WO2021196268A1 - Antibody having neutralizing activity against coronavirus, and use thereof - Google Patents

Antibody having neutralizing activity against coronavirus, and use thereof Download PDF

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Publication number
WO2021196268A1
WO2021196268A1 PCT/CN2020/084478 CN2020084478W WO2021196268A1 WO 2021196268 A1 WO2021196268 A1 WO 2021196268A1 CN 2020084478 W CN2020084478 W CN 2020084478W WO 2021196268 A1 WO2021196268 A1 WO 2021196268A1
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antibody
seq
protein
coronavirus
antigen
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PCT/CN2020/084478
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French (fr)
Chinese (zh)
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郎国竣
邵俊斌
谭永聪
姚航平
张文海
闫鑫甜
胡宇豪
孔超
周蕴华
闫闰
孙兴鲁
吴琪
姚福家
田美
韩晓刚
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三优生物医药(上海)有限公司
上海之江生物科技股份有限公司
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Publication of WO2021196268A1 publication Critical patent/WO2021196268A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention generally relates to antibodies and their uses. More specifically, the present invention relates to antibodies and antigen-binding fragments that specifically recognize the coronavirus spike protein, preparation methods and uses thereof.
  • coronavirus coronavirus
  • 2019novel CoV 2019-nCoV
  • the virus was originally identified in Wuhan, China in December 2019. It can be transmitted from person to person, and the patient appears to be severe Viral pneumonia and respiratory diseases. Since then, the number of cases infected with 2019-nCoV has been increasing. As of March 30, 2020, China has a total of 82,451 confirmed patients infected with the new coronavirus, of which 3311 have died. In addition, there are also nearly 640,000 confirmed patients infected with the new coronavirus in other countries. The number of cases is rising sharply, and the situation is very serious.
  • coronavirus binds to the receptor angiotensin converting enzyme II (also known as ACE2) on the host cell through the spike protein (S protein), and mediates the virus to enter the host cell (Ashour HM, etc.) Human, Insights into the Recent 2019 Novel Coronavirus (SARS-CoV-2) in Light of Past Human Coronavirus Outbreaks, Pathogens, March 4, 2020; 9(3).pii:E186.doi:10.3390/pathogens9030186; Roujian Lu Et al., Genomic characterisation and epidemiology of 2019novel coronavirus: implications for virus origins and receptor binding, www.thelancet.com, published online on January 29, 2020, https://doi.org/10.1016/S0140-6736(20) 30251-8), therefore, it is necessary in the art to develop high-affinity neutralizing antibodies that target the S protein of coronavirus and block its binding to the ACE2 receptor on host cells to effectively prevent and treat such coronaviruses
  • the present inventors have developed a set of human antibodies that specifically bind to the coronavirus S protein with high affinity and inhibit viral infectivity, thereby meeting the above-mentioned needs.
  • the antibody that specifically recognizes the S protein of coronavirus of the present invention can be used to satisfy the above-mentioned needs.
  • the antibody of the present invention has a neutralizing activity to inhibit virus infection of Vero E6 cells, and it protects 50% of cells from 100 TCID 50 attack. Degree) is less than about 0.5 nM, for example, about 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM.
  • the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to the S protein of coronavirus, which comprises
  • the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to the S protein of coronavirus, which comprises
  • SEQ ID NO: 8 The LCDR1 shown or the variant of LCDR1 shown in SEQ ID NO: 8 that does not exceed 2 amino acid changes, the LCDR2 shown in SEQ ID NO: 9 or the LCDR2 shown in SEQ ID NO: 9 does not exceed 2 amino acid changes
  • HCDR1 shown in SEQ ID NO: 11 or a variant with no more than 2 amino acid changes of HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12 or SEQ ID NO: 12 A variant of HCDR2 with no more than 2 amino acid changes, and a HCDR3 shown in SEQ ID NO: 13 or a variant of HCDR3 shown in SEQ ID NO: 13 with no more than 2 amino acid changes;
  • SEQ ID NO: 14 The LCDR1 shown or the variant of LCDR1 shown in SEQ ID NO: 14 that does not exceed 2 amino acid changes, the LCDR2 shown in SEQ ID NO: 15 or the LCDR2 shown in SEQ ID NO: 15 does not exceed 2 amino acid changes
  • the isolated antibody or antigen-binding fragment of the invention comprises
  • HCDR1 shown in SEQ ID NO: 11 HCDR2 shown in SEQ ID NO: 12 and HCDR3 shown in SEQ ID NO: 13
  • LCDR1 shown in SEQ ID NO: 14 and shown in SEQ ID NO: 15 The LCDR2 and the LCDR3 shown in SEQ ID NO: 16.
