WO2022143816A1 - Anticorps à neutralisation croisée à large spectre entièrement humain contre sars-cov-2 et sars-cov et son utilisation - Google Patents

Anticorps à neutralisation croisée à large spectre entièrement humain contre sars-cov-2 et sars-cov et son utilisation Download PDF

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WO2022143816A1
WO2022143816A1 PCT/CN2021/142696 CN2021142696W WO2022143816A1 WO 2022143816 A1 WO2022143816 A1 WO 2022143816A1 CN 2021142696 W CN2021142696 W CN 2021142696W WO 2022143816 A1 WO2022143816 A1 WO 2022143816A1
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antibody
variable region
chain variable
cov
sars
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Chinese (zh)
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孙兵
伊春艳
孙晓玉
凌志洋
张亚光
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中国科学院分子细胞科学卓越创新中心
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A—HUMAN NECESSITIES
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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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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
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    • 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
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
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    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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Definitions

  • the present invention relates to the field of medicine, in particular to anti-SARS-CoV-2 and SARS-CoV broad-spectrum cross-neutralizing antibodies and applications thereof.
  • Monoclonal antibody clinical intervention is very effective in the prevention and treatment of viral infection, and it has been successfully used in clinical practice to prevent respiratory syncytial virus infection.
  • a large number of preclinical and early clinical data show that monoclonal antibodies can effectively prevent and treat infections caused by a variety of viruses. Therefore, the development of preventive and therapeutic neutralizing antibodies is of great value in combating the current novel coronavirus epidemic or future new and sudden coronavirus infections.
  • the spike protein on the surface of coronavirus is an important antigen for inducing neutralizing antibodies, and neutralizing antibodies play a neutralizing role by blocking the binding of the S protein to the receptor or inhibiting the fusion of the virus and the host cell membrane.
  • the S protein consists of two functional domains, S1 and S2. Among them, S1 is responsible for binding to specific receptors on host cells to promote viral infection, and the sequence is highly variable between different coronaviruses.
  • SARS-CoV and SARS-CoV-2 share 76% S protein sequence homology, while RBDs share 73.5% homology, which is lower than that of the full-length S protein, however, Most of the variation exists in the RBM (receptor binding motif) region, and its shared properties are only 47.8%. Therefore, the identity of the conserved core structure outside the RBM in RBD is 84%, which is relatively conserved in SARS-CoV and SARS-CoV-2, which means that the RBD core domain is highly likely to induce SARS-CoV and SARS-CoV-2 broad Spectrum crossover antibodies.
  • the purpose of the present invention is to provide a fully human monoclonal antibody capable of preventing and controlling novel coronavirus (SARS-CoV-2) infection.
  • a first aspect of the present invention provides a heavy chain variable region of an antibody, the heavy chain variable region comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains the RBD binding affinity of SARS-CoV-2 and/or SARS-CoV.
  • the heavy chain variable region further includes a human FR region or a murine FR region.
  • the heavy chain variable regions respectively have the amino acid sequences shown in SEQ ID NO.: 1-12.
  • the second aspect of the present invention provides a heavy chain of an antibody, wherein the heavy chain has the variable region of the heavy chain described in the first aspect of the present invention.
  • the heavy chain of the antibody further includes a heavy chain constant region.
  • the heavy chain constant region is of human origin, mouse origin or rabbit origin.
  • a third aspect of the present invention provides a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains the RBD binding affinity of SARS-CoV-2 and/or SARS-CoV.
  • the light chain variable region further includes a human FR region or a murine FR region.
  • the light chain variable regions respectively have the amino acid sequences shown in SEQ ID NO.: 13-24.
  • the fourth aspect of the present invention provides a light chain of an antibody, the light chain has the light chain variable region according to the third aspect of the present invention.
  • the light chain of the antibody further includes a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • the fifth aspect of the present invention provides an antibody, the antibody has:
  • the antibody has: a heavy chain as described in the second aspect of the invention; and/or a light chain as described in the fourth aspect of the invention.
