WO2019151632A1 - Molécule de liaison ayant une activité neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient - Google Patents

Molécule de liaison ayant une activité neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient Download PDF

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WO2019151632A1
WO2019151632A1 PCT/KR2018/015141 KR2018015141W WO2019151632A1 WO 2019151632 A1 WO2019151632 A1 WO 2019151632A1 KR 2018015141 W KR2018015141 W KR 2018015141W WO 2019151632 A1 WO2019151632 A1 WO 2019151632A1
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seq
region
chain variable
variable region
antibody
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PCT/KR2018/015141
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English (en)
Korean (ko)
Inventor
이수영
이계숙
김철민
송경민
배연진
김우주
정희진
송준영
박만성
노지윤
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(주)셀트리온
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Priority claimed from KR1020180108125A external-priority patent/KR20190093107A/ko
Priority to EP18903480.4A priority Critical patent/EP3747902A4/fr
Priority to JP2020541701A priority patent/JP2021512599A/ja
Priority to TNP/2020/000157A priority patent/TN2020000157A1/en
Priority to JOP/2020/0183A priority patent/JOP20200183A1/ar
Priority to US16/966,248 priority patent/US20220177552A1/en
Application filed by (주)셀트리온 filed Critical (주)셀트리온
Priority to AU2018405442A priority patent/AU2018405442A1/en
Priority to CA3090327A priority patent/CA3090327A1/fr
Priority to EA202091781A priority patent/EA202091781A1/ru
Priority to CN201880088236.1A priority patent/CN111727199A/zh
Priority to BR112020015475-0A priority patent/BR112020015475A2/pt
Publication of WO2019151632A1 publication Critical patent/WO2019151632A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to a binding molecule having a neutralizing activity against Middle East Respiratory Syndrome-Coronavirus (MERS-CoV). More specifically, the present invention relates to a binding molecule having an excellent binding ability to spike protein (S protein) on the surface of MERS-CoV and having a neutralizing effect on MERS-CoV, and for preventing, treating or preventing MERS-CoV infection. Very useful for diagnosis
  • Middle East Respiratory Syndrome-Coronavirus is an infection caused by a coronavirus belonging to beta coronavirus.
  • Middle East Respiratory Syndrome Coronavirus is believed to originate in bats and has been known to enter humans through camels. Although the route of transmission has not been fully defined, it appears that in the Middle East the virus is repeatedly introduced from camels to humans, with limited and non-persistent human-to-human transmission.
  • Middle East Respiratory Syndrome Coronavirus has been reported in 27 countries to date, with 2,078 cases between September 2012 and September 29, 2017, of which 730 died and the death rate was 35.1% (WHO). There are no specific treatments or preventatives, and the role of antiviral agents has not been clearly demonstrated, but given the high mortality and morbidity of MERS-CoV, ribavirin and interferon alpha-2 ⁇ , Active treatment with antiviral drugs, such as the combination of pinavier / ritonavir, is recommended, but there are side effects.
  • Korean Patent Publication No. 10-1593641 discloses an antibody that recognizes MERS-CoV nucleocapsid, a diagnostic composition, kits including the same and MERS-CoV detection method using the same have.
  • the document relates to the determination of MERS-CoV infection using an antibody that specifically binds to the nucleocapsid of MERS-CoV, and thus the neutralizing ability of the antibody against MERS-CoV is unknown.
  • antibodies that have a therapeutic effect on MERS-CoV There is a continuing need for antibodies that have a therapeutic effect on MERS-CoV.
  • the present inventors have developed a binding molecule having a binding capacity to the S protein of MERS-CoV to solve the problems listed above, and completed the present invention by confirming that the binding molecule has a neutralizing effect on MERS-CoV.
  • the problem to be solved by the present invention is to provide a binding molecule having a neutralizing activity to MERS-CoV by binding to the S protein of MERS-CoV.
  • Another object of the present invention is to provide a composition for preventing or treating MERS-CoV comprising the binding molecule.
  • Another problem to be solved by the present invention is to provide a MERS-CoV diagnostic kit comprising the binding molecule.
  • one embodiment of the present invention provides a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of Middle East Respiratory Syndrome-Coronavirus (MERS-CoV). .