  • the isolated antibody or antigen-binding fragment of the invention comprises
  • Heavy chain variable region and light chain variable region wherein the heavy chain variable region comprises the sequence of SEQ ID NO:1 or has at least 90%, 91%, 92%, 93%, 94%, 95% therewith , 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region includes the sequence of SEQ ID NO: 2 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences; or
  • Heavy chain variable region and light chain variable region wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95% therewith , 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region includes the sequence of SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, Sequences that are 95%, 96%, 97%, 98%, or 99% identical.
  • the isolated antibodies of the invention comprise
  • SEQ ID NO: 17 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it, and SEQ ID NO: 18 or a light chain sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; or
  • SEQ ID NO: 19 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it, and SEQ ID NO: 20 or a light chain sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it.
  • the isolated antibodies or antigen-binding fragments of the invention are fully human antibodies.
  • the isolated antibody or antigen-binding fragment of the invention is an IgG1, IgG2, IgG3, or IgG4 antibody; preferably, an IgG1 or IgG4 antibody; more preferably, a human IgG1 or human IgG4 antibody.
  • the antigen-binding fragments of the present invention are Fab, Fab', F(ab') 2 , Fv, single-chain Fv, single-chain Fab, diabody.
  • the present invention provides a multispecific antibody that specifically binds to the coronavirus S protein, which comprises the antibody or antigen-binding fragment described in the first aspect that specifically binds to the epitope of the coronavirus S protein.
  • the present invention provides an antibody combination comprising the antibody or antigen-binding fragment described in the first aspect that specifically binds to the first epitope of the coronavirus S protein, and/or other specific binding to the coronavirus S protein
  • the antibody or antigen-binding fragment of the second epitope, wherein the first epitope and the second epitope may be the same epitope or different epitopes.
  • the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment of the above-mentioned first aspect or the multispecific antibody of the above-mentioned second aspect, a vector containing the nucleic acid (preferably, an expression vector ), a host cell containing the nucleic acid or the vector.
  • the host cell is prokaryotic or eukaryotic, for example, selected from E. coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments, multispecific antibodies.
  • the host cell is a 293 cell or a CHO cell.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment of the present invention, or the multispecific antibody of the present invention.
  • the host cell of the present invention is cultured under the condition of the nucleic acid of the multispecific antibody of the present invention, and the antibody or antigen-binding fragment of the present invention, or the multispecific antibody of the present invention is optionally recovered from the host cell or from the culture medium.
  • the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention, or the antibody combination of the present invention, and a pharmaceutically acceptable carrier.
  • the present invention provides the use of the antibody or antigen-binding fragment of the present invention, or the multispecific antibody of the present invention, for the preparation of drugs for preventing and/or treating coronavirus infections.
  • the coronavirus is 2019-nCoV virus.
  • the present invention provides a method for preventing and/or treating coronavirus infection in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention , The antibody combination of the present invention, or the pharmaceutical composition of the present invention.
  • the coronavirus is 2019-nCoV virus.
  • the antibody of the present invention and the antibody combination of the present invention can effectively block and/or inhibit coronavirus infection, and can be used for the prevention and/or treatment of coronavirus.
  • the present invention provides a kit for detecting coronavirus S protein in a sample, the kit comprising the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention, or the antibody combination of the present invention, with To implement the following steps:
  • the present invention provides antibodies or antibody combinations against coronavirus S protein, which have at least the following beneficial technical effects:
  • the binding of S protein to the receptor ACE2 is the first step for coronavirus to infect the host, and the S1 and S2 subunits of the S protein carry the function of binding receptors and promoting the fusion of the virus envelope and the host cell membrane.
  • the S protein is The ideal target antigen for the development of antibodies with virus-neutralizing activity.
  • S protein is a foreign protein that serves as a host (e.g., human) of coronaviruses (e.g., 2019-n CoV, SARS-CoV). Compared with the receptor protein ACE2, it is not easy to develop antibodies against S protein. There is an organized cross-reaction with subjects, and the safety is higher.