  • the antibody is a specific anti-SARS-CoV-2 and/or SARS-CoV antibody, preferably a specific anti-SARS-CoV-2 and/or SARS-CoV RBD protein of antibodies.
  • the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the antibody is in the form of a drug conjugate.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 1; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 8.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 2; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 9.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 3; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 10.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.:4; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.:11.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.:5; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.:12.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 6; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 13.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 7; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 14.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.: 15, CDR2 as shown in SEQ ID NO.: 22 and as SEQ ID NO.: 29; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:36, CDR2' as the amino acid sequence of KAS and CDR3' as shown in SEQ ID NO.:43.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.: 16, CDR2 as shown in SEQ ID NO.: 23 and as SEQ ID NO.: 30; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:37, CDR2' as the amino acid sequence of GNS and CDR3' as shown in SEQ ID NO.:44.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.: 17, CDR2 as shown in SEQ ID NO.: 24 and as SEQ ID NO.: 31; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:38, the amino acid sequence is CDR2' of DVF and CDR3' as shown in SEQ ID NO.:45.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.: 18, CDR2 as shown in SEQ ID NO.: 25 and as SEQ ID NO.: 32; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:35, CDR2' as the amino acid sequence of WAF and CDR3' as shown in SEQ ID NO.:46.
  • the sixth aspect of the present invention provides a recombinant protein, the recombinant protein has:
  • variable region of the heavy chain according to the first aspect of the present invention the heavy chain according to the second aspect of the present invention, the variable region of the light chain according to the third aspect of the present invention, the variable region of the light chain according to the fourth aspect of the present invention
  • the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.
  • the recombinant protein includes fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • the recombinant protein specifically binds to SARS-CoV-2 and/or the RBD protein of SARS-CoV.
  • a seventh aspect of the present invention provides a CAR construct, wherein the scFv segment of the antigen-binding region of the CAR construct is a receptor-binding region that specifically binds to SARS-CoV-2 and/or SARS-CoV, And the scFv has the variable region of the heavy chain as described in the first aspect of the present invention and the variable region of the light chain as described in the third aspect of the present invention.
  • the eighth aspect of the present invention provides a recombinant immune cell expressing the exogenous CAR construct according to the seventh aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (eg, mice).
  • the ninth aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate comprising:
  • a conjugation moiety conjugated to the antibody moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the conjugate is selected from: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer X-ray tomography) contrast agents, or capable of producing detectable Products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, pre- Drug-activated enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or nanoparticles in any form, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the antibody moiety and the coupling moiety are coupled through chemical bonds or linkers.
  • the tenth aspect of the present invention provides the use of an active ingredient selected from the group consisting of the heavy chain variable region described in the first aspect of the present invention, the heavy chain variable region described in the second aspect of the present invention. chain, light chain variable region according to the third aspect of the invention, light chain according to the fourth aspect of the invention, or antibody according to the fifth aspect of the invention, recombinant according to the sixth aspect of the invention A protein, or a combination thereof, the active ingredient for use in the preparation of a medicament, reagent, assay panel or kit.
  • the reagent, detection plate or kit is used to detect the coronavirus SARS-CoV-2 and/or SARS-CoV.
  • the medicament is used for the treatment or prevention of coronavirus SARS-CoV-2 and/or SARS-CoV infection.
  • the reagent includes a chip and an antibody-coated immune particle.
  • the eleventh aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition contains:
  • an active ingredient selected from the group consisting of a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, and a heavy chain as described in the third aspect of the present invention
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used to prevent and/or treat coronavirus SARS-CoV-2 and/or SARS-CoV infection.
  • a twelfth aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:
  • the polynucleotide has the sequence shown in SEQ ID NO.: 50-56 and/or SEQ ID NO.: 57-63.