  • S protein spike protein
  • MERS-CoV Middle East Respiratory Syndrome-Coronavirus
  • another embodiment of the present invention provides a composition for preventing or treating MERS-CoV comprising the binding molecule.
  • Another embodiment of the present invention provides a MERS-CoV diagnostic kit comprising the binding molecule.
  • One embodiment of the invention relates to neutralizing binding molecules that bind to the S protein of MERS-CoV.
  • One embodiment of the present invention relates to a neutralizing binding molecule, which is any one selected from the group consisting of the binding molecules of the following i) to vi).
  • a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 1, the CDR2 region of SEQ ID NO: 2, and the CDR3 region of SEQ ID NO: 3; And b) a light chain variable region comprising a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 region of SEQ ID NO: 6.
  • a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 7, the CDR2 region of SEQ ID NO: 8, and the CDR3 region of SEQ ID NO: 9; And b) a light chain variable region comprising a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 region of SEQ ID NO: 12. 17.
  • a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 19, the CDR2 region of SEQ ID NO: 20, and the CDR3 region of SEQ ID NO: 21; And b) a light chain variable region comprising a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 region of SEQ ID NO: 24. 16.
  • v) a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 25, the CDR2 region of SEQ ID NO: 26, and the CDR3 region of SEQ ID NO: 27; And b) a light chain variable region comprising a CDR1 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR3 region of SEQ ID NO: 30. 18.
  • the binding molecule comprises antibody 1 to antibody 36 in Table 1 below.
  • the CDRs of the variable regions were determined by conventional methods according to the system devised by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5 th ), National Institutes of Health, Bethesda, MD). (1991)].
  • the CDR numbering used in the present invention used the Kabat method, but other binding molecules including CDRs determined according to other methods such as IMGT method, Chothia method and AbM method are also included in the present invention.
  • One embodiment of the present invention relates to a neutralizing binding molecule, which is any one selected from the group consisting of the binding molecules of the following i) to vi).
  • Binding molecule comprising.
  • Binding molecule comprising.
  • v) a) a heavy chain variable region having at least 95% sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO: 45 and b) a light chain variable region having at least 95% sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 46 Binding molecule comprising.
  • the binding molecule comprises antibody 1 to antibody 6 of Table 2 below.
  • the binding molecule may be, but is not limited to, Fab fragments, Fv fragments, diabodies, chimeric antibodies, humanized antibodies or human antibodies.
  • a complete human antibody that binds to the S protein As used herein, an antibody is used in its broadest sense and is specifically an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody (eg, a bispecific antibody) formed from two or more intact antibodies, and a target. Antibody fragments that exhibit biological activity.
  • Antibodies are proteins produced by the immune system that can recognize and bind specific antigens. In structural terms, the antibody typically has a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains).
  • Each antibody mainly has two regions, a variable region and a constant region.
  • the variable region located at the distal portion of the arm of Y binds to and interacts with the target antigen.
  • the variable region comprises a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen.
  • CDR complementarity determining region
  • the constant region located in the tail of Y is recognized and interacted with by the immune system.
  • Target antigens generally have multiple binding sites called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have one or more corresponding antibodies.
  • the present invention includes functional variants of the binding molecule.
  • Binding molecules are considered functional variants of the binding molecules of the invention provided that the variants can compete with the binding molecules of the invention to specifically bind to MERS-CoV or its S protein, and possess a neutralizing ability for MERS-CoV. do.
  • Functional variants include, but are not limited to, derivatives with substantially similar primary structural sequences, including, for example, in vitro or in vivo modifications, chemicals and / or biochemicals. However, they are not found in the parental monoclonal antibodies of the invention.
  • Such modifications include, for example, acetylation, acylation, covalent linkages of nucleotides or nucleotide derivatives, covalent linkages of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, PEGylation, proteolysis And phosphorylation and the like.
  • Functional variants may optionally be antibodies comprising an amino acid sequence containing substitutions, insertions, deletions or combinations of one or more amino acids in comparison to the amino acid sequence of the parent antibody.
  • functional variants may include truncated forms of amino acid sequences at one or both of the amino terminus or carboxy terminus.
  • Functional variants of the invention may have the same or different, higher or lower binding affinity compared to the parent antibodies of the invention, but can still bind to MERS-CoV or its S protein.