  • coronaviruses e.g., 2019-n CoV, SARS-CoV.
  • antibody drugs Compared to small molecule drugs, antibody drugs have higher specificity, lower toxic effects caused by off-target, and relatively long half-life, lower frequency of medication.
  • the antibody of the present invention is a fully human antibody, which can be clinically treated without humanization, has lower immunogenicity and better druggability.
  • antibodies against the S protein of coronavirus are obtained by screening human natural antibody libraries, in which two antibodies P16-A3 and P17-A11 are tested in vitro by ELISA As determined by the assay, the affinity for S protein is better than that of the control antibody CR3022, and can more significantly block the binding of S protein and ACE2; at the cellular level, the antibody can also significantly block S protein and Vero E6 cells.
  • the antibodies and antibody combinations of the present invention can effectively inhibit the infection of coronaviruses, and have great potential to become effective drugs for the prevention and treatment of such coronaviruses.
  • Figure 1 shows the production method and activity detection method of the fully human antibody targeting the 2019-nCoV coronavirus S protein of the present invention.
  • Figure 2A shows the binding activity of human ACE2-huFc and S protein RBD-mFc.
  • Figure 2B shows the binding activity of human ACE2-His to S protein S1-huFc (also called Spike S1-huFc) or S protein RBD-mFc (also called Spike RBD-mFc).
  • Figure 3A shows the binding ability of the output phage of the antibody to the S protein RBD-mFc in the ELISA assay in the first and second rounds of panning. It can be seen from this figure that each round has better enrichment. VSCM13 helper phage was used as a negative control.
  • Figure 3B shows the ability of the antibody to bind the S protein RBD-mFc in the ELISA assay of the phage output in the second and third rounds of panning. It can be seen from this figure that there is better enrichment in each round, and the best enrichment is 3rd-1 (that is, the No. 1 sample selected in the third round of panning).
  • Figure 4 shows the binding ability of the Fab supernatant of the candidate antibody to the S protein RBD-mFc in the ELISA assay. It can be seen from the figure that the candidate antibody P17-A11 Fab supernatant has good binding ability with S protein RBD-mFc; the candidate antibody P16-A3 Fab supernatant also has a certain binding ability with S protein RBD-mFc.
  • Figure 5 shows the ability of candidate antibodies to block the binding of S protein to ACE2 expressed on cells at the cellular level.
  • the results showed that the antibodies P16-A3 Fab and P17-A11 Fab both showed significant activity to block the binding of S protein to cells.
  • the MFI in the figure represents the average fluorescence intensity.
  • Figure 6 shows the molecular weight and purity identification results of candidate antibodies.
  • the purity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 are all greater than 98%.
  • Figure 7A shows the monomer purity identification result of the candidate antibody P16-A3, and the monomer purity is greater than 98%.
  • Figure 7B shows the monomer purity identification results of the candidate antibody P17-A11, and the monomer purity is greater than 98%.
  • Figure 7C shows the monomer purity identification result of the control antibody CR3022, and its monomer purity is greater than 98%.
  • Figure 8 shows the affinity activity of candidate antibodies specifically binding to protein S based on an ELISA assay.
  • Candidate antibodies P16-A3 and P17-A11 showed excellent and significantly better than the control antibody CR3022's specific binding to S protein activity.
  • the negative antibody in the figure is an isotype human IgG1 antibody.
  • Figure 9 shows the detection of the blocking activity of candidate antibodies based on ELISA.
  • Candidate antibodies P16-A3 and P17-A11 both show excellent ability to block the binding of viral S protein to isolated ACE2 protein, while the control antibody CR3022 has no blocking activity.
  • the negative antibody in the figure is an isotype human IgG1 antibody.
  • Figure 10 shows the grouping of epitopes of candidate antibodies using the double antibody sandwich method of ELISA.
  • the results show that on the RBD domain of the 2019-nCoV coronavirus S protein, antibody P17-A11 and antibody CR3022 bind different epitopes, while antibody P17-A11 and antibody P16-A3 bind the same epitope.
  • Figure 11 shows the grouping of epitopes of candidate antibodies using the competitive method of ELISA.
  • the results show that on the RBD domain of the 2019-nCoV coronavirus S protein, antibody P17-A11 and antibody CR3022 bind different epitopes, while antibody P17-A11 and antibody P16-A3 bind the same epitope.
