WO2022085905A1 - Anticorps se liant spécifiquement à la protéine de spicule du sars-cov-2 et utilisation associée - Google Patents
Anticorps se liant spécifiquement à la protéine de spicule du sars-cov-2 et utilisation associée Download PDFInfo
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- 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
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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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
La présente invention concerne : un anticorps se liant spécifiquement à des spicules de protéine membranaire du nouveau coronavirus SARS-CoV-2, ou un fragment de liaison à l'antigène de celui-ci ; un acide nucléique le codant ; un vecteur d'expression recombinant comprenant l'acide nucléique ; une cellule transformée avec le vecteur ; un procédé de préparation de l'anticorps ou du fragment de liaison à l'antigène de celui-ci ; un conjugué anticorps-médicament (CAM) ou un anticorps multispécifique comprenant l'anticorps ou le fragment de liaison à l'antigène de celui-ci ; une composition pharmaceutique pour la prévention ou le traitement d'une infection par le SARS-CoV-2, une composition pour le diagnostic d'une infection par le SARS-CoV-2, et un kit de diagnostic, comprenant chacun l'anticorps ou le fragment de liaison à l'antigène de celui-ci. L'anticorps ou le fragment de liaison à l'antigène de celui-ci selon la présente invention se lie spécifiquement à la protéine de spicule du SARS-CoV-2 et bloque ainsi complètement la liaison des spicules de protéine membranaire du SARS-CoV-2 à l'enzyme de conversion de l'angiotensine 2 (ACE2) de récepteur sur des surfaces de cellules humaines, ouvrant des applications avantageuses dans la prévention, le traitement ou le diagnostic de nouvelles infections à coronavirus.
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CN111423508A (zh) * | 2020-03-31 | 2020-07-17 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | 一种分离的抗病毒感染的SARS-CoV-2蛋白结合分子 |
CN111647077A (zh) * | 2020-06-02 | 2020-09-11 | 深圳市因诺赛生物科技有限公司 | 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用 |
US10787501B1 (en) * | 2020-04-02 | 2020-09-29 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
EP3715847A1 (fr) * | 2020-02-20 | 2020-09-30 | Euroimmun Medizinische Labordiagnostika AG | Procédé et réactifs pour le diagnostic du sars-cov-2 |
CN111778218A (zh) * | 2020-06-04 | 2020-10-16 | 山东宽和正生物医药有限公司 | 噬菌体展示抗体库及基于其淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体 |
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EP3715847A1 (fr) * | 2020-02-20 | 2020-09-30 | Euroimmun Medizinische Labordiagnostika AG | Procédé et réactifs pour le diagnostic du sars-cov-2 |
CN111423508A (zh) * | 2020-03-31 | 2020-07-17 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | 一种分离的抗病毒感染的SARS-CoV-2蛋白结合分子 |
US10787501B1 (en) * | 2020-04-02 | 2020-09-29 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
CN111647077A (zh) * | 2020-06-02 | 2020-09-11 | 深圳市因诺赛生物科技有限公司 | 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用 |
CN111778218A (zh) * | 2020-06-04 | 2020-10-16 | 山东宽和正生物医药有限公司 | 噬菌体展示抗体库及基于其淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体 |
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