  • the amino acid sequence of the variable region including but not limited to a framework structure, a hypervariable region, particularly, a complementarity-determining region (CDR) of a light or heavy chain, may be modified.
  • the light or heavy chain region comprises three hypervariable regions, including three CDR regions, and a more conserved region, the framework region (FR).
  • Hypervariable regions include amino acid residues from CDRs and amino acid residues from hypervariable loops.
  • Functional variants within the scope of the present invention are about 50% -99%, about 60% -99%, about 80% -99%, about 90% -99%, about 95% -99%, Or about 97% -99% amino acid sequence identity. Gap or Bestfit known to those skilled in the art can be used in computer algorithms to optimally arrange the amino acid sequences to be compared and to define similar or identical amino acid residues.
  • the functional variant may be changed by or obtained by known general molecular biological methods including, but not limited to, a parent antibody or a part thereof by PCR, mutagenesis using oligomeric nucleotides, and partial mutagenesis.
  • a drug may be further attached to the binding molecule.
  • the binding molecule according to the present invention can be used in the form of an antibody-drug conjugate to which a drug is bound.
  • ADCs antibody-drug conjugates
  • immunoconjugates for topical delivery of drugs enables targeted delivery of the drug moiety to infected cells, which, if administered without conjugation of the drug agent, also to normal cells Unacceptable levels of toxicity can result.
  • mAb polyclonal and monoclonal antibodies
  • drug-linking and drug-releasing properties can improve the maximum potency and minimum toxicity of ADCs.
  • another embodiment of the present invention provides a nucleic acid molecule encoding the binding molecule.
  • Nucleic acid molecules of the invention include all nucleic acid molecules in which the amino acid sequence of an antibody provided herein is translated into a polynucleotide sequence, as known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by an open reading frame (ORF), all of which are also included in the nucleic acid molecules of the present invention.
  • ORF open reading frame
  • another embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.
  • Celltrion's unique expression vector MarEx vector see Korean Patent Registration No. 10-1076602
  • commercially widely used pCDNA vectors F, R1, RP1, Col, pBR322, ToL, Ti vectors; Cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Q ⁇ , T-even, T2, T3, T7
  • An expression vector selected from any one selected from the group consisting of plant viruses can be used, but not limited thereto. All expression vectors known to those skilled in the art can be used in the present invention, and when selecting an expression vector, It depends on the property.
  • vectors used by those skilled in the art can be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation upon introduction of the vector into host cells.
  • the introduction method suitable for a vector and a host cell can be selected and used.
  • the vector contains one or more selection markers, but is not limited thereto, and may be selected depending on whether the product is produced using a vector that does not include the selection marker.
  • the selection of the selection marker is chosen by the host cell of interest, which uses methods already known to those skilled in the art and the present invention is not so limited.
  • tag sequences can be inserted and fused onto expression vectors.
  • the tag includes, but is not limited to, hexa-histidine tag, hemagglutinin tag, myc tag, or flag tag, and any tag that facilitates purification known to those skilled in the art can be used in the present invention.
  • another embodiment of the present invention provides a cell line wherein the expression vector is transformed into a host cell to bind to MERS-CoV to produce a binding molecule having a neutralizing ability.
  • the cell line may include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin.
  • the mammalian cells include any one selected from the group consisting of CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, A549 cells, HEK 293 cells and HEK293T cells.
  • One may be used as a host cell, but is not limited thereto, and all cells usable as mammalian host cells known to those skilled in the art are available.
  • the present invention relates to a composition for the prevention or treatment of MERS-CoV infection comprising the binding molecule.
  • the composition of the present invention may include a pharmaceutically acceptable excipient in addition to the binding molecule.
  • Pharmaceutically acceptable excipients are well known to those skilled in the art.
  • composition of the present invention may further comprise at least one other therapeutic or diagnostic agent.
  • interferon, anti-S protein monoclonal antibody, anti-S protein polyclonal antibody, nucleoside analogue, DNA polymerase inhibitor, siRNA agent or therapeutic vaccine may be further added as antiviral drug together with the binding molecule. It may include.
  • compositions comprising the binding molecules of the present invention are in the form of sterile injectable solutions, lyophilized formulations, pre-filled syringe solutions, oral dosage forms, external preparations or suppositories, respectively, according to conventional methods. But may be formulated as,
  • composition comprising the binding molecule of the present invention
  • the method of administration may be divided into oral and parenteral, for example, the route of administration may be intravenous, but is not limited thereto.