  • Figure 12A shows the epitope grouping results of candidate antibodies P16-A3 and P17-A11 based on Fortebio, and the epitopes of antibodies P16-A3 and P17-A11 are the same.
  • Figure 12B shows the epitope grouping results of the candidate antibody P16-A3 and the control antibody CR3022 based on Fortebio. The epitopes of the antibody P16-A3 and the control antibody CR3022 are different.
  • Figure 12C shows the epitope grouping results of the candidate antibody P17-A11 and the control antibody CR3022 based on Fortebio. The epitopes of the antibody P17-A11 and the control antibody CR3022 are different.
  • Figure 13 shows the cell-level detection of candidate antibodies to block the activity of S protein binding on ACE2 naturally expressing cells.
  • Candidate antibodies P16-A3 and P17-A11 both show dose-dependent blocking activity, have excellent blocking effects at the cellular level and are significantly better than the control antibody CR0322.
  • the MFI in the figure represents the average fluorescence intensity.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” when used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence when referring to an antibody variable region that "comprises” a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.
  • Coronaviridae ⁇ -coronavirus
  • the virus particles are spherical or elliptical with a diameter of about 60-220 nm.
  • the virus is a single-stranded positive-strand RNA (+ssRNA) virus.
  • ssRNA positive-strand RNA
  • coronaviruses that are pathogenic to humans, most are related to mild clinical symptoms (Su S, Wong G, Shi W, et al., Epidemiology, genetic recombination, and pathogenesis of coronaviruses.
  • antibody is used in the broadest sense herein and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), as long as they exhibit the desired antigen-binding activity .
  • the antibody can be a complete antibody of any type and subtype (e.g., IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1 and IgA2) (e.g., having two full-length light chains and two full-length heavy chains). chain).
  • the monomer of a complete antibody is a tetrapeptide chain molecule formed by disulfide bonds connecting two full-length light chains and two full-length heavy chains, also known as the monomer of an Ig molecule.
  • Antibody monomer is the basic structure that constitutes an antibody.
  • isolated antibody is intended to refer to an antibody that is substantially free of other antibodies (Ab) with different antigen specificities (for example, an isolated antibody that specifically binds to the coronavirus S protein or an antigen-binding fragment thereof is substantially free of specific Sexually binds to antigens other than the Coronavirus S protein).
  • the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse Phase HPLC) to determine.
  • electrophoresis e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse Phase HPLC
  • blocking antibody As used herein, “blocking antibody”, “neutralizing antibody”, “antibody with neutralizing activity” or “neutralizing antibody” are used interchangeably herein and refer to an antibody that binds to a target antigen or It interacts with it and prevents the target antigen from binding or associating with a binding partner such as a receptor, thereby inhibiting or blocking the biological response that would otherwise occur due to the interaction of the target antigen with a binding partner such as the receptor. In the context of the present invention, it means that the binding of the antibody to the coronavirus S protein results in the inhibition of at least one biological activity of the coronavirus.
  • the neutralizing antibody of the present invention can prevent or block the binding of coronavirus S protein to ACE2.
  • Epitopes refers to an antigenic determinant that interacts with a specific antigen-binding site called a paratope in the variable region of an antibody molecule.
  • a single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects.
  • Epitopes can be formed by contiguous amino acids or discrete amino acids joined by tertiary folding of the protein. Epitopes formed by consecutive amino acids usually remain when exposed to a denaturing solvent, while epitopes formed by tertiary folding usually disappear when treated with a denaturing solvent. Epitopes usually include at least 3, and more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • antigen-binding fragment is a part or section of a complete or complete antibody that has fewer amino acid residues than a complete or complete antibody, which can bind to the antigen or compete with the complete antibody (ie, the complete antibody from which the antigen-binding fragment is derived) Binding antigen.
  • the antigen-binding fragment can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of the intact antibody.
  • Antigen-binding fragments include but are not limited to Fab, Fab', F(ab') 2 , Fv, single-chain Fv (scFv), single-chain Fab, diabody, single domain antibody (sdAb, Nanobody), Camel Ig, Ig NAR, F(ab)' 3 fragment, double-scFv, (scFv) 2 , mini-antibody, bi-functional antibody, tri-functional antibody, tetra-functional antibody, disulfide stabilized Fv protein (“dsFv”) .
  • the term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), hybrid antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof.