  • the thirteenth aspect of the present invention provides a vector, the vector containing the polynucleotide according to the twelfth aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the fourteenth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector according to the thirteenth aspect of the present invention or integrates the vector according to the twelfth aspect of the present invention in the genome polynucleotides.
  • a fifteenth aspect of the present invention provides a method for detecting coronavirus SARS-CoV-2 and/or SARS-CoV in a sample, the method comprising the steps of:
  • the detection is for non-therapeutic and non-diagnostic purposes.
  • the present invention also provides a method for detecting the RBD protein of SARS-CoV-2 and/or SARS-CoV in a sample, the method comprising the steps of:
  • the detection is for non-therapeutic and non-diagnostic purposes.
  • the sixteenth aspect of the present invention provides a detection plate, the detection plate comprises: a substrate (support plate) and a test strip, the test strip contains the antibody according to the fifth aspect of the present invention or the The immunoconjugate according to the ninth aspect of the present invention.
  • a seventeenth aspect of the present invention provides a kit comprising:
  • the kit contains the detection plate according to the sixteenth aspect of the present invention.
  • An eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
  • a nineteenth aspect of the present invention provides a method for treating a novel coronavirus infection, the method comprising: administering the antibody or the antibody-drug conjugate of the antibody according to the fifth aspect of the present invention to a subject in need substance, or CAR-T cells expressing the antibody, or a combination thereof.
  • Figure 1 shows the binding activity of 7 strains of antibodies to SARS-CoV-2 and SARS-CoV verified by ELISA.
  • Figure 2 shows the ability of ELISA to verify the ability of 7 strains of antibodies to block RBD and ACE2.
  • Figure 3 shows the neutralization activity of 7 strains of antibodies against SARS-CoV-2 and SARS-CoV pseudoviruses verified by pseudovirus experiments.
  • Figure 4 shows the microneutralizing activity of RBD-specific antibodies against the SARS-CoV-2 euvirus.
  • Figure 5 shows the affinity of seven antibodies for RBD detected by the OCTET RED 96 instrument.
  • the polyclonal antibody of the present invention targets the conserved regions of SARS-CoV-2 and SARS-CoV RBD, and the mutation frequency is relatively low, which can cope with the problem of drug resistance formed due to the rapid mutation of the coronavirus. On this basis, the present invention has been completed.
  • treating refers to the administration of an internal or external therapeutic agent, comprising the monoclonal antibodies against the coronavirus RBD protein of the present invention, and compositions thereof, to a patient who has one or more disease symptoms and is known to have The therapeutic agent has a therapeutic effect on these symptoms.
  • a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance can, but need not, occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may, but does not necessarily have, one, two or three.
  • Sequence identity refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions.
  • sequence identity between the sequences described in the present invention and the sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,100%.
  • Coronavirus Coronavirus, CoV
  • the coronavirus belongs to the genus Coronavirus in the family Coronaviridae of the order Nidovirales in the systematic taxonomy.
  • Viruses of the genus Coronavirus are RNA viruses with an envelope and a linear single-stranded positive-stranded genome.
  • the coronavirus has a diameter of about 80-120 nm, a methylated cap-like structure at the 5' end of the genome, and a poly(A) tail at the 3' end.
  • the total length of the genome is about 27-32 kb. It is the virus with the largest genome among the known RNA viruses. . It infects only vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cattle, birds.
  • 2019 Novel Coronavirus SARS-CoV-2
  • 2019-nCoV 2019 Novel Coronavirus 2019-nCoV
  • the coronavirus SARS-CoV causes severe acute respiratory syndrome.
  • the multi-strain fully human monoclonal antibody of the present invention targets the relatively conserved core region of the receptor binding region of the coronavirus, so it can broadly bind and neutralize SARS-CoV-2 and SARS-CoV.