  • compositions of the present invention By administering a composition of the present invention to a mammal, including humans, it is possible to prevent or treat diseases caused by MERS-CoV infection and MERS-CoV infection.
  • the dosage of the binding molecule eg, antibody
  • the dosage of the binding molecule depends on the subject being treated, the severity of the disease or condition, the rate of administration and the judgment of the prescribing physician.
  • the present invention relates to a diagnostic kit comprising the binding molecule.
  • the binding molecules of the invention used in diagnostic kits can be detectably labeled.
  • Various methods available for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention.
  • marker types include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds.
  • markers include phosphors (eg, fluresin, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, ⁇ -galactosidase, alkaline phosphatase), radioisotopes (eg, 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (eg dioxetane, luminol or acridinium). Labeling methods such as covalent binding of enzymes or biotinyl groups, iodide methods, phosphorylation methods, biotinylation methods and the like are well known in the art.
  • Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzyme reactions, and the like. Commonly used detection assays include radioisotopes or non-radioisotope methods. These include Western blotting, overlay-assay, Radioimmuno Assay (RIA) and Immunity Radioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (CIA) and Chemiluminoluminescent Immune Assay).
  • radioisotopes or non-radioisotope methods include Western blotting, overlay-assay, Radioimmuno Assay (RIA) and Immunity Radioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (CIA) and Chemiluminoluminescent Immun
  • the diagnostic kit of the present invention can be used to detect the presence of MERS-CoV by contacting a sample with the binding molecule and then confirming the reaction.
  • the sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, lung tissue, mucus, respiratory tissue and saliva of the subject, but is not limited thereto. It is possible.
  • It provides a kit for diagnosing, preventing or treating a disease caused by MERS-Cov comprising a.
  • the kit container may include a solid carrier.
  • Antibodies of the invention may be attached to a solid carrier, which solid carrier may be porous or nonporous, planar or nonplanar.
  • the present invention also provides a method for diagnosing, preventing or treating a disease caused by MERS-CoV infection, comprising administering a therapeutically effective amount of the composition to a subject having a disease resulting from MERS-CoV infection. do.
  • the method of diagnosing, preventing, or treating may further comprise administering an anti-viral drug, a viral entry inhibitor or a virus adhesion inhibitor.
  • binding molecule refers to an antigen-binding that is an intact immunoglobulin, or an immunoglobulin that binds to an antigen, including a monoclonal antibody such as a chimeric, humanized or human monoclonal antibody. Contains fragments. For example, it refers to a variable domain, an enzyme, a receptor, a protein including an immunoglobulin fragment that competes with an intact immunoglobulin in binding to a spike protein of MERS-CoV. Regardless of the structure, the antigen-binding fragment binds to the same antigen recognized by intact immunoglobulins.
  • An antigen-binding fragment may comprise two or more continuations of an amino acid sequence of an antibody, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, At least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, 150 Peptides or polypeptides comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.
  • Antigen-binding fragments are especially Fab, F (ab '), F (ab') 2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent Single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of immunoglobulin sufficient to bind a particular antigen to a polypeptide, and the like.
  • the fragments may be produced synthetically or by enzymatic or chemical digestion of complete immunoglobulins or may be produced genetically by recombinant DNA techniques. Production methods are well known in the art.
  • the term “pharmaceutically acceptable excipient” refers to an inert material that is combined into an active molecule, such as a drug, agent or antibody, to produce an acceptable or convenient dosage form.
  • Pharmaceutically acceptable excipients are nontoxic or are excipients that are acceptable to the recipient for their intended use, at least in the doses and concentrations in which the toxicity is used, and with other components of the formulation including drugs, preparations or binding powders. It is compatible.
  • the term “therapeutically effective amount” refers to the amount of the binding molecule of the invention effective for prophylaxis or treatment before or after exposure of MERS-CoV.
  • the binding molecule of the present invention has a neutralizing effect by having an excellent binding ability of the SERS of MERS-CoV, it is very useful for the prevention, treatment or diagnosis of MERS-CoV infection.
  • FIG. 1 is a diagram showing the neutralization capacity of the antibody concentration against the isolated species Mers coronavirus (MERS-CoV / Korea / KNIH / 002_05_2015) by performing a plaque assay with the two selected antibodies.
