의 치료에 사용되고 있는 타겟, 예를 들면, CCR5 수용체, 뉴라미니다아제(neuraminidase), 헤마글루티닌(hemagglutinin), 인터페론류(예컨대, 인터페론 알파) 등에 대한 결합능을 가지는 항체 또는 그 단편과 결합된 형태인 것이 바람직하나, 이에 한정되는 것은 아니다.The multispecific antibody is a target for which the anti-SARS-COV-2 spike antibody according to the present invention is being used in the treatment of a viral disease, such as influenza, herpes, hepatitis B infection, hepatitis C infection, AIDS, for example, CCR5 receptor, neuraminidase (neuraminidase), hemagglutinin (hemagglutinin), interferons (eg, interferon alpha) having a binding ability to the antibody or a fragment thereof having a binding form, but is not limited thereto. .
다중특이적 항체에 속하는 항체들은 scFv 기반 항체, Fab 기반 항체 및 IgG 기반 항체 등으로 구분할 수 있다. 이중특이적 항체의 경우 두 개의 신호를 동시에 억제 또는 증폭시킬 수 있기 때문에 하나의 신호를 억제/증폭하는 경우보다 더욱 효과적일 수 있으며, 각각의 신호를 각각의 신호억제제로 처리했을 경우와 비교하면, 저용량 투약이 가능하며, 동일한 시간 및 공간에서의 두 개의 신호를 억제/증폭시킬 수 있다.Antibodies belonging to multispecific antibodies may be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of a bispecific antibody, since it can inhibit or amplify two signals at the same time, it can be more effective than the case of inhibiting / amplifying one signal. Low dose dosing is possible, and both signals can be suppressed/amplified in the same time and space.
이중특이적 항체의 제조 방법은 널리 공지되어 있다. 전통적으로, 이중특이적 항체의 재조합 생산은 두 개의 중쇄가 상이한 특이성을 가지는 조건에서 두 개의 면역글로불린 중쇄/경쇄 쌍의 공동 발현을 근간으로 한다.Methods for making bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy/light chain pairs under conditions in which the two heavy chains have different specificities.
scFv를 기반으로 하는 이중특이적 항체의 경우, 상이한 scFv들의 VL과 VH를 각기 서로 조합하여 혼성 scFv를 이종이량체(heterodimer) 형태로 제조하여 디아바디(diabody)를 만들 수 있고, 상이한 scFv를 서로 연결해서 탠덤(tendem) ScFv를 제조할 수 있으며, Fab의 CH1과 CL을 각각의 scFv의 말단에 발현시켜 이종이량체성 미니항체(miniantibody)를 제조할 수 있고, Fc의 동종이량체성 도메인인 CH3 도메인의 일부 아미노산을 치환하여 'knob into hole' 형태의 이종이량체 구조로 변경시켜, 이들 변경된 CH3 도메인을 상이한 각각의 scFv 말단에 발현시킴으로써 이종이량체성 scFv 형태의 미니바디(minibody)를 제조할 수 있다.In the case of an scFv-based bispecific antibody, a hybrid scFv can be prepared in the form of a heterodimer by combining the VL and VH of different scFvs, respectively, to make a diabody, and different scFvs can be combined with each other. By linking, a tandem ScFv can be prepared, and by expressing CH1 and CL of the Fab at the ends of each scFv, a heterodimeric miniantibody can be prepared, and the homodimeric domain of Fc By substituting some amino acids in the CH3 domain to change to a 'knob into hole' type heterodimer structure, and expressing these altered CH3 domains at different scFv ends, a heterodimeric scFv type minibody was prepared. can do.
Fab를 기반으로 하는 이중특이적 항체의 경우, 특정 항원에 대한 개별 Fab'를 이황화결합 또는 매개체를 이용해서 서로 조합하여 이종이량체성 Fab 형태로 제조할 수 있고, 특정 Fab의 중쇄 또는 경쇄의 말단에 상이한 항원에 대한 scFv를 발현시킴으로써 항원 결합가(valency)를 2개로 하거나, Fab과 scFv 사이에 경첩부위(hinge region)를 둠으로써 동종이량체 형태로 4개의 항원결합가를 가지도록 제조할 수 있다. 또한, Fab의 경쇄 말단과 중쇄 말단에 상이한 항원에 대한 scFv를 융합시킴으로써 항원에 대한 결합가를 3개로 만든 이중표적 바이바디(bibody) 및 Fab의 경쇄말단과 중쇄말단에 상이한 scFv를 각각 융합시킴으로써 항원에 대한 결합가를 3개로 가지도록 한 삼중표적 바이바디 형태로 제조할 수 있고, 상이한 Fab 3개를 화학적으로 접합시켜도 수득할 수 있다.In the case of a bispecific antibody based on a Fab, individual Fab's directed against a specific antigen can be combined with each other using a disulfide bond or a mediator to form a heterodimeric Fab, and the ends of the heavy or light chains of the specific Fab It can be prepared to have two antigen valencies by expressing scFvs for different antigens in the . In addition, by fusing scFvs for different antigens to the light and heavy chain ends of the Fab, a dual-target bibody with three antigen binding values and different scFvs to the light and heavy chain ends of the Fab are fused to the antigen, respectively. It can be prepared in the form of a triple-targeted bibody so as to have three binding valencies, and can also be obtained by chemically conjugating three different Fabs.
IgG를 기반으로 하는 이중특이적 항체의 경우, 마우스와 렛트 하이브리도마를 다시 교잡함으로써 하이브리드 하이브리도마, 일명 쿼드로마(quadromas)를 제조하여 이중특이적 항체를 생산하는 방법이 알려져 있다. 또한, 경쇄 부분은 공유하면서, 상이한 중쇄에 대해서 Fc의 CH3 동종이량체성 도메인의 일부 아미노산을 변형시켜 이종이량체 형태로 제작한 이른 바 'Holes and Knob' 형태로 이중특이적 항체를 제조할 수도 있다. 상이한 2종의 scFv를 IgG의 경쇄와 중쇄의 가변 도메인 대신 불변(constant) 도메인에 각각 융합 발현시켜 동종이량체 형태의 (scFv)4-IgG를 제조할 수도 있다. In the case of an IgG-based bispecific antibody, a method for producing a bispecific antibody by re-crossing a mouse and a rat hybridoma to produce a hybrid hybridoma, aka quadromas, is known. In addition, while sharing the light chain portion, it is also possible to prepare a bispecific antibody in the so-called 'Holes and Knob' form by modifying some amino acids of the CH3 homodimeric domain of Fc with respect to different heavy chains to form a heterodimer. there is. (scFv)4-IgG in a homodimeric form can also be prepared by fusion-expressing two different scFvs in constant domains instead of the variable domains of the light and heavy chains of IgG.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체를 포함하는 약학 조성물Pharmaceutical composition comprising an antibody that specifically binds to SARS-CoV-2 S protein
본 발명의 또 다른 측면은, 상술한 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편, 이를 포함하는 항체-약물 접합체 또는 다중특이적 항체를 포함하는 SARS-CoV-2 감염증의 예방 또는 치료용 약학 조성물을 제공한다. Another aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the above-described SARS-CoV-2 spike protein, an antibody-drug conjugate comprising the same, or SARS-CoV-2 comprising a multispecific antibody It provides a pharmaceutical composition for preventing or treating an infection.
상기 SARS-CoV-2 감염증은 SARS-COV-2의 스파이크 단백질의 발현 또는 과발현과 관련된 것일 수 있으며, SARS-CoV-2에 관한 것은 전술한 바와 같다.The SARS-CoV-2 infection may be related to the expression or overexpression of the spike protein of SARS-COV-2, and the SARS-CoV-2 is as described above.
본 명세서에 사용된 용어, "예방"은 본 발명에 따른 조성물의 투여로 SARS-CoV-2(severe acute respiratory syndrome coronavirus 2; 중증급성호흡기증후군 코로나바이러스 2)에 의해 유발되는 신종 코로나바이러스 감염증(COVID-19)을 억제시키거나 진행을 지연시키는 모든 행위를 의미하며, "치료"는 신종 코로나바이러스 감염증의 억제, 경감 또는 제거를 의미한다.As used herein, the term "prevention" refers to a novel coronavirus infection (COVID) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by administration of the composition according to the present invention. -19) means any action that inhibits or delays the progression, and "treatment" means the suppression, alleviation or elimination of novel coronavirus infection.
본 발명에 있어서, 상기 약학 조성물은 치료적 유효량의 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편, 및 약학적으로 허용되는 첨가제를 포함하는 것을 특징으로 할 수 있다. "약학적으로 허용되는 담체"는 약물을 제제화하거나 또는 안정화시키는 것을 돕기 위해서 활성 성분에 추가될 수 있는 물질이고, 환자에게 유의한 독성 효과를 야기하지 않는다.In the present invention, the pharmaceutical composition may include a therapeutically effective amount of an anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable additive. A “pharmaceutically acceptable carrier” is a substance that can be added to an active ingredient to help formulate or stabilize a drug and does not cause significant toxic effects in the patient.
상기 첨가제는 환자를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제 등을 말한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들의 혼합물을 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. The additive refers to a carrier or diluent that does not irritate the patient and does not inhibit the biological activity and properties of the administered compound. Examples of acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and A mixture thereof may be used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
약학적으로 허용되는 담체는 멸균 주사가능한 용액제 또는 분산액제를 즉각 투여용(extemporaneous)으로 제조하기 위한 멸균 수용액 또는 분산액 및 멸균 분말을 포함한다. 조성물은 바람직하게는 비경구 주사용으로 제제화된다. 조성물은 용액제, 마이크로에멀젼제, 리포좀제, 또는 높은 약물 농도에 적합한 기타 주문된 제형으로 제제화될 수 있다. 담체는 예를 들어 물, 에탄올, 폴리올(예를 들어, 글리세롤, 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등) 및 이것들의 적합한 혼합물을 함유하는 용매 또는 분산 매질일 수 있다. 일부 경우에, 조성물 중에 등장화제, 예를 들어 당, 폴리알콜, 예컨대 만니톨, 소르비톨 또는 염화나트륨을 포함시킬 수 있다. 각 제제는 약제학 분야에 잘 알려진 방법들을 이용하여 제조될 수 있다. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for extemporaneous administration. The composition is preferably formulated for parenteral injection. The compositions may be formulated as solutions, microemulsions, liposomes, or other customized formulations suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In some cases, isotonic agents may be included in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride. Each formulation can be prepared using methods well known in the pharmaceutical art.
본 발명에 따른 약학 조성물의 투여량은 특별하게 한정되지는 않으나, 환자의 건강 상태 및 체중, 질환의 중증도, 약제의 종류, 투여경로 및 투여시간을 포함한 다양한 요인에 따라 변경될 수 있다. 본 발명에 따른 약학 조성물은 하루에 1회 또는 다회 용량으로 인간, 쥐(rat), 마우스(mouse), 가축, 등을 포함하는 포유동물 내로 전형적으로 허용된 경구 또는 비경구 경로의 다양한 경로를 통하여 투여될 수 있다. 구체적으로, 구강, 직장내, 국소, 정맥내, 복강내, 근육내, 동맥내, 경피, 비내, 흡입, 안구내, 폐내 또는 피내 경로를 통해 통상적인 방식으로 투여될 수 있지만, 이에 한정되는 것은 아니다. The dosage of the pharmaceutical composition according to the present invention is not particularly limited, but may be changed according to various factors including the patient's health condition and weight, disease severity, drug type, administration route, and administration time. The pharmaceutical composition according to the present invention can be administered in one or multiple doses per day through various routes of oral or parenteral routes typically accepted into mammals including humans, rats, mice, livestock, and the like. may be administered. Specifically, it may be administered in a conventional manner via oral, intrarectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalational, intraocular, intrapulmonary or intradermal routes, but is not limited thereto. not.
본 발명에 따른 약학 조성물은 필요에 따라 볼루스로서 또는 연속 주입에 의해 환자에게 투여될 수 있다. 예를 들어, Fab 단편으로 대표되는 본 발명의 항-SARS-COV-2의 스파이크 항체의 항원 결합 단편의 볼루스 투여는 0.0025 내지 100 ㎎/kg 체중, 0.025 내지 0.25 ㎎/kg, 0.010 내지 0.10 ㎎/kg 또는 0.10 내지 0.50 ㎎/kg의 양일 수 있다. 연속 주입의 경우, Fab 단편으로 대표되는 본 발명의 항-SARS-COV-2의 스파이크 항체의 항원 결합 단편은 0.001 내지 100 ㎎/kg 체중/분, 0.0125 내지 1.25 ㎎/kg/분, 0.010 내지 0.75 ㎎/kg/분, 0.010 내지 1.0 ㎎/kg/분 또는 0.10 내지 0.50 ㎎/kg/분으로 1시간 내지 24시간, 1시간 내지 12시간, 2시간 내지 12시간, 6시간 내지 12시간, 2시간 내지 8시간, 또는 1시간 내지 2시간의 기간 동안 투여될 수 있다. 본 발명의 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편을 투여하는 경우, 투여량은 약 1 내지 10 ㎎/kg 체중, 2 내지 8 ㎎/kg, 또는 5 내지 6 ㎎/kg일 수 있다. 전장 항-SARS-COV-2의 스파이크 항체는 전형적으로 30분 내지 35분의 기간 동안 지속되는 주입을 통해 투여한다. 투여 빈도는 상태의 중증도에 따라 달라진다. 빈도는 1주당 3회 내지 매 1주 또는 2주 마다 1회의 범위일 수 있다.The pharmaceutical composition according to the present invention may be administered to a patient as a bolus or by continuous infusion, if desired. For example, bolus administration of the antigen-binding fragment of the spike antibody of anti-SARS-COV-2 of the present invention represented by the Fab fragment is 0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg /kg or 0.10 to 0.50 mg/kg. In the case of continuous infusion, the antigen-binding fragment of the spike antibody of anti-SARS-COV-2 of the present invention represented by the Fab fragment is 0.001 to 100 mg/kg body weight/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min or 0.10 to 0.50 mg/kg/min, 1 hour to 24 hours, 1 hour to 12 hours, 2 hours to 12 hours, 6 hours to 12 hours, 2 hours to 8 hours, or from 1 hour to 2 hours. When the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof of the present invention is administered, the dosage is about 1 to 10 mg/kg body weight, 2 to 8 mg/kg, or 5 to 6 mg/kg day can The spike antibody of full length anti-SARS-COV-2 is typically administered via infusion lasting for a period of 30 to 35 minutes. The frequency of administration depends on the severity of the condition. The frequency may range from 3 times per week to once every 1 or 2 weeks.
SARS-CoV-2 감염증의 예방 또는 치료 방법How to prevent or treat SARS-CoV-2 infection
본 발명은 또한 상기 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편 또는 상기 다중특이적 항체 또는 항체-약물 접합체의 치료적 유효량을 신종 코로나바이러스 감염증(COVID-19)의 예방 또는 치료를 필요로 하는 환자에게 투여하는 단계를 포함하는, 신종 코로나바이러스 감염증(COVID-19)의 예방 또는 치료 방법에 관한 것이다. 상기 예방 또는 치료 방법은 상기 투여 단계 이전에 상기 질병의 예방 또는 치료를 필요로 하는 환자를 확인하는 단계를 추가로 포함할 수 있다.The present invention also provides a therapeutically effective amount of the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof or the multispecific antibody or antibody-drug conjugate to prevent or treat novel coronavirus infection (COVID-19). It relates to a method for preventing or treating novel coronavirus infection (COVID-19), comprising administering to a patient in need thereof. The prevention or treatment method may further include the step of identifying a patient in need of the prevention or treatment of the disease before the administering step.
경우에 따라서, 상기 항체 또는 이의 항원 결합 단편을 종래의 다른 항바이러스제와 병용함으로써 SARS-COV-2의 스파이크 단백질을 발현하는 세포를 효과적으로 표적화하고, 항바이러스 활성을 증가시킬 수 있다. 상기 항체 또는 이의 항원 결합 단편은 기존의 항바이러스제, 예컨대 에볼라바이러스치료제, HIV(인간면역결핍바이러스)치료제, C형간염치료제, 독감치료제 등일 수 있다. 구체적 약물의 일예로는 칼레트라(kaletra), 렘데시비르(remdesivir), 플라크닐(하이드록시클로로퀸), 레소친(클로로퀸) 등과 함께 사용될 수 있다. 또한, 상기 항바이러스제 이외에 신종 코로나바이러스 감염증(COVID-19)에 치료 효과가 있는 것으로 보고되고 있는 당뇨병 치료제(예컨대, 다파글리플로진(dapagliflozin)), 류마티스 관절염 치료제(예컨대, 아나킨라(anakinra), 토실리주맙(tocilizumab), 사리루맵(sarilumab)), 혈액암 치료제(예컨대, 아칼라브루티닙(acalabrutinib), 다발골수종 치료제(예컨대, 셀리넥서(selinexor)) 등과 함께 사용될 수 있으나, 이에 제한되지는 않는다. 또한, 기타 항체, 예컨대 독감, 헤르페스, B형 감염, C형 감염, 에이즈 등의 치료에 사용되고 있는 타겟, 예를 들면, CCR5 수용체, 뉴라미니다아제(neuraminidase), 헤마글루티닌(hemagglutinin), 인터페론류(예컨대, 인터페론 알파) 등에 대한 결합능을 가지는 항체와 함께 사용될 수 있다. In some cases, by using the antibody or antigen-binding fragment thereof in combination with other conventional antiviral agents, it is possible to effectively target cells expressing the SARS-COV-2 spike protein and increase antiviral activity. The antibody or antigen-binding fragment thereof may be a conventional antiviral agent, such as an Ebola virus treatment agent, an HIV (human immunodeficiency virus) treatment agent, a hepatitis C treatment agent, an influenza treatment agent, and the like. Specific examples of the drug may be used together with kaletra, remdesivir, placknil (hydroxychloroquine), resorcin (chloroquine), and the like. In addition, in addition to the antiviral agent, a treatment for diabetes (eg, dapagliflozin) that is reported to have a therapeutic effect on novel coronavirus infection (COVID-19), a treatment for rheumatoid arthritis (eg, anakinra) , tocilizumab, sarilumab), hematologic cancer therapeutics (eg, acalabrutinib), multiple myeloma therapeutics (eg, selinexor), etc., but is not limited thereto In addition, other antibodies, such as targets used in the treatment of influenza, herpes, hepatitis B, C infection, AIDS, etc., such as CCR5 receptor, neuraminidase, hemagglutinin ( hemagglutinin), interferons (eg, interferon alpha) may be used together with an antibody having a binding ability.
본 발명에 따른 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편, 및 이를 포함하는 약학 조성물은 기존의 항바이러스 치료제와 동시에 투여되거나, 순차적으로 투여될 수 있다.The anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof according to the present invention, and a pharmaceutical composition comprising the same, may be administered simultaneously or sequentially with a conventional antiviral therapeutic agent.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체를 포함하는 진단용 조성물 및 진단 방법Diagnostic composition and diagnostic method comprising an antibody that specifically binds to SARS-CoV-2 S protein
본 발명의 또 다른 측면은, 상술한 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편, 이를 포함하는 항체-약물 접합체 또는 다중특이적 항체를 포함하는 SARS-CoV-2 감염증의 진단용 조성물을 제공한다.Another aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the above-described SARS-CoV-2 spike protein, an antibody-drug conjugate comprising the same, or SARS-CoV-2 comprising a multispecific antibody A composition for diagnosing an infection is provided.
또한, 상기 진단용 조성물을 이용한 신종 코로나바이러스 감염증(COVID-19)의 진단 방법을 제공한다. In addition, there is provided a method for diagnosing novel coronavirus infection (COVID-19) using the diagnostic composition.
본 발명의 진단용 조성물은 목적하는 질환의 진단에 사용되는 주된 수단을 의미하는 것으로, 본 발명의 목적에 따라 SARS-CoV-2를 진단하기 위한 물질들이 포함될 수 있다. 진단 방법은 항체 또는 항체 단편을 샘플과 접촉시키는 것을 포함할 수 있다. 상기 샘플은 객담, 콧구멍, 부비강(sinus cavity), 침샘, 폐, 간, 췌장, 신장, 귀, 눈, 태반, 소화관, 심장, 난소, 뇌하수체, 부신, 갑상선, 뇌 또는 피부로부터 취한 조직, 세포, 소변, 전혈, 혈청, 혈장, 분변, 세포 배양 상등액 또는 파열된 진핵세포일 수 있다.The diagnostic composition of the present invention refers to a main means used for diagnosing a target disease, and according to the purpose of the present invention, substances for diagnosing SARS-CoV-2 may be included. The diagnostic method may comprise contacting the antibody or antibody fragment with a sample. The sample is a tissue, cell taken from sputum, nostril, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin. , urine, whole blood, serum, plasma, feces, cell culture supernatant or ruptured eukaryotic cells.
본 발명에 따른 상기 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편, 상기 항체-약물 접합체, 또는 상기 다중특이적 항체를 통해 시료에서의 SARS-COV-2의 스파이크 단백질 발현 수준을 측정함으로써 신종 코로나바이러스 감염증(COVID-19)을 진단할 수 있다. 발현 수준은 통상적인 면역분석 방법에 따라 측정할 수 있으며, 상기 SARS-COV-2의 스파이크 단백질에 대한 항체를 이용한 방사능면역분석, 방사능면역침전, 면역침전, 면역조직화학염색, ELISA(enzyme-linked immunosorbent assay), 캡처-ELISA, 억제 또는 경쟁 분석, 샌드위치 분석, 유세포 분석(flow cytometry), 면역형광염색 및 면역친화성 정제를 통해 측정할 수 있으나, 이에 한정되는 것은 아니다. 상기 면역분석 결과, 생물학적 시료에서의 SARS-COV-2의 스파이크 단백질 발현이 정상 생물학적 시료(예컨대, 비강, 구강, 혈액, 혈장 또는 혈청으로부터 채취한 시료)보다 높게 나오는 경우에는 신종 코로나바이러스 감염증(COVID-19)인 것으로 진단될 수 있다.Measuring the spike protein expression level of SARS-COV-2 in a sample through the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof, the antibody-drug conjugate, or the multispecific antibody according to the present invention By doing so, it is possible to diagnose the novel coronavirus infection (COVID-19). The expression level can be measured according to a conventional immunoassay method, radioimmunoassay using an antibody against the SARS-COV-2 spike protein, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification, but not limited thereto. As a result of the above immunoassay, when the spike protein expression of SARS-COV-2 in the biological sample is higher than in a normal biological sample (eg, a sample collected from nasal, oral, blood, plasma or serum), novel coronavirus infection (COVID) -19) can be diagnosed.
SARS-CoV-2 S 단백질에 특이적으로 결합하는 항체를 포함하는 키트A kit comprising an antibody that specifically binds to the SARS-CoV-2 S protein
본 발명의 또 다른 측면은, 상술한 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 포함하는 SARS-CoV-2 스파이크 단백질의 검출 또는 정량용 키트를 제공한다. 즉, 상기 항체 또는 이의 항원 결합 단편을 포함하는 SARS-CoV-2 감염증의 진단용 조성물 포함하는 진단용 키트를 제공한다. Another aspect of the present invention provides a kit for detecting or quantifying a SARS-CoV-2 spike protein comprising an antibody or antigen-binding fragment thereof that specifically binds to the above-described SARS-CoV-2 spike protein. That is, there is provided a diagnostic kit comprising a composition for diagnosis of SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof.
본 발명에 따른 상기 키트는 당업자에게 알려진 종래의 제조방법에 의해 제조될 수 있으며, 버퍼, 안정화제, 불활성 단백질 등을 더 포함할 수 있다. 또한, 본 발명에 따른 항-SARS-COV-2의 스파이크 항체 또는 이의 항원 결합 단편, 또는 이를 포함하는 항체-약물 접합체 또는 다중특이적 항체 및 검출가능한 시그널을 발생시키는 레이블(label)을 포함할 수 있다. 상기 레이블은 항체에 결합된 화학물질(예컨대, 바이오틴), 효소(알칼린 포스파타제, β-갈락토시다제, 호스 래디쉬 퍼옥시다제, 루시퍼라제 또는 사이토크롬 P450), 방사능물질(예컨대, C14, I125, P32 및 S35), 형광물질(예컨대, 플루오레신), 발광물질, 화학발광물질(chemiluminescent) 및 FRET(fluorescence resonance energy transfer)를 포함할 수 있으나, 이에 한정되는 것은 아니다. 이때 효소에 대한 기질은 효소로서 알칼린 포스파타아제가 이용되는 경우에는, 기질로서 브로모클로로인돌일 포스페이트(BCIP), 니트로 블루 테트라졸리움(NBT), 나프톨-AS-B1-포스페이트(naphthol-AS-B1-phosphate) 및 ECF(enhanced chemifluorescence)와 같은 발색반응 기질이 이용되고, 호스 래디쉬 퍼옥시다아제가 이용되는 경우에는 클로로나프톨, 아미노에틸카바졸, 디아미노벤지딘, D-루시페린, 루시게닌(비스-N-메틸아크리디늄 니트레이트), 레소루핀 벤질 에테르, 루미놀, 암플렉스 레드 시약(10-아세틸-3,7-디하이드록시페녹사진), HYR(p-phenylenediamine-HCl and pyrocatechol), TMB(tetramethylbenzidine), ABTS(2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-페닐렌디아민(OPD) 및 나프톨/파이로닌, 글루코스 옥시다아제와 t-NBT(nitroblue tetrazolium) 및 m-PMS(phenzaine methosulfate)과 같은 기질이 이용될 수 있으나, 이에 한정되는 것은 아니다.The kit according to the present invention may be prepared by a conventional manufacturing method known to those skilled in the art, and may further include a buffer, a stabilizer, an inactive protein, and the like. In addition, the anti-SARS-COV-2 spike antibody or antigen-binding fragment thereof according to the present invention, or an antibody-drug conjugate or multispecific antibody comprising the same, and a label generating a detectable signal may be included. there is. The label may include a chemical bound to the antibody (eg biotin), an enzyme (alkaline phosphatase, β-galactosidase, horse radish peroxidase, luciferase or cytochrome P450), a radioactive material (eg, C14, I125, P32, and S35), a fluorescent material (eg, fluorescein), a light emitting material, a chemiluminescent material, and a fluorescence resonance energy transfer (FRET), but are not limited thereto. In this case, the substrate for the enzyme is bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate when alkaline phosphatase is used as the enzyme. -B1-phosphate) and ECF (enhanced chemifluorescence) are used, and when horse radish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis -N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS A substrate such as (phenzaine methosulfate) may be used, but is not limited thereto.