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable domains of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases form a partially folded structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be grouped into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific to a single antigenic site. Also, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characteristically obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • the present invention also includes monoclonal antibodies having the corresponding amino acid sequences of the SARS-CoV-2 and the RBD protein monoclonal antibodies of SARS-CoV, and the RBD protein monoclonal antibodies of the SARS-CoV-2 and SARS-CoV. Monoclonal antibodies to antibody variable region chains, and other proteins or protein conjugates and fusion expression products having these chains.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having light and heavy chains containing hypervariable regions (complementarity determining regions, CDRs), as long as the The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light and heavy chains of the invention.
  • immunoconjugate and fusion expression product having light and heavy chains containing hypervariable regions (complementarity determining regions, CDRs), as long as the The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light and heavy chains of the invention.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the anti-SARS-CoV-2 and A conjugate formed by the combination of a monoclonal antibody or its fragment of the RBD protein of SARS-CoV.
  • the present invention also includes cell surface markers or antigens that bind to the anti-SARS-CoV-2 and SARS-CoV RBD protein monoclonal antibodies or fragments thereof.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies.
  • binding fragments encompassed by the term "antigen-binding fragment of an antibody” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising A bivalent fragment of two Fab fragments linked by a disulfide bridge on the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv consisting of the VH and VL domains of the one-armed antibody Fragment.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the present invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10-7 M, eg, about less than 10-8 M, 10-9 M, or 10-10 M or less.
  • KD affinity
  • antigenic determinant refers to a discrete three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the term "murine antibody” in the present invention is a monoclonal antibody against SARS-CoV-2 and the RBD protein of SARS-CoV prepared according to the knowledge and skills in the art.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to the grafting of murine CDR sequences into a human antibody variable region framework, i.e. a different type of human germline antibody Antibody produced in framework sequences.
  • Humanized antibodies can overcome the heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.
  • antibodies may be monospecific, bispecific, trispecific, or more multispecific.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the heavy chain variable region of the antibody comprises three complementarity determining region CDRs as shown in Table A below:
  • amino acid sequences of the heavy chain variable regions are shown in SEQ ID NO.: 1-7, respectively.
  • nucleic acid coding sequences of the heavy chain variable region are respectively as SEQ ID NO.: 50(28-26K), 51(25-F7), 52(25-D9), 53(25- E8), 54 (28-9K), 55 (25-F4), 56 (25-C10).
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
  • VL light chain variable region
  • the light chain variable region of the antibody according to the present invention has the CDRs of the complementarity determining region selected from the following table B:
  • amino acid sequences of the light chain variable regions are shown in SEQ ID NO.: 8-14, respectively.
  • nucleic acid coding sequences of the light chain variable region are respectively as SEQ ID NO.: 57(28-26K), 58(25-F7), 59(25-D9), 60(25- E8), 61 (28-9K), 62 (25-F4), 63 (25-C10).
  • the light chain of the antibody includes the above-mentioned light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin.
  • the function of the antibody of the present invention is determined by the specific gene sequences of the light chain and heavy chain variable region genes of the antibody, and can specifically bind to SARS-CoV-2 and the RBD protein of SARS-CoV, and can prevent SARS-CoV-2 or SARS-CoV-2 or SARS-CoV infects susceptible cells.
  • this antibody variable region gene or complementarity determining region (CDR) gene different forms of genetically engineered antibodies can be engineered and produced in any expression system using prokaryotic and eukaryotic cells.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to antibodies that specifically bind to SARS-CoV-2 and the RBD protein of SARS-CoV, such as Proteins or polypeptides with a heavy chain variable region (amino acid sequence shown in SEQ ID NO.: 1-7) and/or a light chain variable region (amino acid sequence shown in SEQ ID NO.: 8-14) . They may or may not contain the starting methionine.
  • the antibody is a mouse or human-mouse chimeric monoclonal antibody against SARS-CoV-2 and the RBD protein of SARS-CoV, and its heavy chain constant region and/or light chain constant region can be is a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region or light chain constant region of human IgG1, IgG2 and the like.