  • Figure 2 is a human lung tissue infection model (ex vivo) to evaluate the neutralization capacity of the antibody to infect the human lung tissue with the Korean isolated species Mers coronavirus and the antibody cultured after tissue culture, virus titer through plaque measurement Is a diagram showing.
  • Figure 3a shows the results of quantitave PCR for the evaluation of animal treatment efficacy using an animal model capable of MERS coronavirus infection and proliferation (hDPP4 (human dipeptidyl peptidase 4 receptor overexpressing mouse) and the antibody binding to the virus) The figure shown.
  • hDPP4 human dipeptidyl peptidase 4 receptor overexpressing mouse
  • Figure 3b is a plaque assay experiment for the evaluation of animal treatment efficacy using an animal model capable of infection and proliferation of mers coronavirus (human dipeptidyl peptidase 4 receptor overexpressing mouse) and the antibody bound to the virus Is a diagram showing.
  • mers coronavirus human dipeptidyl peptidase 4 receptor overexpressing mouse
  • Figure 4a shows the results of performing quantitave PCR to evaluate the prophylactic efficacy of the antibody against MERS coronavirus using an animal model capable of MERS coronavirus infection and proliferation (human dipeptidyl peptidase 4 receptor overexpressing mouse) Degrees (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • Figure 4b shows the results of the plaque assay experiment to evaluate the preventive effect of the antibody against MERS coronavirus using an animal model capable of MERS coronavirus infection and proliferation (hDPP4 (human dipeptidyl peptidase 4 receptor overexpressing mouse)) Figures shown (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • FIG. 5 shows the histological changes of mouse lungs to evaluate the prophylactic efficacy of antibodies against MERS coronavirus using an animal model capable of MERS coronavirus infection and proliferation (hDPP4 (human dipeptidyl peptidase 4 receptor overexpressing mouse)). It is a figure shown
  • FIG. 6A is a diagram showing the extent of mouse weight loss to evaluate the therapeutic efficacy of antibodies against MERS coronavirus using an animal model capable of MERS coronavirus infection and proliferation (human dipeptidyl peptidase 4 receptor overexpressing mouse) (* P ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • Figure 6b is a diagram showing the survival rate of the mouse to evaluate the therapeutic efficacy of the antibody against MERS coronavirus using an animal model capable of MERS coronavirus infection and proliferation (hDPP4 (human dipeptidyl peptidase 4 receptor overexpressing mouse) ( * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • hDPP4 human dipeptidyl peptidase 4 receptor overexpressing mouse
  • Figure 6c shows the results of quantitave PCR to evaluate the therapeutic efficacy of antibodies against MERS coronavirus using an animal model capable of MERS coronavirus infection and proliferation (human dipeptidyl peptidase 4 receptor overexpressing mouse) Degrees (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • Figure 6d is a plaque assay experiment to evaluate the efficacy of the antibody against MERS coronavirus using an animal model capable of MERS coronavirus infection and proliferation (hDPP4 (human dipeptidyl peptidase 4 receptor overexpressing mouse)) Figures shown (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • PBMC peripheral blood mononuclear cells
  • RNA from TriBol Reagent (Invitrogen) in PBMC isolated in Example 1 cDNA was synthesized using the SuperScript TM III First-Strand cDNA synthesis system (Invitrogen, USA).
  • variable regions of the light and heavy chains of an antibody using high fidelity Taq polymerase (Roche) and degenerative primer set (IDT) from the synthesized cDNA by polymerase chain reaction (PCR). It was. The variable region fragments of the separated light and heavy chains were randomly combined into a single sequence by overlap PCR method, amplified into scFv-type genes, digested with restriction enzymes, and digested with 1% agarose gel electrophoresis. The scFv was isolated using a gel extraction kit (Qiagen) method.
  • Phage vectors were also cleaved with the same restriction enzymes, isolated, mixed with the scFv gene, added with T4 DNA ligase (New England Biolab) and reacted at 16 ° C. for at least 12 hours.
  • the reaction solution was mixed with ER2738 soluble cells and transformed by electroporation.
  • the transformed ER2738 was incubated for at least 12 hours after shaking culture and VCSM13 helper phage (Agilent Technologies).