  • chimeric antibodies e.g., humanized murine antibodies
  • hybrid antibodies e.g., bispecific antibodies
  • antigen-binding fragments thereof for a more detailed description, please see: Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical
  • the terms “whole antibody”, “full-length antibody”, “full antibody” and “whole antibody” are used interchangeably herein to refer to at least two heavy chains (HC) and two Light chain (LC) glycoprotein.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • Mammalian heavy chains are classified into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ . Mammalian light chains are classified as lambda or kappa.
  • Immunoglobulins containing alpha, delta, epsilon, gamma, and mu heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM.
  • the complete antibody forms a "Y" shape.
  • the stem of Y is composed of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchains) are formed in the hinge.
  • the heavy chains ⁇ , ⁇ , and ⁇ have a constant region composed of three tandem (in a row) Ig domains, and a hinge region for increasing flexibility; the heavy chains ⁇ and ⁇ have a constant region composed of four immunoglobulin domains Area.
  • Each arm of Y includes the variable region and the first constant region of a single heavy chain that are bound to the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the light chain variable region and the heavy chain variable region respectively comprise a "framework" region with three hypervariable regions (also referred to as “complementarity determining regions” or “CDRs”) intervening.
  • CDRs complementarity determining regions
  • “Complementarity determining region” or “CDR region” or “CDR” or “hypervariable region” (herein can be used interchangeably with hypervariable region “HVR”) is an antibody variable domain that is hypervariable in sequence and A structurally defined loop ("hypervariable loop") and/or a region containing antigen contact residues ("antigen contact point”) is formed.
  • CDR is mainly responsible for binding to antigen epitopes.
  • the CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. For example, the residue ranges of CDR regions using Kabat and Chothia numbering under different assignment systems are shown in Table A below.
  • the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • CDR of the antibody of the present invention can be artificially evaluated and determined according to any scheme in the art or a combination thereof.
  • the term "CDR” or "CDR sequence” encompasses CDR sequences determined in any of the above-mentioned ways.
  • the sequences of the framework regions of different light chains or heavy chains have relative preservation in species (such as humans).
  • the framework region of the antibody (which is the combined framework region of the light chain and the heavy chain) is used to locate and align the CDRs in a three-dimensional space.
  • CDR is mainly responsible for binding to the epitope of the antigen.
  • Antibodies with different specificities that is, different combination sites for different antigens have different CDRs.
  • the CDRs are different between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDR are called specificity determining residues (SDR).
  • a “monoclonal antibody” is an antibody produced by a single clone of B lymphocytes or by cells in which the light chain and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from a fusion of myeloma cells and immune spleen cells.
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • the double-chain Fv species is composed of a dimer of a heavy chain variable domain and a light chain variable domain in a tight non-covalent association.
  • a heavy-chain variable domain and a light-chain variable domain can be covalently linked through a flexible peptide linker, so that the light chain and the heavy chain can be similar to the double-chain Fv category
  • the "dimeric" structure is associated.
  • the three hypervariable regions (HVR) of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer.
  • the six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific to the antigen) has the ability to recognize and bind to the antigen, but the affinity is lower than the complete binding site.
  • the Fab fragment contains the variable domain of the heavy chain and the variable domain of the light chain and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • the difference between the Fab' fragment and the Fab fragment is that several residues are added to the carboxyl end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of an antibody.
  • Fab'-SH is the name of Fab' herein, in which the cysteine residue of the constant domain carries a free thiol group.
  • F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.
  • the term "specifically binds" or “binding” used when talking about antigens and antibodies means that the antibody forms a complex with an antigen that is relatively stable under physiological conditions.
  • Methods for determining whether an antibody specifically binds to an antigen include, for example, surface plasmon resonance assays, MSD assays (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2): p.270-278), ForteBio affinity assay (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs, 2013.5(2): p.270-8) and so on.
  • the "specific binding" coronavirus S protein antibody of the present invention is measured in the ForteBio affinity assay at at least about 10 -8 M, preferably 10 -9 M; more preferably 10 -10 M, and still more preferably A K D of 10 -11 M, more preferably 10 -12 M binds to the S protein, thereby blocking or inhibiting the binding of the coronavirus S protein to its receptor ACE2 and subsequent membrane fusion.