  • variable regions which are separated into four framework regions (FRs), four
  • FRs framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the present invention includes those molecules having CDR-bearing monoclonal antibody light and heavy chain variable regions, as long as their CDRs are greater than 90% (preferably greater than 95%, optimally 98%) of the CDRs identified herein above) homology.
  • the present invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a polypeptide having the binding activity of SARS-CoV-2 and the RBD protein of SARS-CoV and comprising the above-mentioned CDR regions.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • substitution with amino acids of similar or similar properties generally does not alter the function of the protein.
  • addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
  • the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the present invention also includes fragments of the antibodies of the present invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best produced by amino acid substitutions according to Table C.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • the coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO.: 50-63 or a degenerate variant.
  • a "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes the same amino acid sequence as the polypeptide of the present invention, but differs from the coding region sequence shown in SEQ ID NO.: 50-63 .
  • Polynucleotides encoding the mature polypeptides of the invention include: coding sequences encoding only the mature polypeptide; coding sequences and various additional coding sequences for the mature polypeptide; coding sequences (and optional additional coding sequences) for the mature polypeptide and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%. And, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.: 1-7 and/or SEQ ID NO.: 8-14.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
  • Therapeutic agents that can be conjugated include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptides, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind to the RBD protein molecules of the coronaviruses SARS-2-CoV and SARS-CoV, and thus can be used to prolong the half-life of the drug, and in addition, other therapeutic agents can be used at the same time.
  • the pharmaceutical composition of the present invention contains the above-mentioned monoclonal antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 10 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • Antibodies of the invention can be used in detection applications, eg, in detection of samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples and biopsy specimens.
  • biopsy as used in the present invention shall include all kinds of biopsies known to those skilled in the art. Biopsies used in the present invention may thus include tissue samples prepared, for example, by endoscopic methods or needle or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or its fragment) of the present invention.
  • the kit further includes a container, instructions for use, buffers, and the like.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the fully human monoclonal antibody of the present invention can broadly bind and neutralize SARS-CoV-2 and SARS-CoV.
  • the fully human monoclonal antibody of the present invention targets the relatively conserved core region of the receptor binding region (RBD) of SARS-CoV-2 and SARS-CoV (the mutation frequency of this region is low), and through different mechanism to play a neutralizing role.
  • RBD receptor binding region
  • the different antibodies isolated by the present invention can form a cocktail therapy to enhance the antiviral effect and solve the problem of drug resistance caused by the continuous mutation of the virus.
  • the present invention is a fully human monoclonal antibody, does not contain mouse-derived parts, has lower immunogenicity and higher safety for humans, and can avoid human anti-mouse and other species-derived antibodies mediated immunity Rejection.
  • the fully human monoclonal antibody of the present invention can be applied to the prevention and treatment of SARS-2-CoV and SARS-CoV infection-related diseases, and as a tool for clinical diagnosis.
  • the following describes the preparation of a fully human monoclonal antibody capable of neutralizing the RBD protein of the coronaviruses SARS-CoV-2 and SARS-CoV and the analysis process of the antibody characteristics.
  • Example 1 Obtaining antibody genes and antibody expression by single-cell RT-PCR method
  • PBMC peripheral blood mononuclear cells
  • Peripheral blood was drawn from healthy volunteers, using conventional Ficoll-Paque (manufactured by (CEDARLANE) density gradient centrifugation to obtain more than 10 7 peripheral blood mononuclear cells (PBMC).
  • CEDARLANE peripheral blood mononuclear cells
  • BD Horizon TM Fixable Viability Stain 780 was used to remove dead cells, and specific B cells were obtained by flow cytometry to 96-well RT-PCR plates. One cell was drilled to obtain SARS-CoV-2 RBD protein-specific memory B cells.