시료와 항체가 반응함으로써 나타내는 시그널의 세기를 분석하여 신종 코로나바이러스 감염증(COVID-19)을 진단할 수 있다. 진단을 위해 사용되는 효소의 활성 측정 또는 시그널의 측정은 당업계에 공지된 다양한 방법에 따라 실시될 수 있으며, 이를 통해 SARS-COV-2의 스파이크 단백질 발현을 정성적 또는 정량적으로 분석할 수 있다.A novel coronavirus infection (COVID-19) can be diagnosed by analyzing the strength of the signal displayed by the reaction between the sample and the antibody. Measurement of the activity or signal of an enzyme used for diagnosis may be performed according to various methods known in the art, through which the spike protein expression of SARS-COV-2 may be qualitatively or quantitatively analyzed.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예 1. 항원 발현 및 정제Example 1. Antigen expression and purification
실시예 1.1. 항원 단백질 발현 벡터 제작Example 1.1. Antigen protein expression vector construction
SARS-CoV-2-RBD를 클로닝하기 위해, SARS-CoV-2(2019-nCoV) 스파이크 S1 유전자 ORF cDNA(Sino biological, Cat: VG40591-CF) 및 세포외 도메인만을 얻기 위해 5'과 3'에 제한효소 Sfi I/Nhe I 사이트가 포함된 SARS-CoV-2-RBD에 대한 프라이머 쌍(표 1)을 이용하여 중합효소연쇄반응(polymerase chain reaction, PCR)을 실시하였다. 얻어진 PCR 산물과 N293F 벡터를 이용하여 SARS-CoV-2-RBD의 아미노-말단에 6×His 또는 인간 Fc(hFc)가 융합된 단백질을 각각 발현하는 발현 벡터들을 제작하였다(도 1).To clone SARS-CoV-2-RBD, SARS-CoV-2 (2019-nCoV) spike S1 gene ORF cDNA (Sino biological, Cat: VG40591-CF) and extracellular domain only 5' and 3' A polymerase chain reaction (PCR) was performed using a primer pair (Table 1) for SARS-CoV-2-RBD containing restriction enzyme Sfi I/Nhe I sites. Expression vectors each expressing a protein in which 6×His or human Fc (hFc) was fused to the amino-terminus of SARS-CoV-2-RBD were prepared using the obtained PCR product and the N293F vector ( FIG. 1 ).
SARS-CoV-2-RBD 클로닝을 위한 프라이머 정보Primer information for SARS-CoV-2-RBD cloning
명칭designation 5'→3' 서열5'→3' sequence 서열번호SEQ ID NO:
SARS-CoV-2-RBD-FSARS-CoV-2-RBD-F CGTGTTCAGCCTACCGAGAGCCGTGTTCAGCCTACCGAGAGC 307307
SARS-CoV-2-RBD-RSARS-CoV-2-RBD-R GAAGTTCACGCATTTGTTCTTGAAGTTCACGCATTTGTTCTT 308308
실시예 1.2. 항원 단백질의 발현 및 정제Example 1.2. Expression and purification of antigenic proteins
형질감염(transfection)은 최적화된 조건에서 PEI(polyethylenimine; Aldrich, Cat. 408727)를 이용하여 수행하였다. 인간 HEK293F 세포를 ㎖당 5×105 개가 되도록 조정하여 배지(#Freestyle 293 AGT type; AG100009P1, Thermo.)에 접종하고 1×106 cells/㎖이 될 때까지 배양하였다. 상기 실시예 1.1.에서 얻은 각 발현 벡터와 PEI를 섞어 폴리플렉스(polyplex)를 형성하게 한 후, 상기 세포에 첨가하여 형질감염시킨 다음 발프로산(VPA, valproate, Sigma-P4543) 1 nM을 첨가한 후 6일 동안 더 배양하였다. 발현된 SARS-CoV-2-RBD를 protein A agarose 또는 Ni-NTA 비드를 사용하여 1차 정제하였고, 1차 정제된 단백질을 Superdex 200(1.5 ㎝×100 ㎝) 젤 여과 크로마토그래피를 이용하여 정제하였다. 정제된 단백질의 순도를 SDS-PAGE와 크기 배제 크로마토그래피(TSK-GEL G-3000 SWXL Size-exclusion chromatography(SEC) (Tosoh))를 이용하여 확인하였으며, 단백질의 순도는 95% 이상이었다(도 2). SARS-CoV-2-RBD가 기능을 유지하는지의 여부를 ACE2와의 상호작용(interaction)으로 확인하였다.Transfection (transfection) was performed using PEI (polyethylenimine; Aldrich, Cat. 408727) under optimized conditions. Human HEK293F cells were adjusted to 5×10 5 per ml, inoculated into a medium (#Freestyle 293 AGT type; AG100009P1, Thermo.), and cultured until 1×10 6 cells/ml. Each of the expression vectors obtained in Example 1.1 and PEI were mixed to form a polyplex, and then added to the cells for transfection and then 1 nM of valproic acid (VPA, valproate, Sigma-P4543) was added. After that, it was further cultured for 6 days. The expressed SARS-CoV-2-RBD was first purified using protein A agarose or Ni-NTA beads, and the first purified protein was purified using Superdex 200 (1.5 cm × 100 cm) gel filtration chromatography. . The purity of the purified protein was confirmed using SDS-PAGE and size exclusion chromatography (TSK-GEL G-3000 SWXL Size-exclusion chromatography (SEC) (Tosoh)), and the purity of the protein was 95% or more (Fig. 2). ). Whether or not SARS-CoV-2-RBD maintains function was confirmed by interaction with ACE2.
실시예 2. SARS-CoV-2 스파이크에 대한 인간 항체의 선별Example 2. Selection of Human Antibodies Against SARS-CoV-2 Spike
실시예 2.1. 면역 라이브러리(Immune Library)의 제작Example 2.1. Construction of Immune Library
충남대학교 감염내과를 통해 20명의 Covid-19 확진 환자로부터 동의 하에 PBMC를 입수하였다. PBMC로부터 total RNA를 분리하고, 이를 이용하여 RT-PCR을 진행함으로써 cDNA를 제조하였다. 얻어진 cDNA를 면역 라이브러리 제작에 사용하였다. 중쇄 가변부와 경쇄 가변부를 증폭하기 위해 상기 cDNA로 PCR을 진행하였다. 염기서열 분석을 통해 중쇄 가변영역과 경쇄 가변영역의 다양성을 확인한 다음, scFv(중쇄 가변부와 경쇄 가변부의 연결)를 만들기 위해 다시 PCR을 진행하였다. 서열분석을 통해 중쇄 가변부와 경쇄 가변부의 연결이 잘 되었는지 확인하였다. 면역 라이브러리를 만들기 위해, 정제된 scFv를 파지 벡터(pYG100)에 삽입하여 전기천공 형질전환용 세포인 ElectroTen-Blue 세포(Agilent, Cat #200159)와 함께 2.50 kV, 1 pulse에서 전기 충격을 준 다음, 세포를 SOBCG 한천배지(2%(w/v) 박토트립톤, 1.0% (w/v) 박토-이스트, 0.05%(w/v) NaCl, 5 mM MgCl2, 10 mM 글루코스, 34 ㎍/㎖ 클로람페니콜, 15g 박토-아가)에 도말하여 37℃에서 16시간 동안 배양하였다. 다음날, 얻어진 라이브러리 콜로니의 염기서열을 확인하여 면역 라이브러리의 다양성을 확인하였다.PBMCs were obtained with consent from 20 confirmed Covid-19 patients through the Department of Infectious Diseases, Chungnam National University. Total RNA was isolated from PBMC, and cDNA was prepared by RT-PCR using this. The obtained cDNA was used to construct an immune library. PCR was performed with the cDNA to amplify the heavy chain variable region and the light chain variable region. After confirming the diversity of the heavy chain variable region and the light chain variable region through nucleotide sequence analysis, PCR was performed again to make scFv (connection of the heavy chain variable region and the light chain variable region). Through sequencing, it was confirmed whether the heavy chain variable region and the light chain variable region were well connected. To make an immune library, the purified scFv was inserted into a phage vector (pYG100) and electroporated together with ElectroTen-Blue cells (Agilent, Cat #200159) for transformation at 2.50 kV, 1 pulse, followed by electric shock. Cells were cultured on SOBCG agar medium (2% (w/v) bactotryptone, 1.0% (w/v) bacto-yeast, 0.05% (w/v) NaCl, 5 mM MgCl 2 , 10 mM glucose, 34 μg/ml chloramphenicol, 15 g bacto-agar) and incubated at 37° C. for 16 hours. The next day, the nucleotide sequence of the obtained library colony was confirmed to confirm the diversity of the immune library.
실시예 2.2. 바이오패닝Example 2.2. biopanning
7.5×1010의 다양성을 가진 인간 scFv 라이브러리(Ymax®-ABL; (주)와이바이오로직스))와 상기 실시예 2.1.에서 제작한 면역 라이브러리를 대장균에 감염시킨 후, 얻어진 대장균을 30℃에서 16시간 배양하였다. 배양액을 원심 분리하여 상층액을 PEG(polyethylene glycol)로 농축한 다음, 이를 PBS(phosphate buffered saline) 완충용액에 녹여 인간 항체 라이브러리 파지를 준비하였다.After infecting the human scFv library (Ymax ® -ABL; YBiologics Co., Ltd.) with a diversity of 7.5×10 10 and the immune library prepared in Example 2.1. time incubation. The culture medium was centrifuged, and the supernatant was concentrated with PEG (polyethylene glycol), and then dissolved in phosphate buffered saline (PBS) buffer to prepare human antibody library phages.
실시예 1에서 제조한 SARS-CoV-2-RBD-hFc와 SARS-CoV-2-RBD-His 단백질 항원 각 50 ㎍을 면역흡착튜브(Immunosorb tube)에 코팅한 후 블로킹(blocking)을 수행하였다. 이 면역흡착튜브에 상기와 같이 준비된 라이브러리 파지를 넣고 실온에서 2시간 반응시킨 다음, 1× PBST(PBS + tween-20)와 1× PBS로 세척한 후, 100 mM TAE와 Tris-HCl(pH7.5) 용액을 차례로 처리하여 항원에 특이적으로 결합한 scFv-파지들만을 용출(elution)시켰다. 용출된 파지를 다시 대장균에 감염시켜 증폭시키는 패닝과정을 통해 양성 파지의 풀(pool)을 얻고, 첫 번째 라운드의 패닝에서 증폭된 파지를 가지고 PBST 세척 단계에서 횟수만 늘리고, 나머지는 동일한 방법으로 2라운드와 3라운드 패닝을 수행하였다. 50 μg of each of the SARS-CoV-2-RBD-hFc and SARS-CoV-2-RBD-His protein antigens prepared in Example 1 were coated on an Immunosorb tube, followed by blocking. The library phages prepared as described above were put into the immunosorbent tube, and reacted for 2 hours at room temperature, washed with 1× PBST (PBS + tween-20) and 1× PBS, and then 100 mM TAE and Tris-HCl (pH7. 5) The solution was sequentially treated to elute only scFv-phages specifically binding to the antigen. A pool of positive phages is obtained through the panning process in which the eluted phages are again infected with E. coli and amplified, and the number of times is increased only in the PBST washing step with the phages amplified in the first round of panning, and the rest are performed in the same manner as 2 Round and 3 rounds of panning were performed.
그 결과, 표 2와 같이 3라운드 패닝에서 항원에 결합한 파지 수가 input(항원과 반응시킬 때의 파지 수) 대비 output(항원과 결합시킨 뒤 세척 이후 용출했을 때의 파지 수)이 다소 증가였음을 확인하였다. As a result, as shown in Table 2, it was confirmed that the number of phages bound to the antigen in the 3rd round panning increased slightly compared to the input (the number of phages when reacted with the antigen) compared to the output (the number of phages when eluted after binding to the antigen after washing) did
구체적으로, 표 2에 패닝에 따른 항체의 역가를 비교하여 나타내었다.Specifically, it is shown in Table 2 by comparing the titers of the antibodies according to the panning.
패닝 횟수에 따른 항체의 역가 비교Comparison of antibody titers according to the number of panning
패닝 횟수number of pans InputInput OutputOutput
1회1 time 2.0×1013 2.0×10 13 3.6×107 3.6×10 7
2회 Episode 2 1.2×1013 1.2×10 13 7.0×107 7.0×10 7
3회3rd time 2.0×1013 2.0×10 13 2.0×109 2.0×10 9
실시예 2.3. 단일클론 선별Example 2.3. monoclonal selection
폴리 파지 ELISA를 통해 결합능이 큰 것으로 확인된 3라운드 패닝의 양성 파지 풀로부터 단일클론(monoclone)을 선별하였고, 이들을 96-딥 웰 플레이트(96-deep well plate)에서 키워 헬퍼 파지를 감염하여 배양한 다음, 상층액에 존재하는 모노 scFv-파지를 항원이 코팅된 면역-플레이트에 옮겨 direct ELISA를 수행하였다. 이때, SARS-CoV-2-RBD에 대한 모노 파지 ELISA와 비특이적 항원 대조군인 ITGA6-Fc 단백질에 대한 ELISA도 동시에 수행하여, 얻어진 양성 파지 클론이 SARS-CoV-2-RBD에 특이적인지도 확인하였다.Monoclones were selected from the positive phage pool of 3 round panning confirmed to have high binding ability through poly phage ELISA, and they were grown in 96-deep well plates to infect and culture helper phages. Next, direct ELISA was performed by transferring the mono-scFv-phage present in the supernatant to an antigen-coated immune-plate. At this time, monophage ELISA for SARS-CoV-2-RBD and ELISA for ITGA6-Fc protein, which is a non-specific antigen control, were simultaneously performed to confirm whether the obtained positive phage clone was specific for SARS-CoV-2-RBD.
그 결과, 도 3에서와 같이, 모노 scFv-파지 클론들은 SARS-CoV-2-RBD에만 결합능이 강함을 확인하였다.As a result, as shown in FIG. 3 , it was confirmed that the mono scFv-phage clones had strong binding ability only to SARS-CoV-2-RBD.
실시예 2.4. 단일클론의 염기서열 분석Example 2.4. Monoclonal sequencing analysis
결합능을 기준으로 최종 선별된 15종의 단일클론들에 대해 DNA 정제 키트(Qiagen, 독일)를 사용하여 파지미드(phagemid) DNA를 분리하여 DNA 염기서열을 분석하였다. 중쇄 CDR(Complementarity-determining regions) 및 경쇄 CDR의 아미노산 서열, 중쇄 FR(Framework) 및 경쇄 FR의 아미노산 서열을 각각 하기 표 3 및 표 4에 나타내었고, 중쇄 및 경쇄 가변영역의 아미노산 서열 및 이를 코딩하는 폴리뉴클레오타이드 서열을 각각 하기 표 5 및 표 6에 나타내었다.The phagemid DNA was isolated using a DNA purification kit (Qiagen, Germany) for 15 types of single clones finally selected based on binding ability, and the DNA sequence was analyzed. The amino acid sequences of the heavy chain CDRs (Complementarity-determining regions) and the light chain CDRs, the amino acid sequences of the heavy chain FRs (Framework) and the light chain FRs are shown in Tables 3 and 4, respectively, and the amino acid sequences of the heavy and light chain variable regions and encoding them The polynucleotide sequences are shown in Tables 5 and 6 below, respectively.
단일클론들의 중쇄 및 경쇄 CDR 서열Heavy and light chain CDR sequences of monoclones
항체antibody CDR 서열CDR sequence 서열번호SEQ ID NO:
SA3755SA3755 CDRH1CDRH1 GFTFDDHTGFTFDDHT 1One
CDRH2CDRH2 ISWDGGSTISWDGGST 22
CDRH3CDRH3 TRDASRRPGDGGYDFDVTRDASRRPGDGGYDFDV 33
CDRL1CDRL1 TGTVTRGHWTGTVTRGHW 44
CDRL2CDRL2 DTDDTD 55
CDRL3CDRL3 LLSYSDSRVLLSYSDSRV 66
SA3779SA3779 CDRH1CDRH1 GFTFSSYAGFTFSSYA 77
CDRH2CDRH2 ISYDGSNKISYDGSNK 88
CDRH3CDRH3 ARRGHYYDSSGYLYARRGHYYDSSGYLY 99
CDRL1CDRL1 QRIATYQRIATY 1010
CDRL2CDRL2 AASAAS 1111
CDRL3CDRL3 QQSYAIPYTQQSYAIPYT 1212
SA3827SA3827 CDRH1CDRH1 GFTFSSYAGFTFSSYA 1313
CDRH2CDRH2 ISYDGSNKISYDGSNK 1414
CDRH3CDRH3 ARRGHYYDSSGFLYARRGHYYDSSGFLY 1515
CDRL1CDRL1 QSISTYQSISTY 1616
CDRL2CDRL2 AASAAS 1717
CDRL3CDRL3 QQSYNTPRTQQSYNTPRT 1818
SA3830SA3830 CDRH1CDRH1 GFNFDEYSGFNFDEYS 1919
CDRH2CDRH2 IKYNSNTIIKYNSNTI 2020
CDRH3CDRH3 ARDAMRGGDFDHARDAMRGGDFDH 2121
CDRL1CDRL1 QSISDWQSISDW 2222
CDRL2CDRL2 KASKAS 2323
CDRL3CDRL3 QQYYSTTWTQQYYSTTWT 2424
SA3838SA3838 CDRH1CDRH1 GYIFTELSGYIFTELS 2525
CDRH2CDRH2 FDPEEGKTFDPEEGKT 2626
CDRH3CDRH3 AKIGHLGDFWYAKIGHLGDFWY 2727
CDRL1CDRL1 TSDIGSYDYTSDIGSYDY 2828
CDRL2CDRL2 GVTGVT 2929
CDRL3CDRL3 ATYTSSATYVATYTSSATYV 3030
SA3856SA3856 CDRH1CDRH1 GFTVTSAYGFTVTSAY 3131
CDRH2CDRH2 IYGGGSTIYGGGST 3232
CDRH3CDRH3 TGPYPGRYDYTGPYPGRYDY 3333
CDRL1CDRL1 QDIRTSQDIRTS 3434
CDRL2CDRL2 DASDAS 3535
CDRL3CDRL3 QQYKTVPLTQQYKTVPLT 3636
SA3902SA3902 CDRH1CDRH1 GYAFSYYHGYAFSYYH 3737
CDRH2CDRH2 INPGSGGTINPGSGGT 3838
CDRH3CDRH3 AGSEQPHYYANGAYRVAGSEQPHYYANGAYRV 3939
CDRL1CDRL1 QSISSYQSISSY 4040
CDRL2CDRL2 EVSEVS 4141
CDRL3CDRL3 QQYDTQQYDT 4242
SA4040SA4040 CDRH1CDRH1 GYTFTGYYGYTFTGYY 4343
CDRH2CDRH2 INPNSGGTINNPSGGT 4444
CDRH3CDRH3 ARDQAFSMVRGVTDYARDQAFSMVRGVTDY 4545
CDRL1CDRL1 SSDVGGYNYSSDVGGYNY 4646
CDRL2CDRL2 EVTEVT 4747
CDRL3CDRL3 SSYAGDNTWISSYAGDNTWI 4848
SA4043SA4043 CDRH1CDRH1 VITVSSNYVITVSSNY 4949
CDRH2CDRH2 IYPGGSTIYPGST 5050
CDRH3CDRH3 ARDLDIVGGMDVARDLDIVGGMDV 5151
CDRL1CDRL1 QSISRWQSISRW 5252
CDRL2CDRL2 AASAAS 5353
CDRL3CDRL3 QQYDSYPRTQQYDSYPRT 5454
SA4044SA4044 CDRH1CDRH1 GFTVSSNYGFTVSSNY 5555
CDRH2CDRH2 IYSGGSTIYSGGST 5656
CDRH3CDRH3 ARDLPQFDGFDYARDLPQFDGFDY 5757
CDRL1CDRL1 NIGSKSNIGSKS 5858
CDRL2CDRL2 SDTSDT 5959
CDRL3CDRL3 QVWDSTTDHVVQVWDSTTDHVV 6060
SA4050SA4050 CDRH1CDRH1 GYTFTGYFGYTFTGYF 6161
CDRH2CDRH2 INPNSGGTINNPSGGT 6262
CDRH3CDRH3 ARVPHLSGYYYEGVLGYFDYARVPHLSGYYYEGVLGYFDY 6363
CDRL1CDRL1 SIDIGSYNFSIDIGSYNF 6464
CDRL2CDRL2 DVSDVS 6565
CDRL3CDRL3 SSYTSSSLWVSSYTSSSLWV 6666
SA4053SA4053 CDRH1CDRH1 GFTFSSYWGFTFSSYW 6767
CDRH2CDRH2 IKQDGSEKIKQDGSEK 6868
CDRH3CDRH3 ARDVGRGYCSSTSCYRFGGREDFFDYARDVGRGYCSSTSCYRFGGREDFFDY 6969
CDRL1CDRL1 FSNVGNNFFSNVGNNF 7070
CDRL2CDRL2 DNNDNN 7171
CDRL3CDRL3 GTWDTSLSGVVGTWDTSLSGVV 7272
SA4055SA4055 CDRH1CDRH1 VITVSSNYVITVSSNY 7373
CDRH2CDRH2 IYPGGSTIYPGST 7474
CDRH3CDRH3 ARDLDIVGGMDVARDLDIVGGMDV 7575
CDRL1CDRL1 SSDVGDYNLSSDVGDYNL 7676
CDRL2CDRL2 DVSDVS 7777
CDRL3CDRL3 SSYTSSTTVVSSYTSSTTVV 7878
SA4056SA4056 CDRH1CDRH1 VITVSSNYVITVSSNY 7979
CDRH2CDRH2 IYPGGSTIYPGST 8080
CDRH3CDRH3 ARDLDIVGGMDVARDLDIVGGMDV 8181
CDRL1CDRL1 QSISRWQSISRW 8282
CDRL2CDRL2 AASAAS 8383
CDRL3CDRL3 QQYDSYPRTQQYDSYPRT 8484
SA4057SA4057 CDRH1CDRH1 VITVSSNYVITVSSNY 8585
CDRH2CDRH2 IYPGGSTIYPGST 8686
CDRH3CDRH3 ARDLDIVGGMDVARDLDIVGGMDV 8787
CDRL1CDRL1 SSNIGAPHDSSNIGAPHD 8888
CDRL2CDRL2 ANNANN 8989
CDRL3CDRL3 QSYDSSLRAYVQSYDSSLRAYV 9090
단일클론들의 중쇄 및 경쇄 FR 서열Heavy and light chain FR sequences of monoclones
항체antibody FR 서열FR sequence 서열번호SEQ ID NO:
SA3755SA3755 FRH1FRH1 QMQLVESGGGVVQPGGSLRLSCAASQMQLVESGGGVVQPGGSLRLSCAAS 9191
FRH2FRH2 MHWVRQAPGKGLEWVSLMHWVRQAPGKGLEWVSL 9292
FRH3FRH3 YYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYC 9393
FRH4FRH4 WGQGTQVTVSSWGQGTQVTVSS 9494
FRL1FRL1 QAVVTQEPSLTVSPGGTVTLTCGSSQAVVTQEPSLTVSPGGTVTLTCGSS 9595
FRL2FRL2 PYWFQQKPGQAPRTLIYPYWFQQKPGQAPRTLIY 9696
FRL3FRL3 NKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYCNKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYC 9797
FRL4FRL4 FGTGTKVTVLFGTGTKVTVL 9898
SA3779SA3779 FRH1FRH1 QVQLVESGGGVVQPGRSLRLSCAASQVQLVESGGGVVQPGRSLRLSCAAS 9999
FRH2FRH2 MHWVRQAPGKGLEWVAVMHWVRQAPGKGLEWVAV 100100
FRH3FRH3 YYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 101101
FRH4FRH4 WGHGTLITVSSWGHGTLITVSS 102102
FRL1FRL1 DIQMTQSPSSLSASVGDRVTITCRASDIQMTQSPSSSLSASVGDRVTITCRAS 103103
FRL2FRL2 LHWYQQKPGRAPKLLIYLHWYQQKPGRAPKLLIY 104104
FRL3FRL3 SLQTGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCSLQTGVPSRFSGSGSGTDFTLTINSLQPEDFATYYC 105105
FRL4FRL4 FGQGTKLEIKFGQGTKLEIK 106106
SA3827SA3827 FRH1FRH1 QVQLVESGGTLVQPGRSLRLSCAASQVQLVESGGTLVQPGRSLRLSCAAS 107107
FRH2FRH2 MHWVRQAPGKGLEWVAVMHWVRQAPGKGLEWVAV 108108
FRH3FRH3 YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 109109
FRH4FRH4 WGQGTLVTVSSWGQGTLVTVSS 110110
FRL1FRL1 DIQMTQSPSSLSASVGDRVTITCRASDIQMTQSPSSSLSASVGDRVTITCRAS 111111
FRL2FRL2 LSWYQQKPGKAPKLLIYLSWYQQKPGKAPKLLIY 112112
FRL3FRL3 NLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCNLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYC 113113
FRL4FRL4 FGQGTKVEIKFGQGTKVEIK 114114
SA3830SA3830 FRH1FRH1 QVQLVESGGGLIQPGRSLSLSCAASQVQLVESGGGLIQPGRSLSLSCAAS 115115
FRH2FRH2 MHWVRQRPGKGLEWVSGMHWVRQRPGKGLEWVSG 116116
FRH3FRH3 LYADSVKGRFTVSRDNSKNSLYLQMNSLRAEDTAVYYCLYADSVKGRFTVSRDNSKNSLYLQMNSLRAEDTAVYYC 117117
FRH4FRH4 WGQGTLITVSSWGQGTLITVSS 118118
FRL1FRL1 DIQMTQSPSYLSASAGDRVTITCRASDIQMTQSPSYLSASAGDRVTITCRAS 119119
FRL2FRL2 LAWYQHKPGKAPKLLIYLAWYQHKPGKAPKLLIY 120120
FRL3FRL3 RLENGVPSRFRGSGSATDFTLTISSLQAEDVAVYYCRLENGVPSRFRGSGSATDFTLTISSLQAEDVAVYYC 121121
FRL4FRL4 FGQGTKVDIKFGQGTKVDIK 122122
SA3838SA3838 FRH1FRH1 QVQLVESGAEVKKPGASVRVSCKISQVQLVESGAEVKKPGASVRVSCKIS 123123
FRH2FRH2 IHWVRQAPGKGLEWMGGIHWVRQAPGKGLEWMGG 124124
FRH3FRH3 IYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC 125125
FRH4FRH4 WGQGTLITVSSWGQGTLITVSS 126126
FRL1FRL1 QSALTQPASVSGSPGQSITISCTGTQSALTQPASVSGSPGQSITISCTGT 127127
FRL2FRL2 VSWYQQHPGKAPKLIIYVSWYQQHPGKAPKLIIY 128128
FRL3FRL3 RRPSGVSNRFSGSKSGNTASLTISRLLTEDEAEYYCRRPSGVSNRFSGSKSGNTASLTISRLLTEDEAEYYC 129129
FRL4FRL4 FGTGTKVTVLFGTGTKVTVL 130130
SA3856SA3856 FRH1FRH1 QMQLVESGGGLIQPGGSLRLSCAASQMQLVESGGGLIQPGGSLRLSCAAS 131131
FRH2FRH2 MSWVRQAPGKGLEWVSIMSWVRQAPGKGLEWVSI 132132
FRH3FRH3 YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 133133
FRH4FRH4 WGRGTLVTVSSWGRGTLVTVSS 134134
FRL1FRL1 DIQMTQSPSSLSASVGDRVIITCRASDIQMTQSPSSSLSASVGDRVIITCRAS 135135
FRL2FRL2 LAWFQQRLGEAPRSLIYLAWFQQRLGEAPRSLIY 136136
FRL3FRL3 SLQNGVPSKFRGSGSGTDFTLTISSLQPEDFATYYCSLQNGVPSKFRGSGSGTDFTLTISSLQPEDFATYYC 137137
FRL4FRL4 FGGGTKVEIKFGGGTKVEIK 138138
SA3902SA3902 FRH1FRH1 QVQLVQSGAEVRQPGASVKVSCKTSQVQLVQSGAEVRQPGASVKVSCKTS 139139
FRH2FRH2 IHWVRQAPGQGLEWMGLIHWVRQAPGQGLEWMGL 140140
FRH3FRH3 NYAQKFQGRVTLTRDTSTSTVYMDLTGLRSEDTAVYYCNYAQKFQGRVTLTRDTSTSTVYMDLTGLRSEDTAVYYC 141141
FRH4FRH4 WGQGTLVTVSSWGQGTLVTVSS 142142
FRL1FRL1 DIQMTQSPSTLSASVGDRVTITCRASDIQMTQSPSTLSASVGDRVTITCRAS 143143
FRL2FRL2 LNWYQQKPGKAPKLLIYLNWYQQKPGKAPKLLIY 144144
FRL3FRL3 TLESGVPSRFSGSRSGTEFTLTISSLQPDDFATYYCTLESGVPSRFSGSRSGTEFTLTISSLQPDDFATYYC 145145
FRL4FRL4 FGGGTKVEIKFGGGTKVEIK 146146
SA4040SA4040 FRH1FRH1 EVQLVQSGAEVKKPGASVKVSCKASEVQLVQSGAEVKKPGASVKVSCKAS 147147
FRH2FRH2 MHWARQAPGQGLEWMGWMHWARQAPGQGLEWMGW 148148
FRH3FRH3 NYAQKFQGRVTMTRDTSITTAYMELSRLTSDDTAVYYCNYAQKFQGRVTMTRDTSITTAYMELSRLTSDDTAVYYC 149149
FRH4FRH4 WGQGTLVTVSSWGQGTLVTVSS 150150
FRL1FRL1 QSALTQPPSASGSPGQSVIISCTGSQSALTQPPSASGSPGQSVISCTGS 151151
FRL2FRL2 VAWYQQYPGAAPKLIISVAWYQQYPGAAPKLIIS 152152
FRL3FRL3 KRPSGVPDRFSGSKSGNTASLTISGLQAEDEANYYCKRPSGVPDRFSGSKSGNTASLTISGLQAEDEANYYC 153153
FRL4FRL4 FGGGTKLTVLFGGGTTLVL 154154
SA4043SA4043 FRH1FRH1 QVQLQESGGGLVQPGGSLRLSCAASQVQLQESGGGLVQPGGSLRLSCAAS 155155
FRH2FRH2 MSWVRQAPGKGLEWVSVMSWVRQAPGKGLEWVSV 156156
FRH3FRH3 YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC 157157
FRH4FRH4 WGQGTTVTVSSWGQGTTVTVSS 158158
FRL1FRL1 DIQMTQSPSTLSASVGDRVTITCRASDIQMTQSPSTLSASVGDRVTITCRAS 159159
FRL2FRL2 LAWYQQKPGQAPKLLIYLAWYQQKPGQAPKLLIY 160160
FRL3FRL3 TLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCTLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC 161161
FRL4FRL4 FGQGTKVDIKFGQGTKVDIK 162162
SA4044SA4044 FRH1FRH1 EVQLVQSGGGLIQPGGSLRLSCAASEVQLVQSGGGLIQPGGSLRLSCAAS 163163
FRH2FRH2 MSWVRQAPGKGLEWVSVMSWVRQAPGKGLEWVSV 164164
FRH3FRH3 YYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYC 165165
FRH4FRH4 WGQGTLVTVSSWGQGTLVTVSS 166166
FRL1FRL1 QSALTQPPSLSVAPGRTARITCGGNQSALTQPPSLSVAPGRTARITCGGN 167167
FRL2FRL2 VNWYQHMPGQAPVLVIYVNWYQHMPGQAPVLVIY 168168
FRL3FRL3 DRPSGIPERVSGSKSGNTATLSISRVEAGDEADYYCDRPSGIPERVSGSKSGNTATLSISRVEAGDEADYYC 169169
FRL4FRL4 FGGGTKLTVLFGGGTTLVL 170170
SA4050SA4050 FRH1FRH1 QVQLVESGAEVKKPGASVKVSCKASQVQLVESGAEVKKPGASVKVSCKAS 171171
FRH2FRH2 IHWVRQAPGQGLEWMGWIHWVRQAPGQGLEWMGW 172172
FRH3FRH3 NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC 173173
FRH4FRH4 WGQGTLVTVSSWGQGTLVTVSS 174174
FRL1FRL1 QSALTQPASVSGSPGQSITISCTGTQSALTQPASVSGSPGQSITISCTGT 175175
FRL2FRL2 VSWYQRHPGKAPRLLIYVSWYQRHPGKAPRLIY 176176
FRL3FRL3 DRPSGVSNRFSGSKSANTASLTISGLQAEDEADYYCDRPSGVSNRFSGSKSANTASLTISGLQAEDEADYYC 177177
FRL4FRL4 FGGGTKLTVLFGGGTTLVL 178178