  • Binding affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, as used herein, "binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X to its partner Y can usually be expressed by the binding and dissociation equilibrium constant (K D ). Affinity can be measured by common methods known in the art, including those known in the prior art and those described herein.
  • the term “competition” in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding protein or neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay:
  • the antigen-binding protein to be detected for example, an antibody or immunological functional fragment thereof
  • prevents or inhibits for example, reduces
  • a reference antigen-binding protein for example, a ligand or a reference antibody
  • a common antigen for example, S protein or Its fragment
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253.
  • the assay involves the use of purified antigen bound to a solid surface or cell with either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Usually the tested antigen binding protein is present in excess.
  • the antigen binding proteins identified by competition assays include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding that binds to adjacent epitopes that are sufficiently close to the binding epitope of the reference antigen binding protein Proteins, the two epitopes sterically hinder each other from binding. Additional details on the methods used to determine competitive binding are provided in the examples herein.
  • variable refers to a heavy chain variable region or a light chain variable region that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, including heavy or light chain variants.
  • the modified antigen-binding protein of the body basically retains the biological characteristics of the pre-modified antigen-binding protein.
  • the antigen binding protein containing the sequence of the variable heavy chain variable region or the variable region of the light chain retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein before modification. It should be understood that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region.
  • the antigen binding protein of the present disclosure contains 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the heavy chain variable region described herein The amino acid sequence of the variable region of the heavy chain.
  • the antigen binding protein of the present disclosure includes 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the light chain variable region described herein The amino acid sequence of the variable region of the light chain.
  • the percent homology can be in the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology can be limited to the framework region, and the sequence corresponding to the CDR is the same as the heavy chain variable region and/or light chain.
  • the CDRs disclosed herein have 100% identity within the variable region.
  • the term "CDR variant” refers to a CDR that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, wherein the modified antigen binding protein comprising the CDR variant substantially retains the pre-modification The biological characteristics of antigen binding proteins.
  • the antigen binding protein containing the variant CDR retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein before modification. It should be understood that each CDR that can be modified can be modified alone or in combination with another CDR.
  • the modification is a substitution, especially a conservative substitution.
  • Humanized antibody refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, and the remaining immunoglobulin portion of the humanized antibody is derived from one (or more) humans Immunoglobulin.
  • framework support residues can be changed to retain binding affinity (see, for example, Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al., Bio/Technology, 9: 421 (1991)).
  • Suitable human accepting antibodies may be antibodies selected from conventional databases such as Los Alamos database and Swiss protein database by homology with the nucleotide and amino acid sequence of the donor antibody.
  • Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody may be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDR.
  • a suitable acceptor antibody capable of providing constant or variable framework regions of the light chain can be selected in a similar manner. It should be noted that the acceptor antibody heavy chain and light chain need not be derived from the same acceptor antibody.
  • polynucleotide or “nucleic acid” used interchangeably herein refers to a chain of nucleotides of any length, and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.
  • the sequences are aligned for optimal comparison purposes (for example, the first and second amino acid sequences or nucleic acid sequences may be used for optimal alignment. Gaps can be introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • Mathematical algorithms can be used to achieve sequence comparison between two sequences and calculation of percent identity.
  • the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used. Available), use Blossum 62 matrix or PAM250 matrix and gap weight 16, 14, 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6, to determine the difference between two amino acid sequences Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 are used. Length weights 1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossom 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
  • vector refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors related to cationic flocculants, DNA or RNA expression encapsulated in liposomes Vectors and certain eukaryotic cells, such as producer cells.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the offspring may not be exactly the same as the parent cell in terms of nucleic acid content, but they may contain mutations. This document includes mutant progeny that have the same function or biological activity as the cells screened or selected in the initially transformed cells.
  • the present invention also relates to a method for producing a monoclonal antibody, the method comprising culturing the host cell of the present invention to produce the above-mentioned monoclonal antibody of the present invention.
  • subject refers to animals in need of alleviation, prevention and/or treatment of diseases or conditions such as viral infections, preferably mammals, and more preferably humans. Mammals also include, but are not limited to, farm animals, race animals, pets, primates, horses, dogs, cats, mice, and rats. The term includes human subjects who have coronavirus infection or are at risk of coronavirus infection.
  • administering the antibody of the present invention or the pharmaceutical composition or product of the present invention to a subject in need thereof means administering an effective amount of the antibody or pharmaceutical composition or product or the like.