  • RBD protein was expressed by mammalian cell CHO expression system; refer to Invitrogen ExpiCHO-S TM Expression System manual; RBD protein sequence refer to nCoV-SH01 (GenBank: MT121215.1), and the whole gene synthesis was carried out in Shanghai Jierui Company. The whole gene synthesis was carried out in Shanghai Jierui Company, and it was constructed on the expression vector of invitrogen pcDNA3.1;
  • Biotin labeling of RBD protein No-Weigh Sulfo-NHS-LC-Biotin (purchased from PIERCE, refer to EZ-Link Sulfo-NHS-LC-Biotin Biotin Labeling Protocol of PIERCE Company) 10mM reagent; Two markers, FITC-CD19 and APC-IgG, were purchased from BD Bioscience; SA Streptavidin PE and streptavidin BV 421 (purchased from BD) were used to detect biotin-labeled SARS-CoV-2 RBD protein;
  • PBMC cells are grouped into groups, experimental group + control group, markers are added according to the number of cells, stained in the dark, labeled, resuspended in PBS, and filtered through a 40 ⁇ m BD falcon filter;
  • the obtained single memory B cells were obtained by RT-PCR to obtain cDNA, and then the variable region of the antibody gene was obtained by nested-PCR, and the agarose nucleic acid gel was run to recover and sequence the gel blocks where the heavy and light chains could be paired.
  • Antibody gene sequences were retrieved through IgBLAST (https://www.ncbi.nlm.nih.gov/projects/igblast/). Next, the antibody genes were linked to corresponding IgH, Ig ⁇ and Ig ⁇ expression vectors through AgeI and SalI restriction sites, AgeI and BsiwI restriction sites, AgeI and XhoI restriction sites, respectively.
  • the fully human antibody expression vectors IgH, Ig ⁇ and Ig ⁇ (respectively express antibody heavy chain, kappa chain, lambda chain) were donated by Patrick Wilson laboratory, and the vector sequences are shown in NCBI GenBank: FJ475055, FJ475056 and FJ517647.
  • CHO cells were transiently transfected for fully human antibody expression.
  • ExpiCHO-S TM cells were split to a final density of 3 x 106 - 4 x 106 viable cells/mL, and the cells were allowed to grow overnight.
  • viable cell density and percent viability were determined. The cell density should reach approximately 7 x 10 6 -10 x 10 6 viable cells/mL. Viability should be 95–99% before proceeding with transfection.
  • Cells were diluted to a final density of 6 x 106 viable cells/mL.
  • Expi Fectamine TM CHO/plasmid DNA complexes were prepared using pre-chilled reagents (4°C).
  • Antibodies were purified using Protein G Agarose 4FF media (purchased from GE). First, the collected CHO cell suspension was centrifuged at 4000rpm at 4°C for 30min, and the collected supernatant was filtered with a 0.45um filter for purification. Take a gravity-type spin column, add Protein G Agarose 4FF packing, stabilize the packing with 3 column volumes of 20% ethanol, then equilibrate the column with 5 column volumes of binding buffer, load the sample, and use 10 column volumes of binding The column was equilibrated with buffer, and finally the column was eluted with 3 column volumes of elution buffer, and neutralization buffer was added to the eluted antibody solution to make the pH of the eluted sample reach about 7.5. The antibody solution was dialyzed three times in 5L 1 ⁇ PBS, and the antibody was concentrated and stored at -80°C.
  • Single-cell RT-PCR and Nested-PCR methods were used to obtain matching antibody heavy and light chain variable region genes. Agarose gels were recovered and sequenced. Compare the sequencing results at http://www.ncbi.nlm.nih.gov/igblast and http://www.imgt.org/ to obtain antibody germline gene information and antibody heavy and light chain gene hypervariable regions information, and carry out expression vector construction and subsequent expression purification. Finally, through this technology, multiple fully human monoclonal antibodies that can broadly neutralize SARS-CoV-2 and SARS-CoV were successfully obtained.
  • amino acid sequence of the heavy chain variable region of the fully human antibody of the present invention is as follows, wherein the sequences of heavy chain amino acids CDR1, CDR2, and CDR3 are marked with underlining in order.