SA4053SA4053 FRH1FRH1 EVQLVQSGGGLVQPGGSLRLSCADSEVQLVQSGGGLVQPGGSLRLSCADS 179179
FRH2FRH2 MSWVRQVPGKGLEWVANMSWVRQVPGKGLEWVAN 180180
FRH3FRH3 YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC 181181
FRH4FRH4 WGQGTLVTVSSWGQGTLVTVSS 182182
FRL1FRL1 QLVLTQPPSVSAAPGQRVTISCSGSQLVLTQPPSVSAAPGQRVTISCSGS 183183
FRL2FRL2 VSWYRHLPGTAPELLIYVSWYRHLPGTAPELLIY 184184
FRL3FRL3 QRPSGIPDRFSASKSGTSATLAITGLHTGDEADYYCQRPSGIPDRFSASKSGTSATLAITGLHTGDEADYYC 185185
FRL4FRL4 FGGGTKLTVLFGGGTTLVL 186186
SA4055SA4055 FRH1FRH1 EVQLVQSGGGLVQPGGSLRLSCAASEVQLVQSGGGLVQPGGSLRLSCAAS 187187
FRH2FRH2 MSWVRQAPGKGLEWVSVMSWVRQAPGKGLEWVSV 188188
FRH3FRH3 YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC 189189
FRH4FRH4 WGQGTTVTVSSWGQGTTVTVSS 190190
FRL1FRL1 QSALTQPASVSGSPGQSITISCTGTQSALTQPASVSGSPGQSITISCTGT 191191
FRL2FRL2 VSWYQQHPGKAPKVMIYVSWYQQHPGKAPKVMIY 192192
FRL3FRL3 HRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCHRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC 193193
FRL4FRL4 FGGGTKLTVLFGGGTTLVL 194194
SA4056SA4056 FRH1FRH1 QVQLQESGGGLVQPGGSLRLSCAASQVQLQESGGGLVQPGGSLRLSCAAS 195195
FRH2FRH2 MSWVRQAPGKGLEWVSVMSWVRQAPGKGLEWVSV 196196
FRH3FRH3 YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC 197197
FRH4FRH4 WGQGTTVTVSSWGQGTTVTVSS 198198
FRL1FRL1 DIQMTQSPSTLSASVGDRVTITCRASDIQMTQSPSTLSASVGDRVTITCRAS 199199
FRL2FRL2 LAWYQQKPGQAPKLLIYLAWYQQKPGQAPKLLIY 200200
FRL3FRL3 TLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCTLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC 201201
FRL4FRL4 FGQGTKVDIKFGQGTKVDIK 202202
SA4057SA4057 FRH1FRH1 EVQLVQSGGGLVQPGGSLRLSCAASEVQLVQSGGGLVQPGGSLRLSCAAS 203203
FRH2FRH2 MSWVRQAPGKGLEWVSVMSWVRQAPGKGLEWVSV 204204
FRH3FRH3 YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC 205205
FRH4FRH4 WGQGTTVTVSSWGQGTTVTVSS 206206
FRL1FRL1 QLVLTQPPSVSGAPGQRVTISCTGSQLVLTQPPSVSGAPGQRVTISCTGS 207207
FRL2FRL2 VHWYQQLPGTAPRLLIYVHWYQQLPGTAPRLLIY 208208
FRL3FRL3 NRPSGVPDRFSGSKSGTSASLAITGLQDEDEADYYCNRPSGVPDRFSGSKSGTSASLAITGLQDEDEADYYC 209209
FRL4FRL4 FGTGTKVTVLFGTGTKVTVL 210210
단일클론들의 중쇄 및 경쇄 가변영역 아미노산 서열Monoclonal heavy and light chain variable region amino acid sequences
항체antibody 가변영역 아미노산 서열variable region amino acid sequence 서열번호SEQ ID NO:
SA3755SA3755 중쇄heavy chain QMQLVESGGGVVQPGGSLRLSCAAS GFTFDDHT MHWVRQAPGKGLEWVSL ISWDGGST YYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYC TRDASRRPGDGGYDFDV WGQGTQVTVSSQMQLVESGGGVVQPGGSLRLSCAAS GFTFDDHT MHWVRQAPGKGLEWVSL ISWDGGST YYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYC TRDASRRPGDGGYDFDV WGQGTQVTVSS 211211
경쇄light chain QAVVTQEPSLTVSPGGTVTLTCGSS TGTVTRGHW PYWFQQKPGQAPRTLIY DTD NKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYC LLSYSDSRV FGTGTKVTVLQAVVTQEPSLTVSPGGTVTLTCGSS TGTVTRGHW PYWFQQKPGQAPRTLIY DTD NKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYC LLSYSDSRV FGTGTKVTVL 212212
SA3779SA3779 중쇄heavy chain QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYA MHWVRQAPGKGLEWVAV ISYDGSNK YYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRGHYYDSSGYLY WGHGTLITVSSQVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYA MHWVRQAPGKGLEWVAV ISYDGSNK YYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRGHYYDSSGYLY WGHGTLITVSS 213213
경쇄light chain DIQMTQSPSSLSASVGDRVTITCRAS QRIATY LHWYQQKPGRAPKLLIY AAS SLQTGVPSRFSGSGSGTDFTLTINSLQPEDFATYYC QQSYAIPYT FGQGTKLEIKDIQMTQSPSSLSASVGDRVTITCRAS QRIATY LHWYQQKPGRAPKLLIY AAS SLQTGVPSRFSGSGSGTDFTLTINSLQPEDFATYYC QQSYAIPYT FGQGTKLEIK 214214
SA3827SA3827 중쇄heavy chain QVQLVESGGTLVQPGRSLRLSCAAS GFTFSSYA MHWVRQAPGKGLEWVAV ISYDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRGHYYDSSGFLY WGQGTLVTVSSQVQLVESGGTLVQPGRSLRLSCAAS GFTFSSYA MHWVRQAPGKGLEWVAV ISYDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRGHYYDSSGFLY WGQGTLVTVSS 215215
경쇄light chain DIQMTQSPSSLSASVGDRVTITCRAS QSISTY LSWYQQKPGKAPKLLIY AAS NLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYC QQSYNTPRT FGQGTKVEIKDIQMTQSPSSLSASVGDRVTITCRAS QSISTY LSWYQQKPGKAPKLLIY AAS NLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYC QQSYNTPRT FGQGTKVEIK 216216
SA3830SA3830 중쇄heavy chain QVQLVESGGGLIQPGRSLSLSCAAS GFNFDEYS MHWVRQRPGKGLEWVSG IKYNSNTI LYADSVKGRFTVSRDNSKNSLYLQMNSLRAEDTAVYYC ARDAMRGGDFDH WGQGTLITVSSQVQLVESGGGLIQPGRSLSLSCAAS GFNFDEYS MHWVRQRPGKGLEWVSG IKYNSNTI LYADSVKGRFTVSRDNSKNSLYLQMNSLRAEDTAVYYC ARDAMRGGDFDH WGQGTLITVSS 217217
경쇄light chain DIQMTQSPSYLSASAGDRVTITCRAS QSISDW LAWYQHKPGKAPKLLIY KAS RLENGVPSRFRGSGSATDFTLTISSLQAEDVAVYYC QQYYSTTWT FGQGTKVDIKDIQMTQSPSYLSASAGDRVTITCRAS QSISDW LAWYQHKPGKAPKLLIY KAS RLENGVPSRFRGSGSATDFTLTISSLQAEDVAVYYC QQYYSTTWT FGQGTKVDIK 218218
SA3838SA3838 중쇄heavy chain QVQLVESGAEVKKPGASVRVSCKIS GYIFTELS IHWVRQAPGKGLEWMGG FDPEEGKT IYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC AKIGHLGDFWY WGQGTLITVSSQVQLVESGAEVKKPGASVRVSCKIS GYIFTELS IHWVRQAPGKGLEWMGG FDPEEGKT IYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC AKIGHLGDFWY WGQGTLITVSS 219219
경쇄light chain QSALTQPASVSGSPGQSITISCTGT TSDIGSYDY VSWYQQHPGKAPKLIIY GVT RRPSGVSNRFSGSKSGNTASLTISRLLTEDEAEYYC ATYTSSATYV FGTGTKVTVLQSALTQPASVSGSPGQSITISCTGT TSDIGSYDY VSWYQQHPGKAPKLIIY GVT RRPSGVSNRFSGSKSGNTASLTISRLLTEDEAEYYC ATYTSSATYV FGTGTKVTVL 220220
SA3856SA3856 중쇄heavy chain QMQLVESGGGLIQPGGSLRLSCAAS GFTVTSAY MSWVRQAPGKGLEWVSI IYGGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TGPYPGRYDY WGRGTLVTVSSQMQLVESGGGLIQPGGSLRLSCAAS GFTVTSAY MSWVRQAPGKGLEWVSI IYGGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TGPYPGRYDY WGRGTLVTVSS 221221
경쇄light chain DIQMTQSPSSLSASVGDRVIITCRAS QDIRTS LAWFQQRLGEAPRSLIY DAS SLQNGVPSKFRGSGSGTDFTLTISSLQPEDFATYYC QQYKTVPLT FGGGTKVEIKDIQMTQSPSSLSASVGDRVIITCRAS QDIRTS LAWFQQRLGEAPRSLIY DAS SLQNGVPSKFRGSGSGTDFTLTISSLQPEDFATYYC QQYKTVPLT FGGGTKVEIK 222222
SA3902SA3902 중쇄heavy chain QVQLVQSGAEVRQPGASVKVSCKTS GYAFSYYH IHWVRQAPGQGLEWMGL INPGSGGT NYAQKFQGRVTLTRDTSTSTVYMDLTGLRSEDTAVYYC AGSEQPHYYANGAYRV WGQGTLVTVSSQVQLVQSGAEVRQPGASVKVSCKTS GYAFSYYH IHWVRQAPGQGLEWMGL INPGSGGT NYAQKFQGRVTLTRDTSTSTVYMDLTGLRSEDTAVYYC AGSEQPHYYANGAYRV WGQGTLVTVSS 223223
경쇄light chain DIQMTQSPSTLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY EVS TLESGVPSRFSGSRSGTEFTLTISSLQPDDFATYYC QQYDT FGGGTKVEIKDIQMTQSPSTLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY EVS TLESGVPSRFSGSRSGTEFTLTISSLQPDDFATYYC QQYDT FGGGTKVEIK 224224
SA4040SA4040 중쇄heavy chain EVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYY MHWARQAPGQGLEWMGW INPNSGGT NYAQKFQGRVTMTRDTSITTAYMELSRLTSDDTAVYYC ARDQAFSMVRGVTDY WGQGTLVTVSSEVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYY MHWARQAPGQGLEWMGW INPNSGGT NYAQKFQGRVTMTRDTSITTAYMELSRLTSDDTAVYYC ARDQAFSMVRGVTDY WGQGTLVTVSS 225225
경쇄light chain QSALTQPPSASGSPGQSVIISCTGS SSDVGGYNY VAWYQQYPGAAPKLIIS EVT KRPSGVPDRFSGSKSGNTASLTISGLQAEDEANYYC SSYAGDNTWI FGGGTKLTVLQSALTQPPSASGSPGQSVIISCTGS SSDVGGYNY VAWYQQYPGAAPKLIIS EVT KRPSGVPDRFSGSKSGNTASLTISGLQAEDEANYYC SSYAGDNTWI FGGGTKLTVL 226226
SA4043SA4043 중쇄heavy chain QVQLQESGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSSQVQLQESGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSS 227227
경쇄light chain DIQMTQSPSTLSASVGDRVTITCRAS QSISRW LAWYQQKPGQAPKLLIY AAS TLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYDSYPRT FGQGTKVDIKDIQMTQSPSTLSASVGDRVTITCRAS QSISRW LAWYQQKPGQAPKLLIY AAS TLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYDSYPRT FGQGTKVDIK 228228
SA4044SA4044 중쇄heavy chain EVQLVQSGGGLIQPGGSLRLSCAAS GFTVSSNY MSWVRQAPGKGLEWVSV IYSGGST YYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYC ARDLPQFDGFDY WGQGTLVTVSSEVQLVQSGGGLIQPGGSLRLSCAAS GFTVSSNY MSWVRQAPGKGLEWVSV IYSGGST YYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYC ARDLPQFDGFDY WGQGTLVTVSS 229229
경쇄light chain QSALTQPPSLSVAPGRTARITCGGN NIGSKS VNWYQHMPGQAPVLVIY SDT DRPSGIPERVSGSKSGNTATLSISRVEAGDEADYYC QVWDSTTDHVV FGGGTKLTVLQSALTQPPSLSVAPGRTARITCGGN NIGSKS VNWYQHMPGQAPVLVIY SDT DRPSGIPERVSGSKSGNTATLSISRVEAGDEADYYC QVWDSTTDHVV FGGGTKLTVL 230230
SA4050SA4050 중쇄heavy chain QVQLVESGAEVKKPGASVKVSCKAS GYTFTGYF IHWVRQAPGQGLEWMGW INPNSGGT NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC ARVPHLSGYYYEGVLGYFDY WGQGTLVTVSSQVQLVESGAEVKKPGASVKVSCKAS GYTFTGYF IHWVRQAPGQGLEWMGW INPNSGGT NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC ARVPHLSGYYYEGVLGYFDY WGQGTLVTVSS 231231
경쇄light chain QSALTQPASVSGSPGQSITISCTGT SIDIGSYNF VSWYQRHPGKAPRLLIY DVS DRPSGVSNRFSGSKSANTASLTISGLQAEDEADYYC SSYTSSSLWV FGGGTKLTVLQSALTQPASVSGSPGQSITISCTGT SIDIGSYNF VSWYQRHPGKAPRLIY DVS DRPSGVSNRFSGSKSANTASLTISGLQAEDEADYYC SSYTSSSLWV FGGGTKLTVL 232232
SA4053SA4053 중쇄heavy chain EVQLVQSGGGLVQPGGSLRLSCADS GFTFSSYW MSWVRQVPGKGLEWVAN IKQDGSEK YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDVGRGYCSSTSCYRFGGREDFFDY WGQGTLVTVSSEVQLVQSGGGLVQPGGSLRLSCADS GFTFSSYW MSWVRQVPGKGLEWVAN IKQDGSEK YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDVGRGYCSSTSCYRFGGREDFFDY WGQGTLVTVSS 233233
경쇄light chain QLVLTQPPSVSAAPGQRVTISCSGS FSNVGNNF VSWYRHLPGTAPELLIY DNN QRPSGIPDRFSASKSGTSATLAITGLHTGDEADYYC GTWDTSLSGVV FGGGTKLTVLQLVLTQPPSVSAAPGQRVTISCSGS FSNVGNNF VSWYRHLPGTAPELLIY DNN QRPSGIPDRFSASKSGTSATLAITGLHTGDEADYYC GTWDTSLSGVV FGGGTKLTVL 234234
SA4055SA4055 중쇄heavy chain EVQLVQSGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSSEVQLVQSGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSS 235235
경쇄light chain QSALTQPASVSGSPGQSITISCTGT SSDVGDYNL VSWYQQHPGKAPKVMIY DVS HRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC SSYTSSTTVV FGGGTKLTVLQSALTQPASVSGSPGQSITISCTGT SSDVGDYNL VSWYQQHPGKAPKVMIY DVS HRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC SSYTSSTTVV FGGGTKLTVL 236236
SA4056SA4056 중쇄heavy chain QVQLQESGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSSQVQLQESGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSS 237237
경쇄light chain DIQMTQSPSTLSASVGDRVTITCRAS QSISRW LAWYQQKPGQAPKLLIY AAS TLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYDSYPRT FGQGTKVDIKDIQMTQSPSTLSASVGDRVTITCRAS QSISRW LAWYQQKPGQAPKLLIY AAS TLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYDSYPRT FGQGTKVDIK 238238
SA4057SA4057 중쇄heavy chain EVQLVQSGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSSEVQLVQSGGGLVQPGGSLRLSCAAS VITVSSNY MSWVRQAPGKGLEWVSV IYPGGST YYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYC ARDLDIVGGMDV WGQGTTVTVSS 239239
경쇄light chain QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAPHD VHWYQQLPGTAPRLLIY ANN NRPSGVPDRFSGSKSGTSASLAITGLQDEDEADYYC QSYDSSLRAYV FGTGTKVTVLQLVLTQPPSVSGAPGQRVTISCTGS SSNIGAPHD VHWYQQLPGTAPRLLIY ANN NRPSGVPDRFSGSKSGTSASLAITGLQDEDEADYYC QSYDSSLRAYV FGTGTKVTVL 240240
단일클론들의 중쇄 및 경쇄 가변영역 염기서열Monoclonal heavy and light chain variable region nucleotide sequences
항체antibody 가변영역 뉴클레오타이드 서열variable region nucleotide sequence 서열번호SEQ ID NO:
SA3755SA3755 중쇄heavy chain CAGATGCAGCTGGTAGAGTCTGGGGGAGGTGTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATCATACCATGCACTGGGTCCGCCAAGCTCCGGGGAAGGGTCTGGAGTGGGTCTCTCTTATTAGTTGGGATGGTGGTAGCACATACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACAGCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTACGAGAGATGCTTCACGACGGCCCGGGGATGGTGGCTATGATTTTGACGTCTGGGGCCAGGGAACTCAGGTCACCGTCTCCTCACAGATGCAGCTGGTAGAGTCTGGGGGAGGTGTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATCATACCATGCACTGGGTCCGCCAAGCTCCGGGGAAGGGTCTGGAGTGGGTCTCTCTTATTAGTTGGGATGGTGGTAGCACATACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACAGCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTACGAGAGATGCTTCACGACGGCCCGGGGATGGTGGCTATGATTTTGACGTCTGGGGCCAGGGAACTCAGGTCACCGTCTCCTCA 241241
경쇄light chain CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGCACTGGAACTGTCACCAGAGGTCATTGGCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATTTATGATACAGACAACAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTGACGCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGACTATTACTGCTTGCTCTCCTATAGTGATTCCCGGGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGCACTGGAACTGTCACCAGAGGTCATTGGCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATTTATGATACAGACAACAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTGACGCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGACTATTACTGCTTGCTCTCCTATAGTGATTCCCGGGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA 242242
SA3779SA3779 중쇄heavy chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGCTATGCACTGGGTCCGCCAGGCCCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGCGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGTTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGCGTGGACATTATTATGATAGTAGTGGTTATTTGTATTGGGGCCACGGAACCCTGATCACCGTCTCCTCACAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGCTATGCACTGGGTCCGCCAGGCCCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGCGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGTTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGCGTGGACATTATTATGATAGTAGTGGTTATTTGTATTGGGGCCACGGAACCCTGATCACCGTCTCCTCA 243243
경쇄light chain GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGAATTGCCACCTATTTACATTGGTATCAGCAGAAACCAGGGAGAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAACTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGTCTGCAACCTGAAGATTTTGCGACTTACTACTGTCAACAGAGTTACGCTATCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGAATTGCCACCTATTTACATTGGTATCAGCAGAAACCAGGGAGAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAACTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGTCTGCAACCTGAAGATTTTGCGACTTACTACTGTCAACAGAGTTACGCTATCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 244244
SA3827SA3827 중쇄heavy chain CAGGTGCAGCTGGTGGAGTCTGGGGGAACCTTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGCGTGGGCATTACTATGATAGTAGTGGTTTTTTATATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCACAGGTGCAGCTGGTGGAGTCTGGGGGAACCTTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGCGTGGGCATTACTATGATAGTAGTGGTTTTTTATATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 245245
경쇄light chain GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAGTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATTTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCCACTTACTACTGTCAACAGAGTTACAATACCCCTCGCACTTTTGGCCAAGGGACCAAGGTGGAGATCAAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAGTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATTTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCCACTTACTACTGTCAACAGAGTTACAATACCCCTCGCACTTTTGGCCAAGGGACCAAGGTGGAGATCAAA 246246
SA3830SA3830 중쇄heavy chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATACAGCCTGGCAGGTCCCTGAGCCTCTCGTGTGCAGCCTCTGGATTCAATTTTGATGAGTATTCCATGCACTGGGTCCGACAACGTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATAAAGTACAACAGTAATACCATCCTCTATGCGGACTCTGTGAAGGGCCGCTTCACCGTCTCCCGAGACAACAGCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATGCAATGAGGGGGGGCGATTTTGACCACTGGGGCCAGGGAACCCTGATCACCGTCTCCTCACAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATACAGCCTGGCAGGTCCCTGAGCCTCTCGTGTGCAGCCTCTGGATTCAATTTTGATGAGTATTCCATGCACTGGGTCCGACAACGTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATAAAGTACAACAGTAATACCATCCTCTATGCGGACTCTGTGAAGGGCCGCTTCACCGTCTCCCGAGACAACAGCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATGCAATGAGGGGGGGCGATTTTGACCACTGGGGCCAGGGAACCCTGATCACCGTCTCCTCA 247247
경쇄light chain GACATCCAGATGACCCAGTCTCCATCCTACCTGTCTGCATCTGCAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGACTGGTTGGCCTGGTATCAGCATAAACCAGGAAAAGCCCCTAAGCTCCTCATTTATAAGGCCTCTCGTTTAGAAAATGGGGTCCCATCAAGGTTCCGGGGCAGTGGATCTGCGACAGACTTTACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTACGTGGACGTTCGGCCAAGGGACCAAGGTGGATATCAAAGACATCCAGATGACCCAGTCTCCATCCTACCTGTCTGCATCTGCAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGACTGGTTGGCCTGGTATCAGCATAAACCAGGAAAAGCCCCTAAGCTCCTCATTTATAAGGCCTCTCGTTTAGAAAATGGGGTCCCATCAAGGTTCCGGGGCAGTGGATCTGCGACAGACTTTACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTACGTGGACGTTCGGCCAAGGGACCAAGGTGGATATCAAA 248248
SA3838
SA3838
 		중쇄heavy chain CAGGTGCAGCTGGTAGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAGGGTCTCCTGCAAGATTTCCGGATACATCTTCACTGAATTATCCATACACTGGGTGCGACAGGCTCCTGGAAAGGGGCTTGAGTGGATGGGAGGTTTTGATCCTGAAGAAGGTAAAACAATCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAAATCGGCCACCTGGGGGATTTTTGGTACTGGGGCCAGGGAACCCTGATCACCGTCTCCTCACAGGTGCAGCTGGTAGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAGGGTCTCCTGCAAGATTTCCGGATACATCTTCACTGAATTATCCATACACTGGGTGCGACAGGCTCCTGGAAAGGGGCTTGAGTGGATGGGAGGTTTTGATCCTGAAGAAGGTAAAACAATCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAAATCGGCCACCTGGGGGATTTTTGGTACTGGGGCCAGGGAACCCTGATCACCGTCTCCTCA 249249
경쇄light chain CAGTCTGCCCTGACTCAGCCTGCTTCCGTGTCTGGGTCTCCTGGCCAGTCGATCACCATCTCCTGCACTGGAACCACCAGTGACATTGGTTCTTATGACTATGTCTCCTGGTATCAACAACACCCAGGCAAGGCCCCCAAACTCATCATTTATGGTGTCACTAGGCGGCCCTCGGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTAGGCTCCTGACTGAAGACGAGGCTGAGTATTACTGCGCCACATATACAAGCAGTGCCACTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACAGTCTGCCCTGACTCAGCCTGCTTCCGTGTCTGGGTCTCCTGGCCAGTCGATCACCATCTCCTGCACTGGAACCACCAGTGACATTGGTTCTTATGACTATGTCTCCTGGTATCAACAACACCCAGGCAAGGCCCCCAAACTCATCATTTATGGTGTCACTAGGCGGCCCTCGGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTAGGCTCCTGACTGAAGACGAGGCTGAGTATTACTGCGCCACATATACAAGCAGTGCCACTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA 250250
SA3856SA3856 중쇄heavy chain CAGATGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCACCAGCGCCTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATGGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTACGGGTCCCTATCCCGGCCGCTATGACTACTGGGGCCGGGGAACCCTGGTCACCGTCTCCTCACAGATGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCACCAGCGCCTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATGGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTACGGGTCCCTATCCCGGCCGCTATGACTACTGGGGCCGGGGAACCCTGGTCACCGTCTCCTCA 251251
경쇄light chain GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCATTATCACTTGTCGGGCGAGTCAGGACATTAGAACTTCTTTGGCCTGGTTTCAGCAGAGATTAGGGGAAGCCCCTAGGTCCCTCATCTATGATGCATCCAGTTTGCAGAATGGGGTCCCATCAAAGTTCCGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAATATAAAACTGTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCATTATCACTTGTCGGGCGAGTCAGGACATTAGAACTTCTTTGGCCTGGTTTCAGCAGAGATTAGGGGAAGCCCCTAGGTCCCTCATCTATGATGCATCCAGTTTGCAGAATGGGGTCCCATCAAAGTTCCGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAATATAAAACTGTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 252252
SA3902SA3902 중쇄heavy chain CAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAGGCAGCCTGGGGCCTCAGTGAAAGTTTCCTGCAAGACATCTGGATACGCCTTCTCCTACTACCATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATTAATCAACCCTGGTAGTGGTGGCACAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCTTGACCAGGGACACGTCCACGAGCACAGTCTACATGGACCTAACCGGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGGGTTCTGAGCAACCTCACTACTATGCAAATGGTGCTTACCGAGTCTGGGGTCAGGGAACCCTGGTCACCGTCTCCTCACAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAGGCAGCCTGGGGCCTCAGTGAAAGTTTCCTGCAAGACATCTGGATACGCCTTCTCCTACTACCATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATTAATCAACCCTGGTAGTGGTGGCACAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCTTGACCAGGGACACGTCCACGAGCACAGTCTACATGGACCTAACCGGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGGGTTCTGAGCAACCTCACTACTATGCAAATGGTGCTTACCGAGTCTGGGGTCAGGGAACCCTGGTCACCGTCTCCTCA 253253
경쇄light chain GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGAGGTTTCTACTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATGATACTTTCGGCGGAGGGACCAAGGTGGAAATCAAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGAGGTTTCTACTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATGATACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA 254254
SA4040SA4040 중쇄heavy chain GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTCTCTTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGCGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCACCACAGCCTACATGGAGCTGAGCAGGCTGACATCTGACGACACGGCCGTATATTACTGTGCGAGAGATCAGGCCTTTTCTATGGTTCGGGGAGTCACCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGTCAGGGAGTGCATCCGCCCCAACCCTTGAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTCTCTTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGCGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCACCACAGCCTACATGGAGCTGAGCAGGCTGACATCTGACGACACGGCCGTATATTACTGTGCGAGAGATCAGGCCTTTTCTATGGTTCGGGGAGTCACCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGTCAGGGAGTGCATCCGCCCCAACCCTT 255255