  • the phage library culture prepared in Example 2 was centrifuged to remove host cells, 4% PEG and 0.5M NaCl were added, centrifuged to settle the phage, and the supernatant was removed.
  • the precipitated phage was diluted in 1% BSA / TBS to obtain a phage library, followed by independent panning by binding and dissociation reactions to various MERS-CoV Spike proteins (hereinafter, S protein). ScFv-phage having binding capacity to S protein was isolated.
  • a phage library was placed in an ELISA plate to which the RBD region (residues 367 to 588 on S1 glycoprotein), which is a part of the MERS-CoV S protein, was reacted at room temperature for 2 hours. After removing the reaction solution, the ELISA plate was washed with PBS containing 0.05% tween 20, 60 ⁇ l of 0.1M glycine-HCl (pH 2.2) was added to remove the antigen-binding scFv-phage and 2M Tris (pH 9.1). Neutralized using. Neutralized scFv-phage was infected with ER2738, followed by incubation with helper phage and used for the next panning. A portion of the infected ER2738 was plated onto LB plates prior to loading the secondary phage to obtain colonies the next day.
  • a total of 1,200 colonies formed at each panning were shaken in culture medium containing 96-well deep well plate (Axygen) and shake-cultured at 37 ° C. for at least 12 hours after supplementary phage was added when the OD 600 was 0.7 or more.
  • the culture medium was centrifuged to remove host cells and a supernatant containing scFv-phage was prepared.
  • MERS-CoV S proteins were adsorbed and placed in each well of a blocked 96-well microtiter plate. It was. Each well was washed three times with PBS containing 0.05% Tween 20, and then HRP (horseradish peroxidase) labeled anti-M13 antibody was added and allowed to stand at 37 ° C for 1 hour.
  • HRP horseradish peroxidase
  • the 444 scFv-phages selected in Example 3 were then subjected to shake culture of colonies to obtain DNA, followed by analysis of sequences for antibody variable regions. Among them, 118 scFv-phages, except for clones duplicated as amino acid sequences, were cloned into a vector containing an Fc region to evaluate expression and neutralizing ability in candidate antibody animal cell lines. Fc) form. Transfection reagent was used to transfect and express F2N cells (Korea Patent No. 10-1005967, patent holder: Celltrion) and then MERS-CoV of the antibody fragment (scFv-Fc) using the culture medium. The binding ability of the three S proteins of was confirmed by ELISA.
  • MERS-CoV S proteins were attached to ELISA plates and the expressed antibody fragments were added.
  • Antibodies bound to antigen were selected by washing non-binding antibodies with PBS containing 0.05% Tween 20 and then using anti-human Fc antibodies conjugated with HRP (horseradish peroxidase). As a result, it was confirmed that 111 antibody fragments specifically bind to S proteins of MERS-CoV.
  • 111 antibody fragment cultures expressed by the method of Example 4 were sequentially diluted twice to prepare samples of 12 concentrations.
  • the Saudi isolated species virus (MERS / HCoV / KSA / EMC / 2012) was dissolved in virus stock and diluted to 25 of TCID 50 / well concentration and mixed with the prepared antibody concentration cultures of the 12 different concentrations, respectively. After standing for 2 hours, the cells were transferred to a 96-well culture plate in which Vero cells were cultured, and left at 37 ° C. for 1 hour to induce infection. After removing the antibody fragment culture solution and the virus mixture, the culture medium was placed in each well and incubated for 3 days in a 37 ° C.
  • Antibodies 1 0.022 Antibody 2 ⁇ 0.01 Antibody 3 0.014 Antibody 4 0.010 Antibody 5 0.003 Antibody 6 0.021 Antibody 7 0.014 Antibody 8 0.056 Antibody 9 0.022 Antibody 10 0.061 Antibody 11 0.034 Antibody 12 0.051 Antibody 13 0.050 Antibody 14 0.011 Antibody 15 0.086 Antibody 16 0.016 Antibody 17 0.047 Antibody 18 0.011 Antibodies 19 0.021 Antibody 20 0.023 Antibody 21 0.024 Antibody 22 0.053 Antibody 23 0.019 Antibody 24 0.067 Antibody 25 0.031 Antibody 26 0.011 Antibody 27 0.020 Antibody 28 0.035 Antibody 29 0.048 Antibody 30 0.007 Antibody 31 0.021 Antibody 32 0.060 Antibody 33 0.010 Antibody 34 0.061 Antibody 35 0.033 Antibody 36 0.031 Positive Control Antibody 1 0.09 Positive Control Antibody 2 1.69
  • the antibody sample was diluted, mixed with the same amount of 100 PFU virus, reacted at 37 ° C. for 1 hour, and then infected with a cell line to perform a plaque assay. After incubation for 3 days at 37 ° C., 5% CO 2 incubator, the cells were stained using crystal violet, and the number of plaques formed was compared and analyzed to evaluate the neutralization ability of the antibody sample.