  • amino acid sequence of the light chain variable region of the fully human antibody of the present invention is as follows, wherein the sequences of light chain amino acid CDR1', CDR2' and CDR3' are marked with underlining in order.
  • ELISA was used to detect whether the expressed antibody recognized the RBD protein.
  • Coated RBD protein ELISA plate 0.5 ⁇ g/mL, 100 ⁇ L per well, overnight at 4°C. The next day, the plate was washed 3 times with PBST. Blocked with 2% BSA, 200 ⁇ L per well, 37°C, 2h. Wash the plate 3 times with PBST again.
  • RBD-specific antibodies were tested at 6 concentrations, 3-fold serial dilution, 2 duplicate wells, and the initial test concentration was 30 ⁇ g/ml. Loading, 100 ⁇ L per well, 37°C, 2h. Wash the plate 3 times with PBST.
  • Goat Anti-Human IgG (Fc specific)-Peroxidase antibody (sigma), diluted 1:5000, 100 ⁇ L per well, 37°C, 1h. Wash the plate 3 times with PBST. Add substrate TMB 100 ⁇ L/well to develop color, if the color is lighter, react at 37°C for 15min in the dark. Reactions were stopped by adding 2M H2SO4 , 50 ⁇ L per well. The OD450 was determined and data processed.
  • This experiment was performed using the OCTET RED 96 instrument at the Molecular Platform of the Institute of Biochemical Cells.
  • the full-length SARS-2-CoV S protein plasmid and pNL4-3 plasmid were co-transfected into HEK293T cells in a 10 cm dish, and the medium was changed for 6 hours. The supernatant was collected after 48 hours and diluted with complete medium.
  • the antibodies were serially diluted, mixed with an equal volume of virus, and incubated at 37°C for 1 h. The antibody and virus mixture was transferred into HEK293T cells stably expressing human ACE2. Incubate for 48 hours at 37°C. Remove the supernatant and add lysis buffer to the cells to fully lyse. Luciferase activity was assayed (Promega). Neutralization efficiency was calculated by comparing the luciferase value of the experimental group with the luciferase value of the virus-only control group. Calculated as follows:
  • IC50 values were calculated by Prism software.
  • Seeding cells Take Vero-E6 cells in logarithmic growth phase and seed them into a 96-well plate, 100 ⁇ l per well, 4 ⁇ 10 4 cells per well.
  • Neutralization experiment dilution of the test sample: as shown in Table 1, add 60 ⁇ l of 10 ⁇ g/ml pre-diluted sample in column 1 (rows B-G), and add 60 ⁇ l of virus diluent to the final antibody concentration of 5 ⁇ g/ml), and add the remaining wells in sequence 60 ⁇ l of 3-fold diluted sample.
  • the first row of cell control (Cell Control, CC) was added with 120 ⁇ l of serum-free medium
  • the eighth row of virus control (Virus Control, VC) was added with 60 ⁇ l of serum-free medium.
  • the virus stock titer is 2.5 ⁇ 10 5 TCID50/ml, take 200 ⁇ l of virus stock solution, add 25 ml of serum-free medium, mix well, and dilute the virus to 100 TCID50/50 ⁇ l.
  • Dropping virus vertically drop the virus (except cell control) into a 96-well plate, the sample volume is 60 ⁇ l/well, and the final virus-antibody mixture is 120 ⁇ l.
  • Neutralization Mix the added cell culture plate on a shaker, place it in a 37°C incubator, and neutralize for 1 hour. After neutralization, the supernatant of the culture plate inoculated with cells was aspirated, and then 100 ⁇ l/well of virus-serum mixture was added to it, and cultured in a 37°C CO 2 incubator for 1 hour for infection. After the virus infection, the supernatant from the culture plate was aspirated, and the plaque formation experimental samples were added to the maintenance medium containing 1% methylcellulose (DMEM medium containing 2% FBS), and placed in a 37°C CO2 incubator. Incubate for 72-96 hours.