경쇄light chain CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCATCATCTCCTGCACTGGAAGCAGCAGTGACGTGGGTGGTTATAACTATGTCGCCTGGTACCAACAATACCCAGGCGCAGCCCCCAAACTCATCATTTCTGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCTCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTAATTATTACTGCAGCTCATATGCTGGAGACAACACTTGGATCTTCGGCGGAGGGACCAAGCTGACCGTCCTACAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCATCATCTCCTGCACTGGAAGCAGCAGTGACGTGGGTGGTTATAACTATGTCGCCTGGTACCAACAATACCCAGGCGCAGCCCCCAAACTCATCATTTCTGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCTCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTAATTATTACTGCAGCTCATATGCTGGAGACAACACTTGGATCTTCGGCGGAGGGACCAAGCTGACCGTCCTA 256256
SA4043SA4043 중쇄heavy chain CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCCAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC 257257
경쇄light chain GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATCAGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAGGCCAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATGATAGTTATCCAAGGACGTTCGGCCAAGGGACCAAGGTGGATATCAAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATCAGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAGGCCAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATGATAGTTATCCAAGGACGTTCGGCCAAGGGACCAAGGTGGATATCAAA 258258
SA4044SA4044 중쇄heavy chain GAGGTGCAGCTGGTGCAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGCATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGACCTTCCCCAGTTCGATGGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAACCCTTGAGGTGCAGCTGGTGCAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGCATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGACCTTCCCCAGTTCGATGGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAACCCTT 259259
경쇄light chain CAGTCTGCCCTGACTCAGCCTCCCTCACTGTCCGTGGCCCCAGGAAGGACGGCCAGGATTACCTGTGGAGGAAACAACATTGGAAGTAAAAGTGTTAACTGGTACCAGCACATGCCAGGCCAGGCCCCTGTTTTGGTCATCTATTCTGATACCGACCGGCCCTCAGGGATCCCTGAGCGAGTCTCTGGCTCCAAGTCTGGGAACACGGCCACCCTGAGCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTACTACTGATCATGTGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTACAGTCTGCCCTGACTCAGCCTCCCTCACTGTCCGTGGCCCCAGGAAGGACGGCCAGGATTACCTGTGGAGGAAACAACATTGGAAGTAAAAGTGTTAACTGGTACCAGCACATGCCAGGCCAGGCCCCTGTTTTGGTCATCTATTCTGATACCGACCGGCCCTCAGGGATCCCTGAGCGAGTCTCTGGCTCCAAGTCTGGGAACACGGCCACCCTGAGCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTACTACTGATCATGTGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTA 260260
SA4050SA4050 중쇄heavy chain CAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTTTATCCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTACCTCATCTTAGTGGTTATTACTACGAAGGAGTATTGGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAACCCTTCAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTTTATCCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTACCTCATCTTAGTGGTTATTACTACGAAGGAGTATTGGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAACCCTT 261261
경쇄light chain CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCATTGATATTGGAAGTTATAATTTTGTCTCCTGGTACCAGCGACATCCAGGCAAAGCCCCCCGACTCCTCATTTATGATGTCAGTGATCGGCCCTCAGGGGTCTCTAATCGCTTCTCCGGCTCCAAGTCCGCCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATACAAGCAGCAGTCTTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTACAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCATTGATATTGGAAGTTATAATTTTGTCTCCTGGTACCAGCGACATCCAGGCAAAGCCCCCCGACTCCTCATTTATGATGTCAGTGATCGGCCCTCAGGGGTCTCTAATCGCTTCTCCGGCTCCAAGTCCGCCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATACAAGCAGCAGTCTTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA 262262
SA4053SA4053 중쇄heavy chain GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGACTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTATATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGACGTGGGTAGGGGATATTGTAGTAGTACCAGCTGCTATCGCTTCGGGGGAAGGGAGGATTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCGAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGACTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTATATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGACGTGGGTAGGGGATATTGTAGTAGTACCAGCTGCTATCGCTTCGGGGGAAGGGAGGATTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC 263263
경쇄light chain CAGCTCGTGCTGACTCAGCCGCCCTCCGTGTCTGCGGCCCCCGGACAGAGGGTCACCATCTCCTGCTCTGGAAGCTTCTCCAACGTTGGAAATAATTTTGTCTCGTGGTACCGGCACCTCCCGGGAACAGCCCCCGAACTCCTCATTTATGACAATAATCAGCGACCCTCAGGGATTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGCCATCACCGGACTCCACACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATACCAGCCTGAGTGGTGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTACAGCTCGTGCTGACTCAGCCGCCCTCCGTGTCTGCGGCCCCCGGACAGAGGGTCACCATCTCCTGCTCTGGAAGCTTCTCCAACGTTGGAAATAATTTTGTCTCGTGGTACCGGCACCTCCCGGGAACAGCCCCCGAACTCCTCATTTATGACAATAATCAGCGACCCTCAGGGATTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGCCATCACCGGACTCCACACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATACCAGCCTGAGTGGTGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA 264264
SA4055SA4055 중쇄heavy chain GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCGAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC 265265
경쇄light chain CAGTCTGCCCTGACTCAGCCCGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGATTATAACTTGGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAAGTCATGATTTATGATGTCAGTCATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATACAAGCAGCACTACTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTACAGTCTGCCCTGACTCAGCCCGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGATTATAACTTGGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAAGTCATGATTTATGATGTCAGTCATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATACAAGCAGCACTACTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA 266266
SA4056SA4056 중쇄heavy chain CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCCAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC 267267
경쇄light chain GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATCAGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAGGCCAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATGATAGTTATCCAAGGACGTTCGGCCAAGGGACCAAGGTGGATATCAAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATCAGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAGGCCAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATGATAGTTATCCAAGGACGTTCGGCCAAGGGACCAAGGTGGATATCAAA 268268
SA4057SA4057 중쇄heavy chain GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCGAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGTAATCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGAGGTAGCACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTTGACATCGTGGGGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC 269269
경쇄light chain CAGCTCGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACCGGGAGCAGCTCCAACATCGGGGCACCTCATGATGTACACTGGTACCAGCAACTTCCAGGAACAGCCCCCAGACTCCTCATCTATGCTAACAACAATCGGCCCTCAGGGGTCCCTGACCGCTTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGATGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGTAGCCTGCGTGCTTATGTGTTCGGAACTGGGACTAAGGTCACCGTCCTACAGCTCGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACCGGGAGCAGCTCCAACATCGGGGCACCTCATGATGTACACTGGTACCAGCAACTTCCAGGAACAGCCCCCAGACTCCTCATCTATGCTAACAACAATCGGCCCTCAGGGGTCCCTGACCGCTTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGATGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGTAGCCTGCGTGCTTATGTGTTCGGAACTGGGACTAAGGTCACCGTCCTA 270270
실시예 3. SARS-CoV-2 스파이크 인간 단일클론 항체의 최적화Example 3. Optimization of SARS-CoV-2 Spike Human Monoclonal Antibody
실시예 3.1. 항체 최적화를 위한 라이브러리 제작Example 3.1. Libraries for antibody optimization
LC 셔플링(light chain shuffling; LS) 라이브러리를 제작하기 위해, 모항체(SA3755, SA3779, SA3827, 및 SA3838)의 LC 유전자를 BstX I으로 절단한 다음 벡터로 사용하고, Ymax®-ABL((주)와이바이오로직스)을 BstX I으로 절단하여 인서트로 사용하였다. 리가제로 라이게이션 후, 전기천공 형질전환용 세포를 이용하여 형질 전환을 수행하였다. 사각 접시(square plate)에 형질전환된 세포를 모아 항체 라이브러리를 제조한 결과, 약 1×107의 다양성을 갖는 라이브러리를 얻었고, 염기 서열 분석 결과 HC의 서열은 모두 같으며 LC의 서열이 서로 다른 것을 확인하였다.To construct an LC light chain shuffling (LS) library, the LC genes of the parent antibodies (SA3755, SA3779, SA3827, and SA3838) were digested with BstX I and then used as a vector, and Ymax ® -ABL ((main ) Y Biologics) was cut with BstX I and used as an insert. After ligation with ligase, transformation was performed using cells for electroporation transformation. As a result of preparing an antibody library by collecting transformed cells on a square plate, a library with a diversity of about 1×10 7 was obtained. As a result of nucleotide sequence analysis, all HC sequences are the same and LC sequences are different. confirmed that.
실시예 3.2. 항체 최적화를 위한 바이오패닝Example 3.2. Biopanning for antibody optimization
상기 실시예 1에서 제조한 SARS-CoV-2-RBD-hFc 단백질 항원 50 ㎍을 면역흡착튜브에 코팅한 후 블로킹을 수행하였다. 상기 실시예 3.1.에서 얻은 1×107의 다양성을 가진 LS 셔플링 인간 scFv 라이브러리를 대장균에 감염시킨 후, 대장균을 30℃에서 16 시간 배양하였다. 배양액을 원심 분리하여 상층액을 PEG로 농축한 다음, 이를 PBS 완충용액에 녹여 인간 항체 라이브러리를 준비하였다. 면역흡착튜브에 라이브러리 파지를 넣은 후, 실온에서 2시간 반응시킨 다음, 1× PBS/T와 1× PBS로 세척한 후 항원에 특이적으로 결합한 scFv-파지들만 용출시켰다. 용출된 파지를 다시 대장균에 감염시켜 증폭시키는 패닝 과정을 통해 양성 파지의 풀(pool)을 얻고, 첫 번째 라운드의 패닝(panning)에서 증폭된 파지를 가지고 PBST 세척 단계의 횟수를 늘린 것을 제외하고는 동일한 방법으로 2라운드 패닝을 수행하였다. After coating 50 μg of the SARS-CoV-2-RBD-hFc protein antigen prepared in Example 1 on an immunosorbent tube, blocking was performed. After the LS shuffling human scFv library having a diversity of 1×10 7 obtained in Example 3.1. was infected with E. coli, E. coli was cultured at 30° C. for 16 hours. The culture medium was centrifuged, and the supernatant was concentrated with PEG, and then dissolved in PBS buffer to prepare a human antibody library. After putting library phages in an immunosorbent tube, incubated at room temperature for 2 hours, washed with 1×PBS/T and 1×PBS, and only scFv-phages specifically bound to the antigen were eluted. A pool of positive phages was obtained through the panning process in which the eluted phages were again infected with E. coli and amplified, and the number of PBST washing steps was increased with the amplified phages in the first round of panning. Two rounds of panning were performed in the same manner.
그 결과, 표 7과 같이 2라운드 패닝에서 항원에 결합한 파지 수가 input(항원과 반응시킬 때의 파지 수) 대비 output(항원과 결합시킨 뒤 세척 이후 용출했을 때의 파지 수)이 다소 증가였음을 확인하였다. As a result, as shown in Table 7, it was confirmed that the number of phages bound to the antigen in the second round panning was slightly increased compared to the input (the number of phages when reacted with the antigen) compared to the output (the number of phages when eluted after binding to the antigen after washing) did
구체적으로, 표 7에 패닝에 따른 항체의 역가를 비교하여 나타내었다.Specifically, it is shown in Table 7 by comparing the titers of the antibodies according to the panning.
패닝 횟수에 따른 항체의 역가 비교Comparison of antibody titers according to the number of panning
샘플Sample 패닝 횟수number of pans input input outputoutput
SA3755SA3755 1회1 time 2.0×1012 2.0×10 12 2.0×107 2.0×10 7
2회 Episode 2 1.1×1011 1.1×10 11 2.6×107 2.6×10 7
SA3779 SA3779 1회1 time 5.0×1012 5.0×10 12 3.2×107 3.2×10 7
2회 Episode 2 5.8×1011 5.8×10 11 1.0×108 1.0×10 8
SA3827 SA3827 1회1 time 9.0×1012 9.0×10 12 1.0×108 1.0×10 8
2회 Episode 2 3.4×1011 3.4×10 11 3.1×106 3.1×10 6
SA3838 SA3838 1회1 time 3.0×1012 3.0×10 12 1.0×108 1.0×10 8
2회 Episode 2 1.8×1011 1.8×10 11 4.0×106 4.0×10 6
실시예 3.3. 단일클론 선별Example 3.3. monoclonal selection
폴리 파지 ELISA를 통해 결합능이 큰 것으로 확인된 2라운드 패닝의 양성 파지 풀로부터 단일클론들을 선별하였고, 이들을 이용하여 실시예 2.3.과 동일한 방식으로 direct ELISA를 실시하고 SARS-CoV-2-RBD에 대한 특이성을 확인하였다. 그 결과, 도 4에서와 같이 모노 scFv-파지 클론들은 SARS-CoV-2-RBD에만 결합능이 강함을 확인하였다.Single clones were selected from the positive phage pool of round 2 panning confirmed to have high binding capacity through poly phage ELISA, and direct ELISA was performed in the same manner as in Example 2.3. Specificity was confirmed. As a result, it was confirmed that the mono scFv-phage clones had strong binding ability only to SARS-CoV-2-RBD as shown in FIG. 4 .
실시예 3.4. 단일클론의 염기서열 분석Example 3.4. Monoclonal sequencing analysis
선별된 4종의 단일클론들에 대해 DNA 정제 키트(Qiagen, 독일)를 사용하여 파지미드(phagemid) DNA를 분리하여 DNA 염기서열을 분석하였다. LC 셔플링(shuffling)에 의한 항체 최적화를 진행했기 때문에 중쇄의 서열은 모항체와 같다. 따라서 경쇄 CDR의 아미노산 서열, 경쇄 FR의 아미노산 서열을 각각 하기 표 8 및 표 9에 나타내었고, 경쇄 가변영역의 아미노산 서열 및 이를 코딩하는 폴리뉴클레오타이드 서열을 각각 하기 표 10 및 표 11에 나타내었다.For the selected four types of monoclones, phagemid DNA was isolated using a DNA purification kit (Qiagen, Germany), and DNA sequences were analyzed. Since antibody optimization by LC shuffling was performed, the sequence of the heavy chain is the same as that of the parent antibody. Accordingly, the amino acid sequence of the light chain CDR and the amino acid sequence of the light chain FR are shown in Tables 8 and 9, respectively, and the amino acid sequence of the light chain variable region and the polynucleotide sequence encoding it are shown in Tables 10 and 11, respectively.
경쇄 CDR의 아미노산 서열Amino acid sequence of the light chain CDRs
항체antibody CDR 서열CDR sequence 서열번호SEQ ID NO:
(SA3755AM)
SA4079(SA3755AM)
SA4079 		CDRL1CDRL1 TGTVTRGHWTGTVTRGHW 271271
CDRL2CDRL2 DTDDTD 272272
CDRL3CDRL3 LLSYSDSRVLLSYSDSRV 273273
(SA3779AM)
SA4086(SA3779AM)
SA4086 		CDRL1CDRL1 QSISTYQSISTY 274274
CDRL2CDRL2 AASAAS 275275
CDRL3CDRL3 QQSYNTPRTQQSYNTPRT 276276
(SA3827AM)
SA4114(SA3827AM)
SA4114 		CDRL1CDRL1 QSIDNYQSIDNY 277277
CDRL2CDRL2 AASAAS 278278
CDRL3CDRL3 QQSYSIPRTQQSYSIPRT 279279
(SA3838AM)
SA4118(SA3838AM)
SA4118 		CDRL1CDRL1 TSDIGSYDYTSDIGSYDY 280280
CDRL2CDRL2 GVTGVT 281281
CDRL3CDRL3 SSCTRSSTYVSSCTRSSTYV 282282
경쇄 FR의 아미노산 서열Amino acid sequence of light chain FR
항체antibody FR 서열FR sequence 서열번호SEQ ID NO:
(SA3755AM)
SA4079(SA3755AM)
SA4079 		FRL1FRL1 QAVVTQEPSLTVSPGGTATLTCGSSQAVVTQEPSLTVSPGGTATLTCGSS 283283
FRL2FRL2 PYWFQQKPGQAPRTLIYPYWFQQKPGQAPRTLIY 284284
FRL3FRL3 TKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYCTKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYC 285285
FRL4FRL4 FGTGTKVTVLFGTGTKVTVL 286286
(SA3779AM)
SA4086(SA3779AM)
SA4086 		FRL1FRL1 DIQMTQSPSSLSASVGDRVTITCRASDIQMTQSPSSSLSASVGDRVTITCRAS 287 287
FRL2FRL2
LSWYQQKPGKAPKLLIYLSWYQQKPGKAPKLLIY 288288
FRL3FRL3 NLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCNLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYC 289289
FRL4FRL4 FGQGTKVEIKFGQGTKVEIK 290290
(SA3827AM)
SA4114(SA3827AM)
SA4114 		FRL1FRL1 DIQMTQSPSSLSASVGDRVTITCRAGDIQMTQSPSSSLSASVGDRVTITCRAG 291291
FRL2FRL2 LNWYQQKPGKAPKLLIYLNWYQQKPGKAPKLLIY 292292
FRL3FRL3 TLHSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCTLHSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYC 293293
FRL4FRL4 FGQGTKLEIKFGQGTKLEIK 294294
(SA3838AM)
SA4118(SA3838AM)
SA4118 		FRL1FRL1 QSALTQPASVSGSPGQSITISCTGTQSALTQPASVSGSPGQSITISCTGT 295295
FRL2FRL2 VSWYQQHPGKAPKLIIYVSWYQQHPGKAPKLIIY 296296
FRL3FRL3 RRPSGVSNRFSGSKSGNTASLTISGLQAEDEANYYCRRPSGVSNRFSGSKSGNTASLTISGLQAEDEANYYC 297297
FRL4FRL4 FGTGTKVTVLFGTGTKVTVL 298298
경쇄 가변영역 아미노산 서열light chain variable region amino acid sequence
항체antibody 가변영역 아미노산 서열variable region amino acid sequence 서열번호SEQ ID NO:
(SA3755AM)
SA4079(SA3755AM)
SA4079 		경쇄light chain QAVVTQEPSLTVSPGGTATLTCGSS TGTVTRGHW PYWFQQKPGQAPRTLIY DTD TKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYC LLSYSDSRV FGTGTKVTVLQAVVTQEPSLTVSPGGTATLTCGSS TGTVTRGHW PYWFQQKPGQAPRTLIY DTD TKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYC LLSYSDSRV FGTGTKVTVL 299299
(SA3779AM)
SA4086(SA3779AM)
SA4086 		경쇄light chain DIQMTQSPSSLSASVGDRVTITCRAS QSISTY LSWYQQKPGKAPKLLIY AAS NLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYC QQSYNTPRT FGQGTKVEIKDIQMTQSPSSLSASVGDRVTITCRAS QSISTY LSWYQQKPGKAPKLLIY AAS NLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYC QQSYNTPRT FGQGTKVEIK 300300
(SA3827AM)
SA4114(SA3827AM)
SA4114 		경쇄light chain DIQMTQSPSSLSASVGDRVTITCRAG QSIDNY LNWYQQKPGKAPKLLIY AAS TLHSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QQSYSIPRT FGQGTKLEIKDIQMTQSPSSLSASVGDRVTITCRAG QSIDNY LNWYQQKPGKAPKLLIY AAS TLHSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYC QQSYSIPRT FGQGTKLEIK 301301
(SA3838AM)
SA4118(SA3838AM)
SA4118 		경쇄light chain QSALTQPASVSGSPGQSITISCTGT TSDIGSYDY VSWYQQHPGKAPKLIIY GVT RRPSGVSNRFSGSKSGNTASLTISGLQAEDEANYYC SSCTRSSTYV FGTGTKVTVLQSALTQPASVSGSPGQSITISCTGT TSDIGSYDY VSWYQQHPGKAPKLIIY GVT RRPSGVSNRFSGSKSGNTASLTISGLQAEDEANYYC SSCTRSSTYV FGTGTKVTVL 302302
경쇄 가변영역 염기서열light chain variable region sequence
항체antibody 가변영역 뉴클레오타이드 서열variable region nucleotide sequence 서열번호SEQ ID NO:
(SA3755AM)
SA4079(SA3755AM)
SA4079 		경쇄light chain CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGCCACTCTCACCTGTGGCTCCAGCACTGGAACTGTCACCAGAGGTCATTGGCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATTTATGATACAGACACCAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTGACGCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGACTATTACTGCTTGCTCTCCTATAGTGATTCCCGGGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGCCACTCTCACCTGTGGCTCCAGCACTGGAACTGTCACCAGAGGTCATTGGCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATTTATGATACAGACACCAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTGACGCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGACTATTACTGCTTGCTCTCCTATAGTGATTCCCGGGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA 303303
(SA3779AM)
SA4086(SA3779AM)
SA4086 		경쇄light chain GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAGTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATTTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCCACTTACTACTGTCAACAGAGTTACAATACCCCTCGCACTTTTGGCCAAGGGACCAAGGTGGAGATCAAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAGTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATTTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCCACTTACTACTGTCAACAGAGTTACAATACCCCTCGCACTTTTGGCCAAGGGACCAAGGTGGAGATCAAA 304304
(SA3827AM)
SA4114(SA3827AM)
SA4114 		경쇄light chain GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCAGGCCAGAGCATTGACAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGTCTGCAGCCTGACGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTATCCCTCGAACTTTTGGCCAGGGGACCAAGCTGGAAATCAAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCAGGCCAGAGCATTGACAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGTCTGCAGCCTGACGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTATCCCTCGAACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA 305305
(SA3838AM)
SA4118(SA3838AM)
SA4118 		경쇄light chain CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCACCAGTGACATTGGTTCTTATGACTATGTCTCCTGGTATCAACAACACCCAGGCAAGGCCCCCAAACTCATCATTTATGGTGTCACTAGGCGGCCCTCGGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTAATTATTACTGCAGCTCATGTACACGCAGCAGCACTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCACCAGTGACATTGGTTCTTATGACTATGTCTCCTGGTATCAACAACACCCAGGCAAGGCCCCCAAACTCATCATTTATGGTGTCACTAGGCGGCCCTCGGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTAATTATTACTGCAGCTCATGTACACGCAGCAGCACTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA 306306
실시예 4. SARS-CoV-2 스파이크 인간 단일클론 항체의 생산Example 4. Production of SARS-CoV-2 Spike Human Monoclonal Antibody
실시예 4.1. scFv 형태를 IgG 형태로 전환(conversion)Example 4.1. Conversion of scFv form to IgG form
실시예 2에서 선별된 단일클론 파지 항체를 scFv 형태에서 IgG 형태로 전환하기 위해, 중쇄 가변 영역의 염기서열을 제한효소 SfiI/NheI 사이트를 이용하여 pNATVH((주)와이바이오로직스)에 클로닝하여 N293F HC 벡터를 제조하였고, 경쇄 가변 영역의 염기서열을 제한 효소 SfiI/BsiWI 사이트를 이용하여 pNATVL((주)와이바이오로직스)에 클로닝하여 N293F LC 벡터를 제조하였다.In order to convert the monoclonal phage antibody selected in Example 2 from scFv form to IgG form, the base sequence of the heavy chain variable region was cloned into pNATVH (YBiologics Co., Ltd.) using restriction enzymes SfiI/NheI site to N293F An HC vector was prepared, and the nucleotide sequence of the light chain variable region was cloned into pNATVL (YBiologics Co., Ltd.) using restriction enzymes SfiI/BsiWI site to prepare an N293F LC vector.