  • the genetic information of the selected antibody fragment was converted into a fully human antibody, and the antibody culture was prepared by the method of Example 4, the antigen binding site, antibody expression rate, etc. in the fully human antibody were confirmed, and the virus neutralization ability was evaluated. In total, 18 of 36 complete human antibodies were selected.
  • Example 6 After the 18 antibodies selected in Example 6 were converted into fully human antibodies, the virus neutralization ability against the Saudi isolated virus was evaluated in the same manner as in Example 5). As shown in Table 4, it was confirmed that 16 of the 18 antibodies had better virus neutralizing ability than the positive control antibody 1 (an antibody that binds to the MERS-CoV S protein and is known to have a neutralizing effect).
  • Antibodies 1 0.08 0.16 Antibody 2 0.07 0.07 Antibody 3 0.07 0.38 Antibody 4 0.08 0.12 Antibody 5 0.02 0.04 Antibody 6 0.42 ND Antibody 7 0.06 0.13 Antibody 9 0.08 0.18 Antibody 14 0.15 0.25 Antibody 18 0.19 0.20 Antibody 21 0.11 0.16 Antibody 26 0.08 0.11 Antibody 27 0.24 0.31 Antibody 29 0.39 0.78 Antibody 33 2.43 ND Antibody 34 0.38 ND Antibody 35 0.11 0.20 Antibody 36 ND ND Positive Control Antibody 1 1.75 2.94
  • Example 6 After the 18 antibodies selected in Example 6 were converted into fully human antibodies, the virus neutralization ability of the Korean isolated virus was evaluated in the same manner as in Example 5).
  • the PRNT measurement was carried out as primary (7 antibodies) and secondary (11 antibodies), as shown in Table 18 of 18 species, all 18 antibodies bound to positive control antibodies 1 and 2 (MERS-CoV S protein, and The neutralizing effect was lower than the known antibody) to confirm that it has a better virus neutralizing ability.
  • the physical properties were evaluated in order to select a cell line development target antibody.
  • the physical properties were evaluated for antibody target sites, antibody expression rates, and heat resistance.
  • thermal resistance evaluation simply, dilute the antibody with Sypro Orange (Thermo Fisher Scientific) at the appropriate concentration, mix the sample prepared in a PCR 96 well plate, and use a 7500 Real Time PCR System (Thermo Fisher Sceintific) at 25 ° C. The fluorescence value was measured by setting a melt curve up to 99 ° C. Integrating the evaluation of the neutralizing ability and the results of evaluation of physical properties shown in Table 6, six antibodies shown in Table 5 were selected and cloned into a vector suitable for cell line progression.
  • Example 8 For the six antibodies selected in Example 8, the neutralizing ability of the Saudi isolated species virus and the Jordan isolated species virus (MERS-HCoV / Jordan / 01) was evaluated in the same manner as in Example 5). As shown in Table 7, virus neutralization ability was confirmed to be superior to four positive control antibodies (antibodies that bind to the MERS-CoV S protein and have a neutralizing effect).
  • Plaque assay was performed on the two antibodies selected in Example 9 in the same manner as in Example 5, to obtain the Korean isolated species MERS-Coronavirus (MERS-CoV / Korea / KNIH / 002_05_2015). Neutralizing ability by antibody concentration was evaluated. As a result, it was confirmed that two antibodies had excellent virus neutralizing ability compared to positive control antibody 4 (an antibody that binds to the MERS-CoV S protein and is known to have a neutralizing effect) (FIG. 1).