  • DMEM medium containing 2% FBS maintenance medium containing 1% methylcellulose
  • RBD-specific antibodies can effectively block the binding of RBD to receptor ACE2
  • ACE2-FC recombinant protein was coated on 96 microtiter plates, and different serial dilutions of antibodies were incubated with biotinylated RBD protein before adding onto pre-coated ACE2-FC microplates. Detection was performed with HRP-conjugated streptavidin. As shown in Figure 2, 28-26K, 25-F7 and 25-D9 antibodies all effectively inhibited the binding of SARS-CoV-2 and SARS-CoV RBD to ACE2, and showed a dose-dependent effect. Moreover, the other four antibodies could not block the binding of RBD to ACE2.
  • SARS-CoV-2 and SARS-CoV pseudoviruses were packaged, and SARS-CoV-2 and SARS-CoV pseudoviruses were neutralized on 293T cells stably transfected with human ACE2.
  • experiments showed that 7 strains of antibodies neutralized SARS-CoV-2 and SARS-CoV pseudovirus with different abilities, and the half effective inhibitory concentration IC50 of SARS-CoV-2 pseudovirus was between 0.007-5.4 ⁇ g/ml.
  • the 50% effective inhibitory concentration IC50 of SARS-CoV pseudovirus was between 0.3-25 ⁇ g/ml ( Figure 3).
  • the inventors used single-cell RT-PCR technology to isolate 7 strains of SARS-CoV-2 and SARS-CoV cross-binding antibodies from the peripheral blood PBMCs of recovered volunteers infected with the new coronavirus. These antibodies have undergone the process of somatic hypermutation , has a high affinity for the RBD protein of the new crown. In vitro experiments confirmed that these seven antibodies can effectively inhibit the infection of host cells by SARS-CoV-2, SARS-CoV pseudovirus and SARS-CoV-2 true virus. also. The neutralization mechanisms of these antibodies are different, among which the 28-26K, 25-F7 and 25-D9 tristrain antibodies can effectively block the binding of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2.
  • the other four-clonal antibodies may exert neutralization through other mechanisms. These 7 antibodies are very likely to target the conserved regions of SARS-CoV-2 and SARS-CoV RBD, with relatively low mutation frequency, to deal with the problem of drug resistance due to the rapid formation of SARS-CoV-2 mutation, and to prevent and treat SARS. -CoV-2 has potential application value.

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Abstract

L'invention concerne un anticorps à neutralisation croisée à large spectre contre les coronavirus SARS-CoV-2 et SARS-CoV et son utilisation. En particulier, la présente invention concerne de multiples souches d'anticorps monoclonaux entièrement humains ciblant une protéine RBD de nouveaux coronavirus SARS-CoV-2 et SARS-CoV, une séquence d'acide nucléique codant pour un anticorps et un fragment d'anticorps, et son procédé de préparation. Des expériences in vitro montrent que l'anticorps peut inhiber de manière efficace une infection par des pseudo-virus SARS-CoV-2 et SARS-CoV et du virus eucaryote du SARS-CoV-2 de cellules hôtes, et jouent un rôle de neutralisation au moyen de différents mécanismes, ce qui permet de prévenir et de traiter de manière efficace une infection à coronavirus et de réduire l'incidence de la résistance aux médicaments.
PCT/CN2021/142696 2020-12-31 2021-12-29 Anticorps à neutralisation croisée à large spectre entièrement humain contre sars-cov-2 et sars-cov et son utilisation WO2022143816A1 (fr)

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CN115772218B (zh) * 2022-11-04 2023-08-29 四川大学 两种靶向新冠病毒s蛋白受体结合区的泛变异株广谱中和纳米抗体及其潜在应用
CN116284359A (zh) * 2023-02-11 2023-06-23 四川大学 两种靶向新冠病毒s2亚基茎螺旋表位的广谱中和纳米抗体及其潜在应用

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