실시예 4.2. 인간 단일클론 항체의 생산Example 4.2. Production of human monoclonal antibodies
N293F HC와 N293F LC 벡터를 HEK293F 세포에 동시 형질감염(co-transfection)시키고, 배양 7일차에 배양액을 수거하여 8,000 rpm에서 30분간 원심 분리하여 세포 잔해를 제거해 주었다. 이후 0.22 ㎛ 공극 크기의 보틀 탑 필터 (bottle top filter: Steritop-GP Filter Unit. #SCGPS01RE, Millipore)를 이용하여 여과하였다. 정제를 수행하기 위하여 빈 칼럼(#BR731-1550, Bio-rad)에 4 ㎖의 단백질 A 세파로스 레진 슬러리(KANEKA KanCapATM, Cat. No. KKC20170403_01)를 넣은 후 100 ㎖의 DPBS(#LB001-02)로 레진을 패킹 및 세척하였다. 패킹된 레진에 여과된 배양액을 로딩(loading)하고 1분당 1 ㎖의 속도로 흘려 주었다(#EP-1 Econo pump, Bio-Rad). 150 ㎖의 DPBS로 세척한 후, 10 ㎖의 0.1 M 글라이신(glycine)-HCl(pH 3.3)로 용출하였다. 용출액에 1 M Tris-HCl(pH 9.0)을 10% 첨가하여 pH를 중화시키고, Amicon Ultra-10(#UFC901096, Millipore)를 이용하여 DPBS로 버퍼를 바꾸어 주었다. 이 과정을 약 3회 실시한 후 1 ㎖ 정도로 농축되었을 때 멈추고, Nano-drop으로 농도를 측정하였다.N293F HC and N293F LC vectors were co-transfected into HEK293F cells, and the culture medium was collected on the 7th day of culture and centrifuged at 8,000 rpm for 30 minutes to remove cell debris. Thereafter, it was filtered using a bottle top filter (Steritop-GP Filter Unit. #SCGPS01RE, Millipore) having a pore size of 0.22 μm. In order to perform purification, 4 ml of protein A sepharose resin slurry (KANEKA KanCapA TM , Cat. No. KKC20170403_01) was put into an empty column (#BR731-1550, Bio-rad), and then 100 ml of DPBS (#LB001-02) ) to pack and wash the resin. The filtered culture solution was loaded on the packed resin and flowed at a rate of 1 ml per minute (#EP-1 Econo pump, Bio-Rad). After washing with 150 ml of DPBS, it was eluted with 10 ml of 0.1 M glycine-HCl (pH 3.3). 10% of 1 M Tris-HCl (pH 9.0) was added to the eluate to neutralize the pH, and the buffer was changed to DPBS using Amicon Ultra-10 (#UFC901096, Millipore). After carrying out this process about 3 times, it was stopped when it was concentrated to about 1 ml, and the concentration was measured with a Nano-drop.
실시예 5. SARS-CoV-2 스파이크 인간 단일클론 항체의 특이성Example 5. Specificity of SARS-CoV-2 Spike Human Monoclonal Antibody
실시예 5.1. SARS-CoV-2 스파이크에 대한 항체의 결합 특이성Example 5.1. Binding specificity of antibodies to SARS-CoV-2 spikes
SARS-CoV-2 스파이크 인간 단일클론 항체의 항원 특이성은 HEK293E 세포에 SARS-CoV-2 스파이크를 과발현한 세포 풀을 사용하여 항체의 결합 정도를 유세포 분석기(flow cytometer)로 분석하여 확인하였다.The antigen specificity of the SARS-CoV-2 spike human monoclonal antibody was confirmed by using a cell pool overexpressing the SARS-CoV-2 spike in HEK293E cells and analyzing the degree of binding of the antibody with a flow cytometer.
SARS-CoV-2 스파이크를 과발현하는 형질전환 세포 풀은 SARS-CoV-2 스파이크 세포외 영역(아미노산 서열 M1-P1213, UniProt#P0DTC2)과 EpCAM(epithelial cell adhesion molecule)의 막 횡단부터 세포내 영역(아미노산 서열 A266-A314, UniProt#P16422)을 연결한 키메라(chimeric) 서열을 포함하고 있는 pcDNA3.1 플라스미드를 HEK293E에 형질감염 시킨 후, 200 ㎎/㎖의 Zeocin(#R25001, thermo Fisher Scientific)이 들어있는 선택적 배양 배지에서 선별과정을 수행하였다. 선별과정 후, 세포 표면에서 SARS-CoV-2 스파이크의 발현은 SARS-CoV-2 스파이크도 인식하는 SARS-CoV 스파이크 항체(#40150-T62-CoV2, Sino Biological)를 이용하여 FACS(fluorescence activated cell sorting) 분석을 통해 확인하고 분리하여 사용하였다.The pool of transgenic cells overexpressing the SARS-CoV-2 spike has the SARS-CoV-2 spike extracellular domain (amino acid sequence M1-P1213, UniProt#P0DTC2) and the transmembrane to intracellular domains of the epithelial cell adhesion molecule (EpCAM) ( The pcDNA3.1 plasmid containing the chimeric sequence linked to the amino acid sequence A266-A314, UniProt#P16422) was transfected into HEK293E, and then 200 mg/ml of Zeocin (#R25001, thermo Fisher Scientific) was added. The selection process was performed in a selective culture medium. After the selection process, the expression of SARS-CoV-2 spikes on the cell surface was detected by fluorescence activated cell sorting (FACS) using a SARS-CoV spike antibody (#40150-T62-CoV2, Sino Biological) that also recognizes SARS-CoV-2 spikes. ) was confirmed through analysis and used separately.
SARS-CoV-2 스파이크 인간 단일클론 항체들의 세포 결합을 확인하기 위해 세포는 각 시료당 3×105 개로 준비하여 2 ㎎/㎖의 항체와 4℃에서 30분간 반응시켰다. 그 후 세포들은 2% FBS(fetal bovine serum)가 포함된 PBS로 3회 세척하고, FITC(fluorescein isothiocyanate) 형광물질이 결합된 항-인간 IgG 항체(#FI-3000, Vectorlabs)를 사용하여 4℃에서 30분간 암반응 후, 동일한 세척 과정을 거쳤다. 이후 0.2 ㎖의 2% FBS가 든 PBS로 현탁시킨 후, 유세포 분석기인 CytoFLEX(Beckman coulter, USA)를 사용하여 분석하였다. In order to check the cell binding of SARS-CoV-2 spike human monoclonal antibodies, 3×10 5 cells were prepared for each sample and reacted with 2 mg/ml antibody at 4° C. for 30 minutes. Thereafter, the cells were washed three times with PBS containing 2% fetal bovine serum (FBS), and then at 4°C using an anti-human IgG antibody (#FI-3000, Vectorlabs) conjugated with fluorescein isothiocyanate (FITC). After dark reaction for 30 minutes, the same washing process was performed. After suspension in PBS containing 0.2 ml of 2% FBS, analysis was performed using a flow cytometer, CytoFLEX (Beckman coulter, USA).
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체들은 SARS-CoV-2 스파이크 과발현 HEK293E 세포(HEK293E/CoV-2 스파이크(chimeric))에는 잘 결합하였으나(도 5b 및 도 5d), HEK293E 세포(HEK293E/Mock)에는 결합하지 않았다(도 5a 및 도 5c). 이 결과는 Ymax®-ABL 유래 7종(도 5a 및 도 5b)과 환자-면역 라이브러리(patient-immune library) 유래 8종(도 5c 및 도 5d) 항체들은 HEK293E에 비특이성이 없으며, HEK293E에 발현된 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체들임을 의미한다. As a result, SARS-CoV-2 spike human monoclonal antibodies bound well to SARS-CoV-2 spike-overexpressing HEK293E cells (HEK293E/CoV-2 spike (chimeric)) (FIGS. 5b and 5d), but HEK293E cells (HEK293E) /Mock) did not bind ( FIGS. 5A and 5C ). This result shows that Ymax ® -ABL-derived 7 types ( FIGS. 5a and 5b ) and patient-immune library-derived 8 types ( FIGS. 5c and 5d ) antibodies are non-specific to HEK293E, and expressed in HEK293E It means that these are antibodies that specifically bind to the SARS-CoV-2 spike protein.
실시예 5.2. SARS-CoV-2 스파이크 S1에 대한 항체의 결합 특이성Example 5.2. Binding specificity of antibodies to SARS-CoV-2 spike S1
SARS-CoV-2 스파이크 인간 단일클론 항체가 SARS-CoV-2 스파이크 S1(아미노산 서열 V16-R685, UniProt#P0DTC2) 뿐만 아니라 SARS-CoV 스파이크 S1(아미노산 서열 S14-R667, UniProt#P59594)에도 결합 특이성이 있는지 확인하기 위해 각 단백질의 카복시-말단에 His-tag이 융합된 재조합 단백질들을 사용하여 ELISA(enzyme-linked immunosorbent assay) 분석을 통해 확인하였다. SARS-CoV-2 spike human monoclonal antibody has binding specificity for SARS-CoV-2 spike S1 (amino acid sequence V16-R685, UniProt#P0DTC2) as well as SARS-CoV spike S1 (amino acid sequence S14-R667, UniProt#P59594) In order to confirm whether there is a carboxyl-terminus of each protein, it was confirmed by ELISA (enzyme-linked immunosorbent assay) analysis using recombinant proteins fused with His-tag.
SARS-CoV 스파이크 S1-His(#40150-V08B1, Sino Biological) 또는 SARS-CoV-2 스파이크 S1-His 단백질을 웰 당 200 ng의 농도로 면역-96 마이크로웰 플레이트(immuno-96 microwell plate)에 분주하고 4℃에서 밤새 표면 고정시킨 후, 200 ㎕의 4% 탈지분유가 포함된 PBS-T를 모든 웰에 넣어 37℃에서 1시간 동안 반응시킴으로써 비특이적인 단백질 결합을 차단하였다.SARS-CoV spike S1-His (#40150-V08B1, Sino Biological) or SARS-CoV-2 spike S1-His protein at a concentration of 200 ng per well was aliquoted into an immuno-96 microwell plate. After surface fixation at 4°C overnight, 200 μl of PBS-T containing 4% skim milk powder was put into all wells and reacted at 37°C for 1 hour to block non-specific protein binding.
이후 50 nM의 SARS-CoV-2 스파이크 인간 단일클론 항체 15종을 각 웰에 넣어 37℃에서 1시간 동안 반응시키고, 200 ㎕의 PBS-T로 3회 세척한 뒤에 HRP(horseradish peroxidase)가 결합되어 있는 염소 항-인간 IgG 항체(#ab97225, Abcam)를 1:4,000으로 희석하여 넣고 37℃에서 1시간 반응시켰다. PBS-T로 다시 3회 세척 후, 100 ㎕의 TMB 완충용액(#T0440, Sigma)을 넣어 빛이 없는 조건에서 반응시킨 후, 50 ㎕의 2.5M 황산(H2SO4)으로 반응을 중단시킨 다음 분광 광도계(spectraMax M5 spectrophotometer, Molecular Devices, 미국)를 이용하여 450 ㎚에서 흡광도를 측정하였다. 측정 값들은 GraphPad Prism 8(GraphPad Software, Inc., 미국)를 이용하여 분석하였다.After that, 15 kinds of 50 nM SARS-CoV-2 spike human monoclonal antibody were put into each well and reacted at 37°C for 1 hour, washed 3 times with 200 μl of PBS-T, and then HRP (horseradish peroxidase) was bound. goat anti-human IgG antibody (#ab97225, Abcam) was diluted 1:4,000 and reacted at 37°C for 1 hour. After washing again 3 times with PBS-T, 100 μl of TMB buffer (#T0440, Sigma) was added to react in the absence of light, and then the reaction was stopped with 50 μl of 2.5M sulfuric acid (H 2 SO 4 ). Absorbance was measured at 450 nm using the following spectrophotometer (spectraMax M5 spectrophotometer, Molecular Devices, USA). The measured values were analyzed using GraphPad Prism 8 (GraphPad Software, Inc., USA).
그 결과, Ymax®-ABL 유래 7종(도 6a)과 환자-면역 라이브러리 유래 8종(도 6b)의 SARS-CoV-2 스파이크 인간 단일클론 항체들은 SARS-CoV 스파이크 S1에 결합하지 않았고, 특이적으로 SARS-CoV-2 스파이크 S1에 결합하는 것을 보였다(도 6a 및 도 6b). As a result, the SARS-CoV-2 spike human monoclonal antibodies of Ymax ® -ABL-derived 7 (FIG. 6a) and patient-immune library-derived 8 (FIG. 6B) did not bind to SARS-CoV spike S1, and specific was shown to bind to SARS-CoV-2 spike S1 ( FIGS. 6a and 6b ).
실시예 5.3. SARS-CoV-2 스파이크 인간 단일클론 항체의 항원 친화도(affinity) 확인Example 5.3. Confirmation of antigen affinity (affinity) of SARS-CoV-2 spike human monoclonal antibody
SARS-CoV-2 스파이크 인간 단일클론 항체의 항원에 대한 결합 친화도는 SARS-CoV-2 RBD(아미노산 서열 R319-F541, UniProt#P0DTC2)의 카복시-말단에 마우스 Fc(mFc)-tag이 융합된 재조합 단백질인 SARS-CoV-2 RBD-mFc 항원을 사용하여 Octet　QKe(Fortebio Inc., 미국) 분석 장비를 통해 측정하였다. The binding affinity of the SARS-CoV-2 spike human monoclonal antibody to the antigen is determined by fusion of a mouse Fc (mFc)-tag to the carboxy-terminus of SARS-CoV-2 RBD (amino acid sequence R319-F541, UniProt#P0DTC2). Octet using the recombinant protein SARS-CoV-2 RBD-mFc antigen　It was measured using QKe (Fortebio Inc., USA) analysis equipment.
Octet　QKe를 이용한 항원 항체간 친화도 측정을 위해 AHC(anti-human Fc capture: #18-5060, Fortebio Inc.) 또는 AMC(anti-mouse Fc capture: #18-5088, Fortebio Inc.) 바이오센서를 완충액(#18-1042, Fortebio Inc.)에 10분간 안정화시킨 후에 항원 또는 항체를 고정하고, 고정되지 않은 항원 또는 항체는 완충액으로 5분간 세척하였다. 96-웰 플레이트(#655209, Greiner Bio-One, USA)에 결합을 원하는 항체 또는 항원을 농도별(0.94 nM~60 nM)로 준비하여 5분간 결합(association) 반응을 수행하고, 이어서 5분간 해리(dissociation) 반응을 수행하였다. 모든 실험은 30℃, 1,000 rpm 조건에서 수행하였고, 시간에 따른 결합과 해리 과정 중의 센서그램(sensorgram) 데이터를 수집하여 Octet 데이터 분석 소프트웨어 9.0에 따라 1:1 글로벌 바인딩 피팅 모델(global binding fitting model)을 적용하여 평형 해리상수(equilibrium dissociation constant, KD)를 구하였다.Octet　For affinity measurement between antigen-antibody using QKe, AHC (anti-human Fc capture: #18-5060, Fortebio Inc.) or AMC (anti-mouse Fc capture: #18-5088, Fortebio Inc.) biosensor buffer (#18-1042, Fortebio Inc.) After stabilization for 10 minutes, the antigen or antibody was fixed, and the non-immobilized antigen or antibody was washed with buffer for 5 minutes. Prepare the desired antibody or antigen for binding in a 96-well plate (#655209, Greiner Bio-One, USA) at each concentration (0.94 nM ~ 60 nM), perform an association reaction for 5 minutes, and then dissociate for 5 minutes (dissociation) reaction was performed. All experiments were performed at 30 ° C., 1,000 rpm conditions, and by collecting sensorgram data during the binding and dissociation process over time, a 1:1 global binding fitting model according to Octet data analysis software 9.0. was applied to obtain the equilibrium dissociation constant (K D ).
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체, Ymax®-ABL 유래 7종(도 7a)과 환자-면역 라이브러리 유래 8종(도 7b)의 항체는 SARS-CoV-2 RBD에 대한 항원 친화도(KD)가 0.0284 nM~1.06 nM로 우수하였고, 각 항체들의 값은 표 12와 같다.As a result, SARS-CoV-2 spike human monoclonal antibody, Ymax ® -ABL-derived 7 (Fig. 7a) and patient-immune library-derived 8 (Fig. 7b) antibodies had antigen affinity for SARS-CoV-2 RBD. Figure (K D ) was excellent at 0.0284 nM ~ 1.06 nM, and the values of each antibody are shown in Table 12.
구체적으로, 표 12에 Octet 분석에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 항원 친화도를 정리하여 나타내었다.Specifically, Table 12 summarizes the antigen affinity of the anti-SARS-CoV-2 spike monoclonal antibody by Octet analysis.
Octet 분석에 의한 단일클론 항체의 항원 친화도Antigen Affinity of Monoclonal Antibodies by Octet Assay
항체 라이브러리antibody library 항체antibody SARS-CoV-2 RBD-mFcSARS-CoV-2 RBD-mFc
KD(M)K D (M) Ka(1/Ms)K a (1/Ms) Kdis(1/s)K dis (1/s)
Ymax®-ABL
(A)Ymax ® -ABL
(A) 		SA3755SA3755 1.06×10-9 1.06×10 -9 3.99×105 3.99×10 5 4.24×10-4 4.24×10 -4
SA3779SA3779 1.05×10-9 1.05×10 -9 2.13×105 2.13×10 5 2.23×10-4 2.23×10 -4
SA3827SA3827 4.52×10-10 4.52×10 -10 3.83×105 3.83×10 5 1.73×10-4 1.73×10 -4
SA3830SA3830 7.82×10-10 7.82×10 -10 8.97×105 8.97×10 5 7.02×10-4 7.02×10 -4
SA3838SA3838 1.79×10-10 1.79×10 -10 1.57×105 1.57×10 5 2.81×10-4 2.81×10 -4
SA3856SA3856 8.88×10-11 8.88×10 -11 4.84×105 4.84×10 5 4.30×10-5 4.30×10 -5
SA3902SA3902 3.28×10-10 3.28×10 -10 2.47×105 2.47×10 5 8.12×10-5 8.12×10 -5
환자 면역 라이브러리
(B)Patient Immune Library
(B) 		SA4040SA4040 3.84×10-10 3.84×10 -10 4.71×105 4.71×10 5 1.81×10-4 1.81×10 -4
SA4043SA4043 1.40×10-10 1.40×10 -10 4.50×105 4.50×10 5 6.32×10-4 6.32×10 -4
SA4044SA4044 5.06×10-11 5.06×10 -11 3.75×105 3.75×10 5 1.90×10-4 1.90×10 -4
SA4050SA4050 9.29×10-11 9.29×10 -11 5.03×105 5.03×10 5 4.67×10-5 4.67×10 -5
SA4053SA4053 6.89×10-10 6.89×10 -10 3.16×105 3.16×10 5 2.18×10-4 2.18×10 -4
SA4055SA4055 6.10×10-11 6.10×10 -11 3.87×105 3.87×10 5 2.36×10-5 2.36×10 -5
SA4056SA4056 9.07×10-11 9.07×10 -11 4.77×105 4.77×10 5 4.32×10-5 4.32×10 -5
SA4057SA4057 2.84×10-11 2.84×10 -11 1.75×105 1.75×10 5 4.96×10-6 4.96×10 -6
KD(M), 친화상수(Affinity constant); Ka(1/Ms), 결합률(association rate); Kdis(1/s), 해리율(dissociation rate)K D (M), affinity constant; K a (1/Ms), association rate; K dis (1/s), dissociation rate
실시예 6. SARS-CoV-2 스파이크 인간 단일클론 항체 변이체의 특성Example 6. Characterization of SARS-CoV-2 Spike Human Monoclonal Antibody Variants
실시예 6.1. SARS-CoV-2 스파이크에 대한 항체 변이체의 결합 특이성Example 6.1. Binding specificity of antibody variants for SARS-CoV-2 spikes
SARS-CoV-2 스파이크 인간 단일클론 항체 SA3755, SA3779, SA3827 및 SA3838를 친화도 성숙(affinity maturation, AM)시킨 각각의 변이체인 SA4079, SA4086, SA4114 및 SA4118의 항원 특이성은 실시예 5.1.과 같이 HEK293E 세포에 SARS-CoV-2 스파이크를 과발현한 세포 풀을 사용하여 항체의 결합 정도를 유세포 분석기(flow cytometer)로 분석하여 확인하였다.The antigen specificities of SA4079, SA4086, SA4114 and SA4118, respectively, which were affinity maturation (AM) of the SARS-CoV-2 spike human monoclonal antibodies SA3755, SA3779, SA3827 and SA3838, were HEK293E as in Example 5.1. Using a pool of cells overexpressing the SARS-CoV-2 spike in cells, the degree of binding of the antibody was confirmed by analysis with a flow cytometer.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들은 SARS-CoV-2 스파이크 과발현(over-expression) HEK293E 세포(HEK293E/CoV-2 스파이크(chimeric))에 모항체와 비교하여 유사 또는 우수한 결합을 보였고(도 8b), HEK293E 세포(HEK293E/Mock)에는 결합하지 않았다(도 8a). 이 결과는 SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들(SA4079, SA4086, SA4114, SA4118)이 HEK293E에 비특이성이 없으며, HEK293E에 발현된 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체들임을 의미한다. As a result, SARS-CoV-2 spiked human monoclonal antibody variants showed similar or superior binding compared to the parental antibody to SARS-CoV-2 spiked over-expressing HEK293E cells (HEK293E/CoV-2 spiked (chimeric)). (FIG. 8b), did not bind to HEK293E cells (HEK293E/Mock) (FIG. 8a). These results show that SARS-CoV-2 spike human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) are non-specific to HEK293E, and antibodies that specifically bind to the SARS-CoV-2 spike protein expressed in HEK293E means to take
실시예 6.2. SARS-CoV-2 스파이크 S1에 대한 항체 변이체의 결합 특이성Example 6.2. Binding specificity of antibody variants for SARS-CoV-2 spike S1
SARS-CoV-2 스파이크 인간 단일클론 항체 변이체가 SARS-CoV-2 스파이크 S1에 대해 결합 특이성이 유지되는지 실시예 5.2.와 같이 ELISA 분석을 통해 SARS-CoV 스파이크 S1과 비교하여 확인하였다. Whether the SARS-CoV-2 spike human monoclonal antibody mutant maintains binding specificity for SARS-CoV-2 spike S1 was confirmed by ELISA analysis as in Example 5.2 by comparison with SARS-CoV spike S1.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들(SA4079, SA4086, SA4114, SA4118)은 각각 모항체와 같이 SARS-CoV 스파이크 S1에 결합하지 않았고, 특이적으로 SARS-CoV-2 스파이크 S1에 결합하였다(도 9). As a result, SARS-CoV-2 spike human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) did not bind to SARS-CoV spike S1 like the parent antibody, respectively, and specifically SARS-CoV-2 spike S1 was bound to (FIG. 9).