  • the antibody was not treated in the supernatant at 24, 48 and 72 hours as shown in Table 8 when two antibodies confirmed in Example 10 were reacted with MERS-CoV at 100 ng each. It was confirmed that the growth of the Middle East respiratory syndrome coronavirus compared to the negative control antibody and the positive control antibody (FIG. 2)
  • the effects of MERS coronavirus infection and positive control antibody 4 on hDPP4 TG mice were established using antibodies 3, 5 and negative control antibodies (FIG. 3).
  • the virus was infected with animals and injected intraperitoneally with each antibody and the corresponding dose the following day. After a certain day after infection, the lung tissue of the mouse was extracted, and the amount of the virus was quantified by quantitave PCR (FIG. 3A) and plaque assay (FIG. 3B). Animal infection experiments were previously confirmed to be performing well, and it was previously confirmed that antibody 3 and antibody 5 may have superior or similar therapeutic efficacy compared to positive control antibody 4.
  • the virus was infected with animals for evaluation of therapeutic efficacy and injected intraperitoneally with antibody 5, positive control antibody 4 and negative control antibody each day. After the specific infection date, the weight loss of the mouse and the survival rate of the mice were measured, and lung tissues of the mice were extracted to quantify the amount of the virus (FIG. 6). In evaluating mouse weight loss (FIG. 6A) and mouse survival rate (FIG. 6B), weight loss and lethality were observed only at the lowest dose of negative control antibody and antibody 5 (2 ug). In addition, quantitative PCR (FIG. 6C) and plaque assay (FIG. 6D) quantitative results of the mers coronavirus, it was confirmed that the antibody 5 has a superior or similar therapeutic efficacy compared to the positive control antibody 4.

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Abstract

La présente invention concerne une molécule de liaison ayant une activité neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient (MERS-CoV). Plus particulièrement, la présente invention concerne une molécule de liaison ayant une capacité supérieure à se lier à la protéine S du MERS-CoV et ayant également un effet neutralisant sur le MERS-CoV et est très utile dans la prévention, le traitement ou le diagnostic d'une infection par MERS-CoV.
PCT/KR2018/015141 2018-01-31 2018-11-30 Molécule de liaison ayant une activité neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient WO2019151632A1 (fr)

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BR112020015475-0A BR112020015475A2 (pt) 2018-01-31 2018-11-30 Molécula ligadora tendo atividade de neutralização contra o coronavírus que causa a síndrome respiratória do oriente médio
JP2020541701A JP2021512599A (ja) 2018-01-31 2018-11-30 中東呼吸器症候群コロナウイルスに対して中和活性を有する結合分子
TNP/2020/000157A TN2020000157A1 (en) 2018-01-31 2018-11-30 Binding molecule having neutralizing activity against middle east respiratory syndrome coronavirus
JOP/2020/0183A JOP20200183A1 (ar) 2018-01-31 2018-11-30 جزيء ربط له نشاط تحييد ضد متلازمة الشرق الأوسط التنفسية – فيروس كورونا
US16/966,248 US20220177552A1 (en) 2018-01-31 2018-11-30 Binding Molecule Having Neutralizing Activity Against Middle East Respiratory Syndrome-Coronavirus
EP18903480.4A EP3747902A4 (fr) 2018-01-31 2018-11-30 Molécule de liaison ayant une activité neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient
AU2018405442A AU2018405442A1 (en) 2018-01-31 2018-11-30 Binding molecule having neutralizing activity against middle east respiratory syndrome-coronavirus
CA3090327A CA3090327A1 (fr) 2018-01-31 2018-11-30 Molecule de liaison ayant une activite neutralisante contre le coronavirus du syndrome respiratoire du moyen-orient
EA202091781A EA202091781A1 (ru) 2018-01-31 2018-11-30 Связывающая молекула, обладающая нейтрализующей активностью в отношении коронавируса ближневосточного респираторного синдрома
CN201880088236.1A CN111727199A (zh) 2018-01-31 2018-11-30 针对中东呼吸综合征-冠状病毒具有中和活性的结合分子

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KR10-2018-0108125 2018-09-11
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CN113667010A (zh) * 2020-05-15 2021-11-19 普米斯生物技术(珠海)有限公司 冠状病毒的抗体及其衍生物用途
CN113667010B (zh) * 2020-05-15 2023-07-28 普米斯生物技术(珠海)有限公司 冠状病毒的抗体及其衍生物用途

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