실시예 6.3. SARS-CoV-2 스파이크 인간 단일클론 항체의 항원 친화도(affinity) 확인Example 6.3. Confirmation of antigen affinity (affinity) of SARS-CoV-2 spike human monoclonal antibody
SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들의 항원에 대한 결합 친화도는 실시예 5.3.과 같이 카복시-말단에 마우스 Fc(mFc)-tag이 융합된 재조합 단백질인 SARS-CoV-2 RBD-mFc 항원을 사용하여 Octet　QKe(Fortebio Inc., 미국) 분석 장비를 통해 측정하였다. The antigen binding affinity of SARS-CoV-2 spike human monoclonal antibody variants is SARS-CoV-2 RBD-mFc, a recombinant protein in which a mouse Fc (mFc)-tag is fused to the carboxy-terminus as in Example 5.3. Octet using antigen　It was measured using QKe (Fortebio Inc., USA) analysis equipment.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들(SA4079, SA4086, SA4114, SA4118)은 SARS-CoV-2 RBD에 대한 항원 친화도(KD)가 0.0899 nM~0.157 nM로 우수하였고, 각각 모항체의 항원 친화도(표 12)에 비교하여 증가함을 보였다(도 10 및 표 13).As a result, SARS-CoV-2 spike human monoclonal antibody mutants (SA4079, SA4086, SA4114, SA4118) had excellent antigen affinity (K D ) for SARS-CoV-2 RBD from 0.0899 nM to 0.157 nM, Each showed an increase compared to the antigen affinity of the parent antibody (Table 12) ( FIGS. 10 and 13 ).
구체적으로, 표 13에 Octet 분석에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 항원 친화도를 정리하여 나타내었다.Specifically, Table 13 summarizes the antigen affinity of the anti-SARS-CoV-2 spike monoclonal antibody variants by Octet analysis.
Octet 분석에 의한 단일클론 항체 변이체의 항원 친화도Antigen Affinity of Monoclonal Antibody Variants by Octet Assay
친화도 성숙
(Affinity maturation, AM)Affinity Maturity
(Affinity maturation, AM) 		항체antibody SARS-CoV-2 RBD-mFcSARS-CoV-2 RBD-mFc
KD(M)K D (M) Ka(1/Ms)K a (1/Ms) Kdis(1/s)K dis (1/s)
SA3755AMSA3755AM SA4079SA4079 1.54×10-10 1.54×10 -10 3.73×105 3.73×10 5 5.73×10-5 5.73×10 -5
SA3779AMSA3779AM SA4086SA4086 1.06×10-10 1.06×10 -10 4.71×105 4.71×10 5 4.99×10-5 4.99×10 -5
SA3827AMSA3827AM SA4114SA4114 8.99×10-11 8.99×10 -11 4.33×105 4.33×10 5 3.89×10-5 3.89×10 -5
SA3838AMSA3838AM SA4118SA4118 2.96×10-11 2.96×10 -11 6.95×105 6.95×10 5 2.05×10-5 2.05×10 -5
KD(M), 친화상수(Affinity constant); Ka(1/Ms), 결합률(association rate); Kdis(1/s), 해리율(dissociation rate)K D (M), affinity constant; K a (1/Ms), association rate; K dis (1/s), dissociation rate
실시예 7. SARS-CoV-2 스파이크 인간 단일클론 항체 및 변이체의 항원 결합부위(epitope) 결정Example 7. Determination of antigen binding site (epitope) of SARS-CoV-2 spike human monoclonal antibody and variants
실시예 7.1. SARS-CoV-2 스파이크 항체의 항원 결합 영역 확인Example 7.1. Identification of antigen binding region of SARS-CoV-2 spike antibody
SARS-CoV-2 스파이크 인간 단일클론 항체 및 변이체의 항원 결합 부위(epitope)를 확인하기 위해 먼저 ACE2와 결합하는 SARS-CoV-2 RBD의 RBM 영역에 항체가 결합하는지 여부를 확인하였다. 항체의 SARS-CoV-2 RBM 영역 결합 여부는 표 14와 도 12a와 같이 SARS-CoV-2의 RBD내 RBM 아미노산 영역을 SARS-CoV RBD(아미노산 서열 R306-F527, UniProt#P59594)의 RBM으로 치환한 돌연변이체(SARS-CoV-2_RBM_CoV) 또는 그 반대로 치환한 돌연변이체(SARS-CoV_RBM_CoV-2), SARS-CoV-2 RBD, SARS-CoV RBD의 카복시-말단에 His-tag이 융합된 재조합 융합 단백질들인 SARS-CoV-2_RBM_CoV-His, SARS-CoV_RBM_CoV-2-His, SARAS-CoV-2 RBD-His, SARS-CoV RBD-His(#40150-V08B2, Sino Biological) 항원들을 사용하여 실시예 5.2.와 같이 ELISA 분석으로 확인하였다.In order to confirm the antigen binding site (epitope) of the SARS-CoV-2 spike human monoclonal antibody and variant, it was first confirmed whether the antibody binds to the RBM region of SARS-CoV-2 RBD that binds ACE2. Whether the antibody binds to the SARS-CoV-2 RBM region is determined by substituting the RBM amino acid region in the RBD of SARS-CoV-2 with the RBM of the SARS-CoV RBD (amino acid sequence R306-F527, UniProt#P59594) as shown in Table 14 and FIG. 12A. A recombinant fusion protein in which a His-tag is fused to the carboxy-terminus of one mutant (SARS-CoV-2_RBM_CoV) or vice versa (SARS-CoV_RBM_CoV-2), SARS-CoV-2 RBD, and SARS-CoV RBD Example 5.2 using the SARS-CoV-2_RBM_CoV-His, SARS-CoV_RBM_CoV-2-His, SARAS-CoV-2 RBD-His, SARS-CoV RBD-His (#40150-V08B2, Sino Biological) antigens. It was also confirmed by ELISA analysis.
그 결과, Ymax®-ABL 유래 7종(도 11a)과 환자-면역 라이브러리 유래 8종(도 11b)의 SARS-CoV-2 RBD 인간 단일클론 항체들과 Ymax®-ABL 유래 4종을 최적화한 SA4079, SA4086, SA4114, SA4118 항체 변이체들(도 11c)은 항원 SARS-CoV-2 RBD와 SARS-CoV_RBM_CoV-2에 결합하고, SARS-CoV-2_RBM_CoV 또는 SARS-CoV RBD에는 결합하지 않았다. 이 결과는 SARS-CoV-2 스파이크 인간 단일클론 항체 및 변이체들이 ACE2와 결합하는 SARS-CoV-2 RBD의 RBM 영역에 특이적으로 결합한다는 것을 의미하며, 이부분에 에피토프를 갖는 항체들임을 알 수 있다. As a result, Ymax ® -ABL-derived 7 types (FIG. 11a) and patient-immune library-derived 8 types (FIG. 11b) SARS-CoV-2 RBD human monoclonal antibodies and Ymax ® -ABL-derived SA4079 optimized for 4 types , SA4086, SA4114, SA4118 antibody variants ( FIG. 11C ) bound to the antigens SARS-CoV-2 RBD and SARS-CoV_RBM_CoV-2, but not SARS-CoV-2_RBM_CoV or SARS-CoV RBD. This result means that the SARS-CoV-2 spike human monoclonal antibody and its variants specifically bind to the RBM region of SARS-CoV-2 RBD that binds to ACE2, indicating that antibodies having an epitope in this region. there is.
구체적으로, 표 14에 SARS-CoV-2 또는 SARS-CoV의 RBD 돌연변이 서열을 나타내었다.Specifically, Table 14 shows the RBD mutant sequence of SARS-CoV-2 or SARS-CoV.
SARS-CoV-2 또는 SARS-CoV의 RBD 돌연변이 서열RBD mutant sequence of SARS-CoV-2 or SARS-CoV
재조합 단백질recombinant protein 아미노산 잔기 또는 서열 영역의 치환Substitution of amino acid residues or sequence regions
SARS-CoV-2 RBD-HisSARS-CoV-2 RBD-His R319-F541 wild type(WT)(UniProt#P0DTC2)R319-F541 wild type (WT) (UniProt#P0DTC2)
SARS-CoV-2 RBD-M2-HisSARS-CoV-2 RBD-M2-His F486A, N487A, Y489AF486A, N487A, Y489A
SARS-CoV-2 RBD-M3-HisSARS-CoV-2 RBD-M3-His T500A, N501A, G502A, Y505AT500A, N501A, G502A, Y505A
SARS-CoV-2 RBD-M4-HisSARS-CoV-2 RBD-M4-His S438T, N439R, L441IS438T, N439R, L441I
SARS-CoV-2 RBD-M5-HisSARS-CoV-2 RBD-M5-His S443A, K444T, V445S, G446TS443A, K444T, V445S, G446T
SARS-CoV-2 RBD-M6-HisSARS-CoV-2 RBD-M6-His L452K, L455Y, F456LL452K, L455Y, F456L
SARS-CoV-2 RBD-M7-HisSARS-CoV-2 RBD-M7-His K458H, S459G, N460K, K462RK458H, S459G, N460K, K462R
SARS-CoV-2 RBD-M8-HisSARS-CoV-2 RBD-M8-His T470N, E471V, I472P, Y473F, Q474ST470N, E471V, I472P, Y473F, Q474S
SARS-CoV-2 RBD-M9-HisSARS-CoV-2 RBD-M9-His A475P, G476D, S477G, T478KA475P, G476D, S477G, T478K
SARS-CoV-2 RBD-M10-HisSARS-CoV-2 RBD-M10-His N481T, G482P, V483Δ, E484P, G485A, F486LN481T, G482P, V483Δ, E484P, G485A, F486L
SARS-CoV-2 RBD-M12-HisSARS-CoV-2 RBD-M12-His Q498Y, P499T, N501TQ498Y, P499T, N501T
SARS-CoV-2_RBM_CoV-HisSARS-CoV-2_RBM_CoV-His SARS-CoV RBD M417-T487 서열 영역으로 SARS-CoV-2 RBD T430-N501 서열 영역을 치환Substitution of the SARS-CoV-2 RBD T430-N501 sequence region with the SARS-CoV RBD M417-T487 sequence region
SARS-CoV_RBM_CoV-2-HisSARS-CoV_RBM_CoV-2-His SARS-CoV-2 RBD T430-N501 서열 영역으로 SARS-CoV RBD M417-T487 서열 영역을 치환Substitution of the SARS-CoV RBD M417-T487 sequence region with the SARS-CoV-2 RBD T430-N501 sequence region
SARS-CoV RBD-HisSARS-CoV RBD-His R306-F527 wild type(WT)(UniProt#P59594)R306-F527 wild type (WT) (UniProt#P59594)
실시예 7.2. SARS-CoV-2 스파이크 인간 단일클론 항체의 항원에 대한 결합부위(epitope) 확인Example 7.2. Identification of antigen binding site (epitope) of SARS-CoV-2 spike human monoclonal antibody
SARS-CoV-2 스파이크 인간 단일클론 항체 및 변이체의 항원에 대한 결합부위(epitope) 확인은 ACE2와 결합하는 SARS-CoV-2 RBD의 RBM 아미노산 서열을 표 14와 도 12a(gray box)와 같이 SARS-CoV RBD의 RBM 서열을 참고하여 특정 또는 불특정하게 다른 아미노산으로 치환하고 His-tag을 카복시-말단에 융합한 10종(M2~M10, M12)의 재조합 SARS-CoV-2 RBD 돌연변이체와 SARS-CoV-2 RBD 야생형(wild-type, WT)을 이용하여 ELISA 분석으로 확인하였다. SARS-CoV-2 spike human monoclonal antibody and antigen binding site (epitope) confirmation of the variant, the RBM amino acid sequence of SARS-CoV-2 RBD binding to ACE2 SARS as shown in Table 14 and Figure 12a (gray box) -Recombinant SARS-CoV-2 RBD mutants and SARS- of 10 species (M2 to M10, M12) in which a His-tag is fused to the carboxy-terminus after replacing specific or unspecified amino acids with reference to the RBM sequence of CoV RBD It was confirmed by ELISA analysis using CoV-2 RBD wild-type (wild-type, WT).
그 결과, SARS-CoV-2 스파이크 항체들은 SARS-CoV-2 RBD의 WT과 충분히 결합하는 조건에서 각 재조합 돌연변이체에 결합하지 않거나 매우 낮아지는 항체들이 존재하였으며, 결합하지 않거나 매우 낮아지는 부위(dark gray box)에 대해 그룹화한 결과 도 12b와 같이 9그룹(G1~G9)으로 분류되었다. 결과에서 SARS-CoV-2 스파이크 인간 단일클론 항체 및 변이체들이 재조합 SARS-CoV-2 RBD 돌연변이체들에 결합하지 않거나 매우 낮아진 부위(dark gray box)가 실제 항체들의 항원 결합 부위인 에피토프(epitope)임을 가리킨다. 15종 SARS-CoV-2 스파이크 인간 단일클론 항체들과 4종 항체 변이체들의 각 그룹별 에피토프 서열 부분은 표 14와 도 12의 돌연변이 서열 부분에 해당하는 SARS-CoV-2 RBD의 WT 서열이다.As a result, SARS-CoV-2 spike antibodies did not bind to each recombinant mutant under conditions of sufficient binding to WT of SARS-CoV-2 RBD, or there were antibodies that did not bind or were very low. gray box) were grouped into 9 groups (G1 to G9) as shown in FIG. 12b. As a result, the SARS-CoV-2 spike human monoclonal antibody and the mutants did not bind to the recombinant SARS-CoV-2 RBD mutants, or the region (dark gray box) was the epitope, the antigen-binding site of the actual antibodies. points to The epitope sequence for each group of 15 SARS-CoV-2 spike human monoclonal antibodies and 4 antibody variants is the WT sequence of SARS-CoV-2 RBD corresponding to the mutant sequence of Table 14 and FIG. 12 .
세부적으로 대개의 항체들(SA4053, SA4055, SA4050, SA3779, SA4086(SA3779AM), SA3902, SA3838, SA4118(SA3838AM), SA3827, SA4114(SA3827AM), SA4040, SA4043, SA4056, SA4057, SA3830, SA3856, SA4044)은 M2, M8, M9, M10 부위 전체 또는 부분을 포함하거나 외적으로 다른 부분에 추가적인 에피토프를 갖는 항체들이었다. 또한, 특징적으로 SA3755, SA4079(SA3755AM), SA4053은 M5에, SA4053, SA3830, SA3856은 M6에, SA4044는 M7에, SA4053은 M12 부위에 에피토프를 갖거나 이를 포함하는 항체들이었다. Specifically, most of the antibodies (SA4053, SA4055, SA4050, SA3779, SA4086 (SA3779AM), SA3902, SA3838, SA4118 (SA3838AM), SA3827, SA4114 (SA3827AM), SA4040, SA4043, SA4056, SA4057, SA3830, SA3856, SA4044) were antibodies that contained all or part of the M2, M8, M9, or M10 regions, or had additional epitopes in other regions externally. Also, characteristically, SA3755, SA4079 (SA3755AM), SA4053 had an epitope in M5, SA4053, SA3830, SA3856 was in M6, SA4044 was in M7, and SA4053 was an antibody having or containing an epitope in the M12 region.
실시예 8. SARS-CoV-2 스파이크 인간 단일클론 항체의 중화능 평가Example 8. Evaluation of neutralizing ability of SARS-CoV-2 spike human monoclonal antibody
실시예 8.1. 경쟁 효소면역분석법에 의한 SARS-CoV-2 스파이크 인간 단일클론 항체의 중화능 확인 Example 8.1. Confirmation of neutralizing ability of SARS-CoV-2 spike human monoclonal antibody by competitive enzyme immunoassay
SARS-CoV-2 스파이크 인간 단일클론 항체가 ACE2/SARS-CoV-2 RBD 복합체 또는 ACE2/SARS-CoV-2 스파이크 S1 복합체 형성을 억제할 수 있는지 확인하기 위해, 1.94 nM의 SARS-CoV-2 RBD-mFc 또는 200 nM의 SARS-CoV-2 스파이크 S1-mFc 단백질에 항체들을 500 nM에서부터 1/3 희석 배수로 연속적으로 희석하였다. 그 후, 37℃에서 30분간 반응(pre-incubation) 시킨 후, 항원-항체 혼합물을 ACE2(UniProt#Q9BYF1) 카복시-말단에 His-tag이 융합된 재조합 단백질인 ACE2-His가 한 웰 당 200 ng으로 코팅된 면역-96 마이크로웰 플레이트(immune-96 microwell plate)에 첨가하여 37℃에서 1시간 동안 반응시켰다.To determine whether SARS-CoV-2 spike human monoclonal antibody can inhibit the formation of ACE2/SARS-CoV-2 RBD complex or ACE2/SARS-CoV-2 spike S1 complex, 1.94 nM SARS-CoV-2 RBD Antibodies to -mFc or 200 nM SARS-CoV-2 spike S1-mFc protein were serially diluted from 500 nM to 1/3 dilution fold. Thereafter, after pre-incubation at 37° C. for 30 minutes, the antigen-antibody mixture was prepared with 200 ng per well of ACE2-His, a recombinant protein in which His-tag was fused to the carboxy-terminus of ACE2 (UniProt#Q9BYF1). was added to an immune-96 microwell plate coated with , and reacted at 37° C. for 1 hour.
이후 200 ㎕의 PBS-T로 3회 세척한 뒤에 HRP(horseradish peroxidase)가 결합되어 있는 토끼 항 마우스 IgG 항체(Rabbit anti-Mouse IgG-HPR antibody, Cell signaling)를 1:2,000으로 희석하여 넣고 37℃에서 1시간 반응시켰다. PBS-T로 다시 3회 세척 후, 100 ㎕의 TMB 완충용액을 넣어 빛이 없는 조건에서 1분 30초 간 반응시킨 후, 50 ㎕의 2.5 M 황산(H2SO4)으로 반응을 중단시킨 다음, 분광 광도계(spectraMax M5 spectrophotometer, Molecular Devices, 미국)를 이용하여 450 ㎚에서 흡광도를 측정하였다. 측정 결과는 GraphPad Prism 8(GraphPad Software, Inc., 미국)를 이용하여 분석하였다.After washing three times with 200 μl of PBS-T, a rabbit anti-mouse IgG-HPR antibody (Cell signaling) bound to horseradish peroxidase (HRP) was diluted at 1:2,000 and added at 37°C. was reacted for 1 hour. After washing again 3 times with PBS-T, 100 μl of TMB buffer solution was added and reacted for 1 minute and 30 seconds in the absence of light, and then the reaction was stopped with 50 μl of 2.5 M sulfuric acid (H 2 SO 4 ). , absorbance was measured at 450 nm using a spectrophotometer (spectraMax M5 spectrophotometer, Molecular Devices, USA). Measurement results were analyzed using GraphPad Prism 8 (GraphPad Software, Inc., USA).
SARS-CoV-2 스파이크 인간 단일클론 항체의 ACE2/SARS-CoV-2 RBD 또는 ACE2/SARS-CoV-2 스파이크 S1 결합을 저해하는 능력은 SARS-CoV-2 RBD-mFc 또는 SARS-CoV-2 스파이크 S1-mFc의 결합력 감소로 알 수 있고, 그 저해 정도는 항체에 의한 최대 억제의 50%에 이르는데 필요한 시료의 양(IC50)으로 나타낼 수 있다. 실험 결과에서 SARS-CoV-2 스파이크 인간 단일클론 항체들은 농도 의존적으로 ACE2와 SARS-CoV-2 RBD 또는 SARS-CoV-2 스파이크 S1 결합을 효과적으로 저해함을 확인하였다(도 13a 내지 도 13d).The ability of the SARS-CoV-2 spike human monoclonal antibody to inhibit ACE2/SARS-CoV-2 RBD or ACE2/SARS-CoV-2 spike S1 binding was related to the SARS-CoV-2 RBD-mFc or SARS-CoV-2 spike. It can be seen from the decrease in the binding force of S1-mFc, and the degree of inhibition can be expressed as the amount of sample (IC 50 ) required to reach 50% of the maximum inhibition by the antibody. From the experimental results, it was confirmed that the SARS-CoV-2 spike human monoclonal antibodies effectively inhibited ACE2 and SARS-CoV-2 RBD or SARS-CoV-2 spike S1 binding in a concentration-dependent manner ( FIGS. 13a to 13d ).
Ymax®-ABL 유래 7종(도 13a 및 도 13b)과 환자-면역 라이브러리 유래 8종(도 13c 및 도 13d) 항체들의 SARS-CoV-2 RBD에 대한 IC50 값은 각각 0.91 nM~25.08 nM와 0.14 nM~58.27 nM(도 13a 및 도 13c), SARS-CoV-2 스파이크 S1에 대해서는 각각 0.61 nM~4.06 nM와 1.43 nM~22 nM였으며(도 13b 및 도 13d), 대조로 넣어준 수용성 ACE2에 우월한 특성을 보였다. 각 항체들의 IC50 값은 표 15와 같다.The IC 50 values for SARS-CoV-2 RBD of Ymax ® -ABL-derived 7 ( FIGS. 13a and 13b ) and 8 ( FIGS. 13c and 13d ) antibodies from the patient-immune library were 0.91 nM to 25.08 nM, respectively. 0.14 nM to 58.27 nM ( FIGS. 13A and 13C ), and 0.61 nM to 4.06 nM and 1.43 nM to 1.43 nM to 22 nM for SARS-CoV-2 spike S1, respectively ( FIGS. 13B and 13D ). showed superior characteristics. The IC 50 values of each antibody are shown in Table 15.
구체적으로, 표 15에 경쟁 효소면역분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 15 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody by the competitive enzyme immunoassay.
경쟁 효소면역분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능Neutralizing ability of anti-SARS-CoV-2 spike monoclonal antibody by competitive enzyme immunoassay
항체
라이브러리antibody
library 		코팅(Coating)Coating ACE2-HisACE2-His
바인더(Binder)Binder SARS-CoV-2 RBD-mFcSARS-CoV-2 RBD-mFc SARS-CoV-2 스파이크 S1-mFcSARS-CoV-2 Spike S1-mFc
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared IC50(nM)IC 50 (nM) R-squaredR-squared
Ymax®-ABL
(A, B)Ymax ® -ABL
(A, B) 		hIgGhIgG -- -- -- --
SA3755SA3755 7.7367.736 0.99540.9954 4.0614.061 0.99840.9984
SA3779SA3779 11.2511.25 0.98080.9808 4.0314.031 0.99710.9971
SA3827SA3827 0.91360.9136 0.99850.9985 0.60970.6097 0.99220.9922
SA3830SA3830 10.2310.23 0.96480.9648 1.8561.856 0.99280.9928
SA3838SA3838 2.1962.196 0.99220.9922 1.2021.202 0.99470.9947
SA3856SA3856 25.0825.08 0.9840.984 2.1142.114 0.98380.9838
SA3902SA3902 12.4212.42 0.99250.9925 3.8533.853 0.99810.9981
ACE2ACE2 276.3276.3 0.98060.9806 75.2675.26 0.99280.9928
환자 면역
라이브러리
(C, D)patient immunity
library
(C, D) 		hIgGhIgG -- -- -- --
SA3755* SA3755 * 23.1423.14 0.99660.9966 8.2698.269 0.98030.9803
SA4040SA4040 25.2825.28 0.990.99 3.0963.096 0.98550.9855
SA4043SA4043 1.7561.756 0.99910.9991 1.4251.425 0.95840.9584
SA4044SA4044 58.2758.27 0.99240.9924 13.6413.64 0.98810.9881
SA4050SA4050 3.1443.144 0.98650.9865 1.4541.454 0.96380.9638
SA4053SA4053 40.640.6 0.94280.9428 2222 0.99140.9914
SA4055SA4055 0.14390.1439 0.9730.973 1.8761.876 0.94230.9423
SA4056SA4056 1.8581.858 0.99870.9987 1.3981.398 0.95610.9561
SA4057SA4057 4.8094.809 0.99930.9993 2.6312.631 0.98970.9897
SA3755*: Ymax®-ABL 유래 내부참조물질(internal reference)SA3755 * : Ymax ® -ABL-derived internal reference material
실시예 8.2. 세포기반 경쟁 분석법에 의한 SARS-CoV-2 스파이크 인간 단일클론 항체의 중화능 확인 Example 8.2. Confirmation of neutralizing ability of SARS-CoV-2 spike human monoclonal antibody by cell-based competition assay
인간 ACE2를 과발현하는 형질전환 세포 풀은 인간 ACE2를 포함하고 있는 pcDNA3.1 플라스미드를 HEK293E에 형질감염 시킨 후, 200 ㎎/㎖의 Zeocin(#R25001, thermo Fisher Scientific)이 들어있는 선택적 배양 배지에서 선별과정을 수행하였다. 선별과정 후 세포 풀은 염소 항-인간 ACE2 항체(#AF933, R&D system)를 이용한 FACS 분석을 통해 발현 상태를 확인하여 분리하였고, SARS-CoV-2 스파이크 인간 단일클론 항체에 의한 SARS-CoV-2 RBD와 ACE2 결합 억제 중화능 확인을 위해 사용되었다.A pool of transformed cells overexpressing human ACE2 was selected in a selective culture medium containing 200 mg/ml Zeocin (#R25001, thermo Fisher Scientific) after transfection of the pcDNA3.1 plasmid containing human ACE2 into HEK293E. The process was carried out. After the selection process, the cell pool was isolated by confirming the expression status through FACS analysis using goat anti-human ACE2 antibody (#AF933, R&D system), and SARS-CoV-2 by SARS-CoV-2 spike human monoclonal antibody It was used to confirm the neutralizing ability of RBD and ACE2 binding inhibition.
SARS-CoV-2 스파이크 인간 단일클론 항체가 SARS-CoV-2 RBD와 ACE2 결합을 억제할 수 있는지 확인하기 위해, 제조한 인간 ACE2 과발현 HEK293E 세포(HEK293E/ACE2)를 각각 시료당 3×105개로 준비하고, 20 nM(1 ㎍/㎖)의 SARS-CoV-2 RBD-mFc 단백질에 SARS-CoV-2 스파이크 인간 단일클론 항체 또는 대조로 수용체인 ACE2-His를 500 nM에서부터 1/5 희석 배수로 연속적으로 희석하여 4℃에서 30분간 반응(pre-incubation) 시켰다.To confirm that the SARS-CoV-2 spike human monoclonal antibody can inhibit SARS-CoV-2 RBD and ACE2 binding, the prepared human ACE2-overexpressing HEK293E cells (HEK293E/ACE2) were divided into 3×10 5 cells per sample, respectively. Prepared, 20 nM (1 ㎍ / ㎖) of SARS-CoV-2 RBD-mFc protein SARS-CoV-2 spike human monoclonal antibody or the receptor ACE2-His as a control, serially from 500 nM at 1/5 dilution fold. was diluted to and reacted (pre-incubated) at 4°C for 30 minutes.
그 후, 항원-항체 혼합물을 준비한 세포와 4℃에서 30분간 반응시켰다. 그 후, 세포는 2% FBS가 포함된 PBS로 3회 세척하고, SARS-CoV-2 RBD-mFc를 검출할 수 있는 FITC(fluorescein isothiocyanate) 형광물질이 결합된 항-마우스 IgG 항체(#FI-2000, Vectorlabs)를 사용하여 4℃에서 30분간 암반응 후, 동일한 세척 과정을 거쳤다. 이후 0.2 ㎖의 2% FBS가 든 PBS로 현탁시킨 후, 유세포 분석기인 CytoFLEX(Beckman coulter, USA)을 사용하여 분석하였다. Thereafter, the antigen-antibody mixture was reacted with the prepared cells at 4° C. for 30 minutes. Thereafter, the cells were washed 3 times with PBS containing 2% FBS, and an anti-mouse IgG antibody (#FI- 2000, Vectorlabs) after dark reaction at 4° C. for 30 minutes, followed by the same washing process. After suspension in PBS containing 0.2 ml of 2% FBS, analysis was performed using a flow cytometer, CytoFLEX (Beckman coulter, USA).
SARS-CoV-2 스파이크 인간 단일클론 항체의 SARS-CoV-2 RBD와 ACE2 결합을 저해하는 중화능은 ACE2 발현세포에 결합하는 SARS-CoV-2 RBD-mFc의 결합력 감소로 알 수 있고, 그 저해 정도는 항체에 의한 최대 억제의 50%에 이르는데 필요한 시료의 양(IC50)으로 나타낼 수 있다. SARS-CoV-2 RBD-mFc의 결합 정도를 확인하는 MFI(mean fluorescence intensity) 값들과 GraphPad Prism 8 소프트웨어(GraphPad Software, Inc., 미국)를 이용하여 분석한 결과, SARS-CoV-2 스파이크 인간 단일클론 항체들은 농도 의존적으로 SARS-CoV-2 RBD와 ACE2 결합을 저해함을 확인하였다(도 14a 및 도 14b). Ymax®-ABL 유래 7종(도 14a)과 환자-면역 라이브러리 유래 8종(도 14b) 항체들의 SARS-CoV-2 RBD에 대한 IC50 값은 각각 1.75 nM~3.8 nM와 1.69 nM~4.13 nM로서 모두 나노몰 수준으로 우수하였으며, 대조로 처리한 SARS-CoV-2 RBD 수용체인 ACE2에 비교하여 우월함을 확인하였다. 각 항체들의 IC50 값은 표 16과 같다.The neutralizing ability of the SARS-CoV-2 spike human monoclonal antibody to inhibit SARS-CoV-2 RBD and ACE2 binding can be seen from the decrease in the binding affinity of SARS-CoV-2 RBD-mFc binding to ACE2-expressing cells, and its inhibition The degree can be expressed as the amount of sample (IC 50 ) required to reach 50% of the maximum inhibition by the antibody. As a result of analysis using MFI (mean fluorescence intensity) values confirming the binding degree of SARS-CoV-2 RBD-mFc and GraphPad Prism 8 software (GraphPad Software, Inc., USA), SARS-CoV-2 spike human single It was confirmed that the clonal antibodies inhibited SARS-CoV-2 RBD and ACE2 binding in a concentration-dependent manner ( FIGS. 14a and 14b ). The IC 50 values for SARS-CoV-2 RBD of Ymax ® -ABL-derived 7 (Fig. 14a) and 8 (Fig. 14b) antibodies from the patient-immune library were 1.75 nM to 3.8 nM and 1.69 nM to 4.13 nM, respectively. All were excellent at the nanomolar level, and superiority was confirmed compared to ACE2, a SARS-CoV-2 RBD receptor treated as a control. The IC 50 values of each antibody are shown in Table 16.
구체적으로, 표 16에 세포기반 경쟁분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 16 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody by the cell-based competition assay.
세포기반 경쟁분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능Neutralizing ability of anti-SARS-CoV-2 spike monoclonal antibody by cell-based competition assay
항체 라이브러리antibody library 세포cell HEK293E/ACE2HEK293E/ACE2
바인더(Binder)Binder SARS-CoV-2 RBD-mFcSARS-CoV-2 RBD-mFc
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared
Ymax®-ABL
(A)Ymax ® -ABL
(A) 		hIgGhIgG -- --
SA3755SA3755 3.7973.797 0.97830.9783
SA3779SA3779 2.8392.839 0.98590.9859
SA3827SA3827 2.7372.737 0.9840.984
SA3830SA3830 2.5692.569 0.98780.9878
SA3838SA3838 3.3843.384 0.97850.9785
SA3856SA3856 2.672.67 0.98220.9822
SA3902SA3902 1.7451.745 0.97390.9739
ACE2ACE2 28.7128.71 0.99250.9925
환자 면역
라이브러리
(B)patient immunity
library
(B) 		hIgGhIgG -- --
SA3755* SA3755 * 2.2592.259 0.99560.9956
SA4040SA4040 1.5891.589 0.98840.9884
SA4043SA4043 1.6871.687 0.99660.9966
SA4044SA4044 2.2082.208 0.99940.9994
SA4050SA4050 2.9332.933 0.99370.9937
SA4053SA4053 2.2082.208 0.9980.998
SA4055SA4055 2.9052.905 0.97610.9761
SA4056SA4056 2.1942.194 0.99660.9966
SA4057SA4057 4.1284.128 0.99350.9935
ACE2ACE2 16.4216.42 0.92360.9236
SA3755*: Ymax®-ABL 유래 내부참조물질(internal reference)SA3755 * : Ymax ® -ABL-derived internal reference material
실시예 8.3. 세포내 유입 억제 분석법에 의한 SARS-CoV-2 스파이크 단일클론 항체의 중화능 확인Example 8.3. Confirmation of neutralizing ability of SARS-CoV-2 spike monoclonal antibody by intracellular influx inhibition assay
SARS-CoV-2 RBD가 세포 표면에 존재하는 ACE2와 결합하여 세포내로 유입(internalization)될 때, SARS-CoV-2 스파이크 인간 단일클론 항체가 이를 저해할 수 있는지 확인하기 위하여 카복시 말단에 녹색형광단백질(green fluorescent protein, GFP)를 융합하여 재조합한 ACE2를 과발현하는 HEK293E 세포(HEK293E/ACE2-GFP)와 인간 Fc(hFc)가 융합된 재조합 SARS-CoV-2 RBD-hFc 항원을 사용하여 Incucyte® internalization 시험법을 수행하였고, 항원의 세포 내 위치를 확인하기 위하여 Incucyte® live-cell analysis system을 이용하여 분석하였다. When SARS-CoV-2 RBD binds to ACE2 present on the cell surface and enters the cell (internalization), a green fluorescent protein at the carboxy terminus to confirm that the SARS-CoV-2 spike human monoclonal antibody can inhibit it. Incucyte ® internalization using recombinant SARS-CoV-2 RBD-hFc antigen fused with HEK293E cells (HEK293E/ACE2-GFP) overexpressing recombinant ACE2 by fusion (green fluorescent protein, GFP) and human Fc (hFc) The test method was performed and analyzed using the Incucyte ® live-cell analysis system to confirm the intracellular localization of the antigen.
96-웰 세포 배양 마이크로 플레이트(#CT-3595, Corning)에 세포를 웰 당 2×104개로 50 ㎕에 시딩(seeding)하여 18~24시간 동안 배양하였다. SARS-CoV-2 RBD-hFC 항원의 Fc 부분을 Incucyte® Human FabFluor-pH Red Antibody Labeling Reagent를 이용하여 1:2 몰비(molar ratio)로 37℃에서 15분간 암반응시키고, 이를 형광물질로 표지한 후, 2 mM에서부터 1/5 희석 배수로 연속적으로 희석한 SARS-CoV-2 스파이크 인간 단일클론 항체와 혼합하여 3분간 반응시킨 뒤 세포에 처리하였다. Incucyte® Human FabFluor-pH Red Antibody Labeling Reagent가 접합된 SARS-CoV-2 RBD-hFc는 세포 표면 ACE2와 결합하여 세포내로 유입(internalization)되고 세포내 낮은 pH 조건에서 적색(red) 형광을 발광하여 세포내 유입 유무 및 정도를 확인 가능하다. 세포는 IncuCyte ZOOM HD/2CLR System(Essen Biosciences, 미국)를 이용하여 15분~30분 간격으로 24시간 반복하여 현시(phase), 녹색(green), 적색(red) 3가지 채널을 모두 이미징하고, 'Total Red Object Area(㎛2/well)'로 분석하였다. In a 96-well cell culture microplate (#CT-3595, Corning), cells were seeded in 50 μl at 2×10 4 cells per well and cultured for 18 to 24 hours. The Fc portion of the SARS-CoV-2 RBD-hFC antigen was dark-reacted at 37°C for 15 minutes at a molar ratio of 1:2 using Incucyte ® Human FabFluor-pH Red Antibody Labeling Reagent, followed by labeling with a fluorescent material. , was mixed with SARS-CoV-2 spike human monoclonal antibody serially diluted from 2 mM to 1/5 dilution factor, reacted for 3 minutes, and then treated with cells. SARS-CoV-2 RBD-hFc conjugated with Incucyte ® Human FabFluor-pH Red Antibody Labeling Reagent binds to cell surface ACE2 and enters the cell (internalization). It is possible to check the presence and extent of inflow. Cells were imaged using the IncuCyte ZOOM HD/2CLR System (Essen Biosciences, USA) for 24 hours at intervals of 15 to 30 minutes, and all three channels of phase, green, and red were imaged, 'Total Red Object Area (㎛ 2 /well)' was analyzed.
SARS-CoV-2 스파이크 인간 단일클론 항체가 ACE2를 통한 SARS-CoV-2 RBD의 세포 내 유입을 저해하는지는 세포내에서 SARS-CoV-2 RBD 항원의 적색 형광물질 감소로 알 수 있으며, 그 저해 정도는 항체에 의한 최대 억제의 50%에 이르는데 필요한 시료의 양(IC50)으로 나타낼 수 있고, GraphPad Prism 8 소프트웨어를 이용하여 분석하였다.Whether the SARS-CoV-2 spike human monoclonal antibody inhibits the SARS-CoV-2 RBD influx through ACE2 can be known by reducing the red fluorescence of the SARS-CoV-2 RBD antigen in the cell, and the inhibition The degree can be expressed as the amount of sample required to reach 50% of the maximum inhibition by the antibody (IC 50 ), and was analyzed using GraphPad Prism 8 software.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체는 농도 의존적으로 ACE2와의 결합을 통한 SARS-CoV-2 RBD의 세포내 유입을 효과적으로 저해함을 확인하였다(도 15a 및 도 15b). Ymax®-ABL 유래 7종(도 15a)과 환자-면역 라이브러리 유래 8종(도 15b) 항체들의 SARS-CoV-2 RBD에 대한 IC50 값은 각각 11.92 nM~40.02 nM와 12.78 nM~62.72 nM였으며, 각 항체들의 IC50 값은 표 17과 같다.As a result, it was confirmed that the SARS-CoV-2 spike human monoclonal antibody effectively inhibited the intracellular influx of SARS-CoV-2 RBD through binding to ACE2 in a concentration-dependent manner ( FIGS. 15A and 15B ). The IC 50 values for SARS-CoV-2 RBD of Ymax ® -ABL-derived 7 ( FIG. 15A ) and 8 ( FIG. 15B ) antibodies from the patient-immune library were 11.92 nM to 40.02 nM and 12.78 nM to 62.72 nM, respectively. , IC 50 values of each antibody are shown in Table 17.
구체적으로, 표 17에 세포내 유입 억제 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 17 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody by the intracellular influx inhibition assay.
세포내 유입 억제 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능Neutralizing ability of anti-SARS-CoV-2 spike monoclonal antibody by intracellular uptake inhibition assay
항체 라이브러리antibody library 세포cell HEK293E/ACE2-GFPHEK293E/ACE2-GFP
바인더(Binder)Binder SARS-CoV-2 RBD-hFcSARS-CoV-2 RBD-hFc
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared
Ymax®-ABL
(A)Ymax ® -ABL
(A) 		hIgGhIgG -- --
SA3755SA3755 40.0240.02 0.97010.9701
SA3779SA3779 18.9318.93 0.97580.9758
SA3827SA3827 12.8612.86 0.93860.9386
SA3830SA3830 13.5313.53 0.97850.9785
SA3838SA3838 36.0836.08 0.98530.9853
SA3856SA3856 11.9211.92 0.94640.9464
SA3902SA3902 14.8914.89 0.97990.9799
환자 면역
라이브러리
(B)patient immunity
library
(B) 		hIgGhIgG -- --
SA4040SA4040 30.8430.84 0.96690.9669
SA4043SA4043 12.7812.78 0.97930.9793
SA4044SA4044 39.1739.17 0.99220.9922
SA4050SA4050 36.9236.92 0.94620.9462
SA4053SA4053 62.7262.72 0.98680.9868
SA4055SA4055 37.137.1 0.96460.9646
SA4056SA4056 28.1128.11 0.9770.977
SA4057SA4057 28.1428.14 0.97630.9763
실시예 8.4. SARS-CoV-2 바이러스를 사용한 미세중화분석법에 의한 SARS-CoV-2 스파이크 단일클론 항체의 중화능 확인Example 8.4. Confirmation of neutralizing ability of SARS-CoV-2 spike monoclonal antibody by microneutralization assay using SARS-CoV-2 virus
SARS-CoV-2 바이러스에 대한 항체 중화능은 아프리카 초록원숭이 신장 세포(Africa green monkey kidney cell line)인 Vero 세포와 함께 미세중화분석법(microneutralization assay)를 통해 확인하였다. Antibody neutralization ability against SARS-CoV-2 virus was confirmed by microneutralization assay together with Vero cells, an African green monkey kidney cell line.
96-웰 세포 배양 마이크로 플레이트에 Vero 세포를 웰 당 2.5×104개로 12~18시간 동안 배양하여 준비하고, SARS-CoV-2 인간 단일클론 항체를 500 nM에서부터 1/4 희석 배수로 연속적으로 희석하여 10,000 PFU의 SARS-CoV-2 바이러스와 1시간 반응시킨 후, 준비한 세포에 웰 당 5,000 PFU로 48 시간 동안 감염시켰다. 그 후 세포는 PBS로 1회 세척하고 4℃에서 4% 파라포름아마이드(paraformamide)로 고정한 다음 PBS로 세척 후, 0.05% 크리스탈 바이올렛(crystal violet)/메탄올(methanol) 용액을 각 웰에 넣어 항체 중화능에 의해 바이러스에 대한 감염세포 병변(cytopathic effect, CPE)을 보이지 않는 세포들을 염색하였다. 얻어진 이미지는 NIH(National Institute of Health)　ImageJ 프로그램을 이용하여 영역 세기(area intensity)를 구하고, % 생존율(viability)/억제율(inhibition)을 계산하여 GrapPad Prism 5 소프트웨어를 통해 SARS-CoV-2 바이러스에 대한 항체 중화능 IC50 값을 계산하였다.Prepared by culturing Vero cells in a 96-well cell culture microplate at 2.5×10 4 cells per well for 12 to 18 hours, and serially diluting SARS-CoV-2 human monoclonal antibody from 500 nM to 1/4 dilutions. After reacting with 10,000 PFU of SARS-CoV-2 virus for 1 hour, the prepared cells were infected at 5,000 PFU per well for 48 hours. After that, the cells were washed once with PBS, fixed with 4% paraformamide at 4°C, washed with PBS, and then added to each well with 0.05% crystal violet/methanol solution to neutralize the antibody Cells that showed no cytopathic effect (CPE) were stained by the assay. The obtained image was obtained by obtaining the area intensity using the National Institute of Health (NIH) ImageJ program, and calculating the % viability/inhibition to SARS-CoV-2 virus through GrapPad Prism 5 software. Antibody neutralizing capacity IC 50 values were calculated.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체는 농도 의존적으로 Live SARS-CoV-2 바이러스에 대한 감염을 효과적으로 저해함을 확인하였다(도 16a 및 도 16b). Ymax®-ABL 유래 7종(도 16a)과 환자-면역 라이브러리 유래 8종(도 16b) 항체들의 SARS-CoV-2 바이러스에 대한 IC50 값은 각각 8.736 nM~231 nM와 0.7102 nM~346 nM였으며, 각 항체들의 IC50 값은 표 18과 같다.As a result, it was confirmed that the SARS-CoV-2 spike human monoclonal antibody effectively inhibited infection with live SARS-CoV-2 virus in a concentration-dependent manner ( FIGS. 16A and 16B ). The IC 50 values of the Ymax ® -ABL-derived 7 ( FIG. 16A ) and 8 ( FIG. 16B ) antibodies from the patient-immune library against SARS-CoV-2 virus were 8.736 nM to 231 nM and 0.7102 nM to 346 nM, respectively. , IC 50 values of each antibody are shown in Table 18.
구체적으로, 표 18에 미세중화 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 18 summarizes the results of confirming the neutralization ability of the anti-SARS-CoV-2 spike monoclonal antibody by the microneutralization assay.
미세중화 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체의 중화능Neutralization capacity of anti-SARS-CoV-2 spike monoclonal antibody by microneutralization assay
항체 라이브러리antibody library 바이러스virus SARS-CoV-2SARS-CoV-2
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared
Ymax®-ABL
(A)Ymax ® -ABL
(A) 		hIgGhIgG -- --
SA3755SA3755 8.7368.736 0.96980.9698
SA3779SA3779 56.7756.77 0.96820.9682
SA3827SA3827 8.9188.918 0.96950.9695
SA3830SA3830 231231 0.86590.8659
SA3838SA3838 9.3999.399 0.90970.9097
SA3856SA3856 136.7136.7 0.92260.9226
SA3902SA3902 37.3237.32 0.97460.9746
환자 면역
라이브러리
(B)patient immunity
library
(B) 		SA4040SA4040 8.1398.139 0.89820.8982
SA4043SA4043 12.4612.46 0.97640.9764
SA4044SA4044 80.4880.48 0.83980.8398
SA4050SA4050 0.71020.7102 0.96510.9651
SA4053SA4053 346346 0.65460.6546
SA4055SA4055 7.0047.004 0.96860.9686
SA4056SA4056 11.2611.26 0.96910.9691
SA4057SA4057 37.5237.52 0.87890.8789
실시예 9. SARS-CoV-2 스파이크 인간 단일클론 항체 변이체의 중화능 평가Example 9. Evaluation of neutralizing ability of SARS-CoV-2 spike human monoclonal antibody variants
실시예 9.1. 경쟁 효소면역분석법에 의한 SARS-CoV-2 스파이크 인간 단일클론 항체 변이체의 중화능 확인 Example 9.1. Confirmation of neutralizing ability of SARS-CoV-2 spike human monoclonal antibody variants by competitive enzyme immunoassay
SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들이 ACE2와 결합하는 SARS-CoV-2 RBD 또는 SARS-CoV-2 스파이크 S1 결합을 저해할 수 있는지 확인하기 위해 실시예 8.1.과 같은 방법으로 경쟁 효소면역분석법을 수행하였다. In order to determine whether SARS-CoV-2 spike human monoclonal antibody variants can inhibit SARS-CoV-2 RBD or SARS-CoV-2 spike S1 binding binding to ACE2, competitive enzyme immunization was performed in the same manner as in Example 8.1. The assay was performed.
실험 결과로서, SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들(SA4079, SA4086, SA4114, SA4118)은 농도 의존적으로 ACE2와 SARS-CoV-2 RBD 또는 SARS-CoV-2 스파이크 S1 결합을 저해함을 확인하였으며, 각각의 모항체와 유사 또는 우월한 중화 효능을 보였다(도 17a 및 도 17b). SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들의 SARS-CoV-2 RBD에 대한 IC50 값은 0.7086 nM~3.625 nM(도 17a), SARS-CoV-2 스파이크 S1에 대해서는 1.194 nM~2.711 nM 사이였으며(도 17b), 각 모항체와 비교한 항체 변이체들의 IC50 값은 표 19와 같다.As an experimental result, SARS-CoV-2 spike human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) inhibited ACE2 and SARS-CoV-2 RBD or SARS-CoV-2 spike S1 binding in a concentration-dependent manner. It was confirmed, and each parent antibody showed similar or superior neutralizing efficacy ( FIGS. 17A and 17B ). The IC 50 values for SARS-CoV-2 RBD of SARS-CoV-2 spike human monoclonal antibody variants were between 0.7086 nM and 3.625 nM ( FIG. 17A ) and between 1.194 nM and 2.711 nM for SARS-CoV-2 spike S1. (FIG. 17b), IC 50 values of the antibody variants compared with each parent antibody are shown in Table 19.
구체적으로, 표 19에 경쟁 효소면역분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 19 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody mutant by the competitive enzyme immunoassay.
경쟁 효소면역분석법법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능Neutralizing ability of anti-SARS-CoV-2 spike monoclonal antibody variants by competitive enzyme immunoassay
친화도 성숙
(Affinity maturation, AM)Affinity Maturity
(Affinity maturation, AM) 		코팅(Coating)Coating ACE2-HisACE2-His
바인더(Binder)Binder SARS-CoV-2 RBD-mFc(A)SARS-CoV-2 RBD-mFc(A) SARS-CoV-2 스파이크 S1-mFc(B)SARS-CoV-2 Spike S1-mFc(B)
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared IC50(nM)IC 50 (nM) R-squaredR-squared
hIgGhIgG -- -- -- --
ParentParent SA3755SA3755 5.7075.707 0.97400.9740 4.6674.667 0.99790.9979
SA3755AMSA3755AM SA4079SA4079 3.6253.625 0.98790.9879 2.7112.711 0.98820.9882
ParentParent SA3779SA3779 7.9567.956 0.94650.9465 3.5943.594 0.99770.9977
SA3779AMSA3779AM SA4086SA4086 1.2361.236 0.97970.9797 1.2811.281 0.98090.9809
ParentParent SA3827SA3827 0.32100.3210 0.99690.9969 0.90040.9004 0.98070.9807
SA3827AMSA3827AM SA4114SA4114 0.36420.3642 0.99800.9980 1.1941.194 0.96930.9693
ParentParent SA3838SA3838 1.6161.616 0.99820.9982 2.2472.247 0.99440.9944
SA3838AMSA3838AM SA4118SA4118 0.70860.7086 0.99650.9965 2.2762.276 0.98780.9878
실시예 9.2. 세포기반 경쟁 분석법에 의한 SARS-CoV-2 스파이크 인간 단일클론 항체 변이체의 중화능 확인Example 9.2. Confirmation of neutralizing ability of SARS-CoV-2 spike human monoclonal antibody variants by cell-based competition assay
SARS-CoV-2 스파이크 인간 단일클론 항체 변이체가 SARS-CoV-2 RBD와 ACE2 결합을 억제할 수 있는지 확인하기 위해, 실시예 8.2.와 같이 인간 ACE2 과발현 HEK293E 세포(HEK293E/ACE2)와 SARS-CoV-2 RBD-mFc 단백질을 이용한 세포기반 경쟁 분석법을 수행하고 분석하였다. To determine whether the SARS-CoV-2 spike human monoclonal antibody variant can inhibit SARS-CoV-2 RBD and ACE2 binding, as in Example 8.2, human ACE2 overexpressing HEK293E cells (HEK293E/ACE2) and SARS-CoV -2 A cell-based competition assay using RBD-mFc protein was performed and analyzed.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들(SA4079, SA4086, SA4114, SA4118)은 농도 의존적으로 SARS-CoV-2 RBD와 ACE2 결합을 저해하였으며, 각각 모항체와 비교에서 유사 또는 우월한 중화능을 보였다(도 14c). 각 항체 변이체들의 SARS-CoV-2 RBD에 대한 IC50 값은 2.346 nM~1.705 nM 사이로 모두 나노몰 수준으로 우수하였으며, 대조로 처리한 SARS-CoV-2 RBD 수용체인 ACE2에 비교하여 매우 우월함을 확인하였다. 각 모항체와 비교한 항체 변이체들의 IC50 값은 표 20과 같다.As a result, SARS-CoV-2 spike human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) inhibited SARS-CoV-2 RBD and ACE2 binding in a concentration-dependent manner, and were similar or superior to the parent antibody, respectively. It showed neutralizing ability (Fig. 14c). The IC 50 values for SARS-CoV-2 RBD of each antibody variant were all excellent at nanomolar levels between 2.346 nM and 1.705 nM, and it was confirmed that they were very superior compared to ACE2, the SARS-CoV-2 RBD receptor treated as a control. did Table 20 shows the IC 50 values of the antibody variants compared to each parent antibody.
구체적으로, 표 20에 세포기반 경쟁분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 20 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody variants by the cell-based competition assay.
세포기반 경쟁분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능Neutralizing ability of anti-SARS-CoV-2 spike monoclonal antibody variants by cell-based competition assay
친화도 성숙
(Affinity maturation, AM)Affinity Maturity
(Affinity maturation, AM) 		세포cell HEK293E/ACE2HEK293E/ACE2
바인더(Binder)Binder SARS-CoV-2 RBD-mFcSARS-CoV-2 RBD-mFc
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared
hIgGhIgG -- --
ParentParent SA3755SA3755 3.0943.094 0.99430.9943
SA3755AMSA3755AM SA4079SA4079 2.3462.346 0.99250.9925
ParentParent SA3779SA3779 2.6462.646 0.99550.9955
SA3779AMSA3779AM SA4086SA4086 2.4712.471 0.99230.9923
ParentParent SA3827SA3827 2.7122.712 0.98980.9898
SA3827AMSA3827AM SA4114SA4114 2.3942.394 0.98960.9896
ParentParent SA3838SA3838 2.9892.989 0.99230.9923
SA3838AMSA3838AM SA4118SA4118 2.7052.705 0.99310.9931
ACE2ACE2 52.0552.05 0.99350.9935
실시예 9.3. 세포내 유입 억제 분석법에 의한 SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능 확인Example 9.3. Confirmation of neutralizing ability of SARS-CoV-2 spike monoclonal antibody variants by intracellular entry inhibition assay
SARS-CoV-2 RBD가 세포 표면에 존재하는 ACE2와 결합하여 세포내로 유입(internalization)될 때, SARS-CoV-2 스파이크 인간 단일클론 항체 변이체가 각 모항체와 같이 이를 저해할 수 있는지 확인하기 위하여 실시예 8.3.과 같이 재조합한 ACE2-GFP를 과발현하는 HEK293E 세포(HEK293E/ACE2-GFP)와 SARS-CoV-2 RBD-hFc 항원을 이용한 Incucyte® internalization 시험법을 수행하였다.When SARS-CoV-2 RBD binds to ACE2 present on the cell surface and enters the cell (internalization), the SARS-CoV-2 spike human monoclonal antibody variant can inhibit it like each parent antibody. As in Example 8.3. HEK293E cells overexpressing recombinant ACE2-GFP (HEK293E/ACE2-GFP) and Incucyte ® internalization assay using the SARS-CoV-2 RBD-hFc antigen was performed.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체 변이체들(SA4079, SA4086, SA4114, SA4118)은 각 모항체와 같이 농도 의존적으로 ACE2와의 결합을 통한 SARS-CoV-2 RBD의 세포내 유입을 효과적으로 저해함을 확인하였다(도 18). 항체 변이체들의 SARS-CoV-2 RBD에 대한 IC50 값은 24.06 nM~39.97 nM 사이였으며, 각 모항체와 비교한 항체 변이체들의 IC50 값은 표 21과 같다.As a result, SARS-CoV-2 spiked human monoclonal antibody variants (SA4079, SA4086, SA4114, SA4118) effectively inhibited the intracellular influx of SARS-CoV-2 RBD through binding to ACE2 like each parent antibody in a concentration-dependent manner. It was confirmed that inhibition (FIG. 18). The IC 50 values for SARS-CoV-2 RBD of the antibody variants were between 24.06 nM and 39.97 nM, and the IC 50 values of the antibody variants compared to each parent antibody are shown in Table 21.
구체적으로, 표 21에 세포내 유입 억제 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 21 summarizes the results of confirming the neutralizing ability of the anti-SARS-CoV-2 spike monoclonal antibody variant by the intracellular influx inhibition assay.
세포내 유입 억제 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능Neutralizing ability of anti-SARS-CoV-2 spike monoclonal antibody variants by endocytosis inhibition assay
친화도 성숙
(Affinity maturation, AM)Affinity Maturity
(Affinity maturation, AM) 		세포cell HEK293E/ACE2-GFPHEK293E/ACE2-GFP
바인더(Binder)Binder SARS-CoV-2 RBD-hFcSARS-CoV-2 RBD-hFc
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared
hIgGhIgG -- --
ParentParent SA3755SA3755 35.835.8 0.99050.9905
SA3755AMSA3755AM SA4079SA4079 39.9739.97 0.9820.982
ParentParent SA3779SA3779 27.3427.34 0.99040.9904
SA3779AMSA3779AM SA4086SA4086 27.1927.19 0.97960.9796
ParentParent SA3827SA3827 18.2818.28 0.98970.9897
SA3827AMSA3827AM SA4114SA4114 24.0624.06 0.98320.9832
ParentParent SA3838SA3838 42.8342.83 0.98040.9804
SA3838AMSA3838AM SA4118SA4118 31.4131.41 0.98030.9803
실시예 9.4. SARS-CoV-2 바이러스를 사용한 미세중화분석법에 의한 SARS-CoV-2 스파이크 인간 단일클론 항체 변이체의 중화능 확인Example 9.4. Confirmation of neutralizing ability of SARS-CoV-2 spike human monoclonal antibody variants by microneutralization assay using SARS-CoV-2 virus
SARS-CoV-2 스파이크 인간 단일클론 항체 변이체가 각 모항체와 같이 SARS-CoV-2 바이러스에 대한 중화능을 갖는지 확인하기 위하여 실시예 8.4.와 같이 Live SARS-CoV-2 바이러스를 Vero 세포에 72 시간동안 감염 시킨 후 미세중화 분석법(microneutralization assay)을 수행하여 확인하였다.In order to check whether the SARS-CoV-2 spike human monoclonal antibody variant has neutralizing ability against SARS-CoV-2 virus like each parent antibody, Live SARS-CoV-2 virus was injected into Vero cells as in Example 8.4. After infection for a period of time, it was confirmed by performing a microneutralization assay.
그 결과, SARS-CoV-2 스파이크 인간 단일클론 항체 4종(SA3755, SA3779, SA3827, SA3838)을 최적화하여 선별한 항체 변이체들(SA4079, SA4086, SA4114, SA4118)은 각 모항체와 같이 농도 의존적으로 SARS-CoV-2 바이러스의 감염을 효과적으로 중화함을 확인하였다(도 19a 및 도 19b). 항체 변이체들의 SARS-CoV-2 바이러스에 대한 IC50 값은 2.741 nM~12.83 nM 사이였으며, 각 모항체와 비교한 항체 변이체들의 IC50 값은 표 22와 같다.As a result, the antibody variants (SA4079, SA4086, SA4114, SA4118) selected by optimizing the four SARS-CoV-2 spike human monoclonal antibodies (SA3755, SA3779, SA3827, SA3838) were concentration-dependent like each parent antibody. It was confirmed that it effectively neutralized the infection of SARS-CoV-2 virus ( FIGS. 19a and 19b ). The IC 50 values of the antibody variants against SARS-CoV-2 virus were between 2.741 nM and 12.83 nM, and the IC 50 values of the antibody variants compared to each parent antibody are shown in Table 22.
구체적으로, 표 22에 미세중화 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능 확인 결과를 정리하여 나타내었다.Specifically, Table 22 summarizes the results of confirming the neutralization ability of the anti-SARS-CoV-2 spike monoclonal antibody variant by the microneutralization assay.
미세중화 분석법에 의한 항-SARS-CoV-2 스파이크 단일클론 항체 변이체의 중화능Neutralization capacity of anti-SARS-CoV-2 spike monoclonal antibody variants by microneutralization assay
친화도 성숙
(Affinity maturation, AM)Affinity Maturity
(Affinity maturation, AM) 		바이러스virus SARS-CoV-2SARS-CoV-2
항체antibody IC50(nM)IC 50 (nM) R-squaredR-squared
-- hIgGhIgG -- --
-- SA4055* SA4055 * 3.2243.224 0.95910.9591
ParentParent SA3755SA3755 5.1155.115 0.95750.9575
SA3755AMSA3755AM SA4079SA4079 2.7412.741 0.97120.9712
ParentParent SA3779SA3779 45.5745.57 0.95210.9521
SA3779AMSA3779AM SA4086SA4086 12.4512.45 0.93140.9314
-- hIgGhIgG -- --
-- SA4055* SA4055 * 4.5114.511 0.93930.9393
ParentParent SA3827SA3827 7.9367.936 0.92400.9240
SA3827AMSA3827AM SA4114SA4114 12.83012.830 0.93820.9382
ParentParent SA3838SA3838 13.51013.510 0.97360.9736
SA3838AMSA3838AM SA4118SA4118 8.2898.289 0.91890.9189
SA4055*: 내부참조물질(internal reference)SA4055 * : internal reference
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it will be clear to those of ordinary skill in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (14)

  1. 서열번호 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79 및 85로 구성된 군에서 선택되는 중쇄(heavy chain) CDR1;a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79 and 85;
    서열번호 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80 및 86으로 구성된 군에서 선택되는 중쇄 CDR2;a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80 and 86;
    서열번호 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81 및 87로 구성된 군에서 선택되는 중쇄 CDR3를 포함하는 중쇄 가변영역; 및a heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81 and 87; and
    서열번호 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 271, 274, 277 및 280으로 구성된 군에서 선택되는 경쇄(light chain) CDR1;A light chain selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 271, 274, 277 and 280 CDR1;
    서열번호 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 272, 275, 278 및 281로 구성된 군에서 선택되는 경쇄 CDR2;a light chain CDR2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 272, 275, 278 and 281;
    서열번호 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 273, 276, 279 및 282로 구성된 군에서 선택되는 경쇄 CDR3를 포함하는 경쇄 가변영역을 포함하는,SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 273, 276, 279 and 282 comprising a light chain CDR3 selected from the group consisting of comprising a light chain variable region,
    SARS-CoV-2 스파이크(spike) 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편.An antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 spike protein.
  2. 제1항에 있어서, The method of claim 1,
    하기 군에서 선택되는 어느 하나의 CDR 조합을 포함하는, SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편:An antibody or antigen-binding fragment thereof that specifically binds to a SARS-CoV-2 spike protein, comprising any one CDR combination selected from the following group:
    (1) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 서열번호 3의 중쇄 CDR3, 서열번호 4의 경쇄 CDR1, 서열번호 5의 경쇄 CDR2, 서열번호 6의 경쇄 CDR3;(1) heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, light chain CDR3 of SEQ ID NO: 6;
    (2) 서열번호 7의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 서열번호 9의 중쇄 CDR3, 서열번호 10의 경쇄 CDR1, 서열번호 11의 경쇄 CDR2, 서열번호 12의 경쇄 CDR3;(2) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 10, light chain CDR2 of SEQ ID NO: 11, light chain CDR3 of SEQ ID NO: 12;
    (3) 서열번호 13의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 서열번호 15의 중쇄 CDR3, 서열번호 16의 경쇄 CDR1, 서열번호 17의 경쇄 CDR2, 서열번호 18의 경쇄 CDR3;(3) heavy chain CDR1 of SEQ ID NO: 13, heavy chain CDR2 of SEQ ID NO: 14, heavy chain CDR3 of SEQ ID NO: 15, light chain CDR1 of SEQ ID NO: 16, light chain CDR2 of SEQ ID NO: 17, light chain CDR3 of SEQ ID NO: 18;
    (4) 서열번호 19의 중쇄 CDR1, 서열번호 20의 중쇄 CDR2, 서열번호 21의 중쇄 CDR3, 서열번호 22의 경쇄 CDR1, 서열번호 23의 경쇄 CDR2, 서열번호 24의 경쇄 CDR3;(4) heavy chain CDR1 of SEQ ID NO: 19, heavy chain CDR2 of SEQ ID NO: 20, heavy chain CDR3 of SEQ ID NO: 21, light chain CDR1 of SEQ ID NO: 22, light chain CDR2 of SEQ ID NO: 23, light chain CDR3 of SEQ ID NO: 24;
    (5) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2, 서열번호 27의 중쇄 CDR3, 서열번호 28의 경쇄 CDR1, 서열번호 29의 경쇄 CDR2, 서열번호 30의 경쇄 CDR3;(5) heavy chain CDR1 of SEQ ID NO: 25, heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 28, light chain CDR2 of SEQ ID NO: 29, light chain CDR3 of SEQ ID NO: 30;
    (6) 서열번호 31의 중쇄 CDR1, 서열번호 32의 중쇄 CDR2, 서열번호 33의 중쇄 CDR3, 서열번호 34의 경쇄 CDR1, 서열번호 35의 경쇄 CDR2, 서열번호 36의 경쇄 CDR3;(6) heavy chain CDR1 of SEQ ID NO: 31, heavy chain CDR2 of SEQ ID NO: 32, heavy chain CDR3 of SEQ ID NO: 33, light chain CDR1 of SEQ ID NO: 34, light chain CDR2 of SEQ ID NO: 35, light chain CDR3 of SEQ ID NO: 36;
    (7) 서열번호 37의 중쇄 CDR1, 서열번호 38의 중쇄 CDR2, 서열번호 39의 중쇄 CDR3, 서열번호 40의 경쇄 CDR1, 서열번호 41의 경쇄 CDR2, 서열번호 42의 경쇄 CDR3;(7) heavy chain CDR1 of SEQ ID NO: 37, heavy chain CDR2 of SEQ ID NO: 38, heavy chain CDR3 of SEQ ID NO: 39, light chain CDR1 of SEQ ID NO: 40, light chain CDR2 of SEQ ID NO: 41, light chain CDR3 of SEQ ID NO: 42;
    (8) 서열번호 43의 중쇄 CDR1, 서열번호 44의 중쇄 CDR2, 서열번호 45의 중쇄 CDR3, 서열번호 46의 경쇄 CDR1, 서열번호 47의 경쇄 CDR2, 서열번호 48의 경쇄 CDR3;(8) heavy chain CDR1 of SEQ ID NO: 43, heavy chain CDR2 of SEQ ID NO: 44, heavy chain CDR3 of SEQ ID NO: 45, light chain CDR1 of SEQ ID NO: 46, light chain CDR2 of SEQ ID NO: 47, light chain CDR3 of SEQ ID NO: 48;
    (9) 서열번호 49의 중쇄 CDR1, 서열번호 50의 중쇄 CDR2, 서열번호 51의 중쇄 CDR3, 서열번호 52의 경쇄 CDR1, 서열번호 53의 경쇄 CDR2, 서열번호 54의 경쇄 CDR3;(9) heavy chain CDR1 of SEQ ID NO: 49, heavy chain CDR2 of SEQ ID NO: 50, heavy chain CDR3 of SEQ ID NO: 51, light chain CDR1 of SEQ ID NO: 52, light chain CDR2 of SEQ ID NO: 53, light chain CDR3 of SEQ ID NO: 54;
    (10) 서열번호 55의 중쇄 CDR1, 서열번호 56의 중쇄 CDR2, 서열번호 57의 중쇄 CDR3, 서열번호 58의 경쇄 CDR1, 서열번호 59의 경쇄 CDR2, 서열번호 60의 경쇄 CDR3;(10) heavy chain CDR1 of SEQ ID NO: 55, heavy chain CDR2 of SEQ ID NO: 56, heavy chain CDR3 of SEQ ID NO: 57, light chain CDR1 of SEQ ID NO: 58, light chain CDR2 of SEQ ID NO: 59, light chain CDR3 of SEQ ID NO: 60;
    (11) 서열번호 61의 중쇄 CDR1, 서열번호 62의 중쇄 CDR2, 서열번호 63의 중쇄 CDR3, 서열번호 64의 경쇄 CDR1, 서열번호 65의 경쇄 CDR2, 서열번호 66의 경쇄 CDR3;(11) heavy chain CDR1 of SEQ ID NO: 61, heavy chain CDR2 of SEQ ID NO: 62, heavy chain CDR3 of SEQ ID NO: 63, light chain CDR1 of SEQ ID NO: 64, light chain CDR2 of SEQ ID NO: 65, light chain CDR3 of SEQ ID NO: 66;
    (12) 서열번호 67의 중쇄 CDR1, 서열번호 68의 중쇄 CDR2, 서열번호 69의 중쇄 CDR3, 서열번호 70의 경쇄 CDR1, 서열번호 71의 경쇄 CDR2, 서열번호 72의 경쇄 CDR3;(12) heavy chain CDR1 of SEQ ID NO: 67, heavy chain CDR2 of SEQ ID NO: 68, heavy chain CDR3 of SEQ ID NO: 69, light chain CDR1 of SEQ ID NO: 70, light chain CDR2 of SEQ ID NO: 71, light chain CDR3 of SEQ ID NO: 72;
    (13) 서열번호 73의 중쇄 CDR1, 서열번호 74의 중쇄 CDR2, 서열번호 75의 중쇄 CDR3, 서열번호 76의 경쇄 CDR1, 서열번호 77의 경쇄 CDR2, 서열번호 78의 경쇄 CDR3;(13) heavy chain CDR1 of SEQ ID NO: 73, heavy chain CDR2 of SEQ ID NO: 74, heavy chain CDR3 of SEQ ID NO: 75, light chain CDR1 of SEQ ID NO: 76, light chain CDR2 of SEQ ID NO: 77, light chain CDR3 of SEQ ID NO: 78;
    (14) 서열번호 79의 중쇄 CDR1, 서열번호 80의 중쇄 CDR2, 서열번호 81의 중쇄 CDR3, 서열번호 82의 경쇄 CDR1, 서열번호 83의 경쇄 CDR2, 서열번호 84의 경쇄 CDR3;(14) heavy chain CDR1 of SEQ ID NO: 79, heavy chain CDR2 of SEQ ID NO: 80, heavy chain CDR3 of SEQ ID NO: 81, light chain CDR1 of SEQ ID NO: 82, light chain CDR2 of SEQ ID NO: 83, light chain CDR3 of SEQ ID NO: 84;
    (15) 서열번호 85의 중쇄 CDR1, 서열번호 86의 중쇄 CDR2, 서열번호 87의 중쇄 CDR3, 서열번호 88의 경쇄 CDR1, 서열번호 89의 경쇄 CDR2, 서열번호 90의 경쇄 CDR3;(15) heavy chain CDR1 of SEQ ID NO: 85, heavy chain CDR2 of SEQ ID NO: 86, heavy chain CDR3 of SEQ ID NO: 87, light chain CDR1 of SEQ ID NO: 88, light chain CDR2 of SEQ ID NO: 89, light chain CDR3 of SEQ ID NO: 90;
    (16) 서열번호 1의 중쇄 CDR1, 서열번호 2의 중쇄 CDR2, 서열번호 3의 중쇄 CDR3, 서열번호 271의 경쇄 CDR1, 서열번호 272의 경쇄 CDR2, 서열번호 273의 경쇄 CDR3;(16) heavy chain CDR1 of SEQ ID NO: 1, heavy chain CDR2 of SEQ ID NO: 2, heavy chain CDR3 of SEQ ID NO: 3, light chain CDR1 of SEQ ID NO: 271, light chain CDR2 of SEQ ID NO: 272, light chain CDR3 of SEQ ID NO: 273;
    (17) 서열번호 7의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 서열번호 9의 중쇄 CDR3, 서열번호 274의 경쇄 CDR1, 서열번호 275의 경쇄 CDR2, 서열번호 276의 경쇄 CDR3;(17) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO: 8, heavy chain CDR3 of SEQ ID NO: 9, light chain CDR1 of SEQ ID NO: 274, light chain CDR2 of SEQ ID NO: 275, light chain CDR3 of SEQ ID NO: 276;
    (18) 서열번호 13의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 서열번호 15의 중쇄 CDR3, 서열번호 277의 경쇄 CDR1, 서열번호 278의 경쇄 CDR2, 서열번호 279의 경쇄 CDR3; 및 (18) heavy chain CDR1 of SEQ ID NO: 13, heavy chain CDR2 of SEQ ID NO: 14, heavy chain CDR3 of SEQ ID NO: 15, light chain CDR1 of SEQ ID NO: 277, light chain CDR2 of SEQ ID NO: 278, light chain CDR3 of SEQ ID NO: 279; and
    (19) 서열번호 25의 중쇄 CDR1, 서열번호 26의 중쇄 CDR2, 서열번호 27의 중쇄 CDR3, 서열번호 280의 경쇄 CDR1, 서열번호 281의 경쇄 CDR2, 서열번호 282의 경쇄 CDR3.(19) heavy chain CDR1 of SEQ ID NO: 25, heavy chain CDR2 of SEQ ID NO: 26, heavy chain CDR3 of SEQ ID NO: 27, light chain CDR1 of SEQ ID NO: 280, light chain CDR2 of SEQ ID NO: 281, light chain CDR3 of SEQ ID NO: 282.
  3. 제1항에 있어서, The method of claim 1,
    서열번호 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237 및 239로 이루어진 군에서 선택되는 어느 하나의 중쇄 가변영역을 포함하는, SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편.SEQ ID NO: 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237 and 239 comprising any one heavy chain variable region selected from the group consisting of, SARS-CoV -2 An antibody or antigen-binding fragment thereof that specifically binds to the spike protein.
  4. 제1항에 있어서, The method of claim 1,
    서열번호 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 299, 300, 301 및 302로 이루어진 군에서 선택되는 어느 하나의 경쇄 가변영역을 포함하는 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편.Any one light chain variable selected from the group consisting of SEQ ID NOs: 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 299, 300, 301 and 302 An antibody or antigen-binding fragment thereof that specifically binds to a SARS-CoV-2 spike protein comprising a region.
  5. 제1항에 있어서, The method of claim 1,
    하기 군에서 선택되는 어느 하나의 가변영역 조합을 포함하는, SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편:An antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 spike protein, comprising a combination of any one variable region selected from the following group:
    (1) 서열번호 211의 중쇄 가변영역 및 서열번호 212의 경쇄 가변영역;(1) the heavy chain variable region of SEQ ID NO: 211 and the light chain variable region of SEQ ID NO: 212;
    (2) 서열번호 213의 중쇄 가변영역 및 서열번호 214의 경쇄 가변영역;(2) the heavy chain variable region of SEQ ID NO: 213 and the light chain variable region of SEQ ID NO: 214;
    (3) 서열번호 215의 중쇄 가변영역 및 서열번호 216의 경쇄 가변영역;(3) a heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 216;
    (4) 서열번호 217의 중쇄 가변영역 및 서열번호 218의 경쇄 가변영역;(4) a heavy chain variable region of SEQ ID NO: 217 and a light chain variable region of SEQ ID NO: 218;
    (5) 서열번호 219의 중쇄 가변영역 및 서열번호 220의 경쇄 가변영역;(5) a heavy chain variable region of SEQ ID NO: 219 and a light chain variable region of SEQ ID NO: 220;
    (6) 서열번호 221의 중쇄 가변영역 및 서열번호 222의 경쇄 가변영역;(6) a heavy chain variable region of SEQ ID NO: 221 and a light chain variable region of SEQ ID NO: 222;
    (7) 서열번호 223의 중쇄 가변영역 및 서열번호 224의 경쇄 가변영역;(7) a heavy chain variable region of SEQ ID NO: 223 and a light chain variable region of SEQ ID NO: 224;
    (8) 서열번호 225의 중쇄 가변영역 및 서열번호 226의 경쇄 가변영역;(8) a heavy chain variable region of SEQ ID NO: 225 and a light chain variable region of SEQ ID NO: 226;
    (9) 서열번호 227의 중쇄 가변영역 및 서열번호 228의 경쇄 가변영역;(9) a heavy chain variable region of SEQ ID NO: 227 and a light chain variable region of SEQ ID NO: 228;
    (10) 서열번호 229의 중쇄 가변영역 및 서열번호 230의 경쇄 가변영역;(10) a heavy chain variable region of SEQ ID NO: 229 and a light chain variable region of SEQ ID NO: 230;
    (11) 서열번호 231의 중쇄 가변영역 및 서열번호 232의 경쇄 가변영역;(11) a heavy chain variable region of SEQ ID NO: 231 and a light chain variable region of SEQ ID NO: 232;
    (12) 서열번호 233의 중쇄 가변영역 및 서열번호 234의 경쇄 가변영역;(12) a heavy chain variable region of SEQ ID NO: 233 and a light chain variable region of SEQ ID NO: 234;
    (13) 서열번호 235의 중쇄 가변영역 및 서열번호 236의 경쇄 가변영역;(13) a heavy chain variable region of SEQ ID NO: 235 and a light chain variable region of SEQ ID NO: 236;
    (14) 서열번호 237의 중쇄 가변영역 및 서열번호 238의 경쇄 가변영역;(14) a heavy chain variable region of SEQ ID NO: 237 and a light chain variable region of SEQ ID NO: 238;
    (15) 서열번호 239의 중쇄 가변영역 및 서열번호 240의 경쇄 가변영역;(15) a heavy chain variable region of SEQ ID NO: 239 and a light chain variable region of SEQ ID NO: 240;
    (16) 서열번호 211의 중쇄 가변영역 및 서열번호 299의 경쇄 가변영역;(16) a heavy chain variable region of SEQ ID NO: 211 and a light chain variable region of SEQ ID NO: 299;
    (17) 서열번호 213의 중쇄 가변영역 및 서열번호 300의 경쇄 가변영역;(17) a heavy chain variable region of SEQ ID NO: 213 and a light chain variable region of SEQ ID NO: 300;
    (18) 서열번호 215의 중쇄 가변영역 및 서열번호 301의 경쇄 가변영역; 및 (18) a heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 301; and
    (19) 서열번호 219의 중쇄 가변영역 및 서열번호 302의 경쇄 가변영역.(19) the heavy chain variable region of SEQ ID NO: 219 and the light chain variable region of SEQ ID NO: 302.
  6. 제1항 내지 제5항 중 어느 한 항의 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 코딩하는 핵산.A nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein of any one of claims 1 to 5.
  7. 제6항에 따른 핵산을 포함하는 재조합 발현 벡터.A recombinant expression vector comprising the nucleic acid according to claim 6 .
  8. 제7항에 따른 재조합 발현 벡터로 형질전환된 세포.A cell transformed with the recombinant expression vector according to claim 7.
  9. (i) 제8항에 따른 세포를 배양하는 단계; 및 (ii) 얻어진 세포 배양액으로부터 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 회수하는 단계를 포함하는, SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편의 제조 방법.(i) culturing the cell according to claim 8; and (ii) recovering an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein from the obtained cell culture medium. A method for producing an antigen-binding fragment thereof.
  10. 제1항 내지 제5항 중 어느 한 항의 항체 또는 이의 항원 결합 단편 및 약물을 포함하는 항체-약물 접합체(antibody-drug conjugate, ADC).Claims 1 to 5, wherein any one of the antibody or antigen-binding fragment thereof and an antibody comprising a drug-drug conjugate (antibody-drug conjugate, ADC).
  11. 제1항 내지 제5항 중 어느 한 항의 항체 또는 이의 항원 결합 단편을 포함하는 다중특이적 항체(multispecific antibody).A multispecific antibody comprising the antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof.
  12. 제1항 내지 제5항 중 어느 한 항의 항체 또는 이의 항원 결합 단편, 제10항의 항체-약물 접합체 또는 제11항의 다중특이적 항체를 포함하는 SARS-CoV-2 감염증의 예방 또는 치료용 약학 조성물.Claims 1 to 5 of any one of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of claim 10, or a pharmaceutical composition for preventing or treating SARS-CoV-2 infection comprising the multispecific antibody of claim 11.
  13. 제1항 내지 제5항 중 어느 한 항의 항체 또는 이의 항원 결합 단편을 포함하는 SARS-CoV-2 감염증의 진단용 조성물.A composition for diagnosis of SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.
  14. 제1항 내지 제5항 중 어느 한 항의 항체 또는 이의 항원 결합 단편을 포함하는 SARS-CoV-2 스파이크 단백질의 검출 또는 정량용 키트.A kit for detecting or quantifying a SARS-CoV-2 spike protein comprising the antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof.
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