  • the term "effective amount” means the amount of a drug or agent that elicits a biological or pharmaceutical response of a tissue, system, animal, or human, for example, which is sought by a researcher or clinician.
  • therapeutically effective amount refers to an amount that causes an improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or reduces the rate of progression of the disease or condition, compared to a corresponding subject who did not receive the amount. ⁇ The amount. The term also includes within its scope an amount effective to enhance normal physiological functions.
  • Coronavirus targeted by the antibody of the present invention its structure and the way to enter the host cell
  • Coronavirus (including SARS-CoV and the newly discovered 2019-nCoV) is a spherical single-stranded positive-stranded RNA virus, which is characterized by a spike protein protruding from the surface of the virion (Barcena, M. et al., Cryo-electron Tomography of mouse hepatitis virus: Insights into the structure of the coronavirion.Proc.Natl.Acad.Sci.USA2009,106,582–587).
  • the spherical shape of the virus particles and the protrusions of the spikes make the coronavirus look like a crown under the electron microscope and it is named the coronavirus.
  • Coronavirus is an enveloped virus (the envelope is derived from the lipid bilayer of the host cell membrane), which is mainly composed of viral structural proteins (such as spike protein (Spike, S), membrane protein (Membrane, M), A virus structure formed by membrane protein (Envelope, E) and nucleocapsid protein (Nucleocapsid, N), in which S protein, M protein, and E protein are all embedded in the viral envelope, and N protein interacts with viral RNA and is located in the virus The core of the particle forms the nucleocapsid (Fehr, AR et al., Coronaviruses: An overview of their replication and pathogenesis. Methods Mol. Biol. 2015, 1282, 1-23).
  • S protein is a highly glycosylated protein that can form homotrimeric spikes on the surface of virus particles and mediate the virus into host cells.
  • 2019-nCoV is a single-stranded positive-stranded RNA virus with a membrane structure and a size of 80-120nm.
  • the genome length is about 29.9kb.
  • This virus is between the genome sequence of SARS-CoV, which belongs to the ⁇ -coronavirus genus of the coronavirus family.
  • the homology is 80%.
  • the open reading frame (ORF) of the viral genome, ORF1a and ORF1b account for 2/3 of the genome, expressing hydrolases and enzymes related to replication and transcription, such as cysteine protease (PLpro) and serine protease (3CLpro).
  • RNA-dependent RNA polymerase RdRp
  • helicase Hel
  • S spike protein
  • E envelope protein
  • M membrane protein
  • N Nucleocapsid protein
  • the N protein wraps the viral genome to form a nucleoprotein complex
  • the E protein and M protein are mainly involved in the assembly process of the virus
  • the S protein is mainly mediated by binding to host cell receptors. The invasion of the virus determines the host specificity of the virus. After sequence comparison, it is found that the S protein of 2019-nCoV virus and SARS-CoV virus has a similarity of 75%.
  • ACE2 is also the receptor protein for 2019-nCoV to infect the human body into the cell. It is expected that high-affinity neutralizing antibodies that target the coronavirus S protein and block its binding to the ACE2 receptor can effectively prevent and treat coronavirus (for example, 2019-n CoV, SARS-CoV) infections.
  • coronavirus for example, 2019-n CoV, SARS-CoV
  • antibody against coronavirus S protein refers to the antibody of the present invention that can bind to the coronavirus S protein (for example, 2019-n CoV S protein, SARS-CoV S protein) with sufficient affinity.
  • the antibody can be used as a diagnostic, preventive and/or therapeutic agent targeting the S protein of coronavirus.
  • the antibodies and antigen-binding fragments of the present invention specifically bind to the coronavirus S protein with high affinity.
  • the antibody of the present invention is a blocking antibody or a neutralizing antibody, wherein the antibody can bind to the coronavirus S protein and block the binding of the coronavirus S protein to ACE2.
  • the blocking antibody or neutralizing antibody can be used to prevent coronavirus infection and/or treat individuals infected with coronavirus.
  • the coronavirus S protein antibody of the present invention specifically binds to the coronavirus S protein, which comprises
  • amino acid change is an addition, deletion or substitution of an amino acid, for example, the amino acid change is a conservative amino acid substitution.
  • the coronavirus S protein antibody of the present invention binds to mammalian coronavirus S protein, such as human coronavirus S protein, monkey coronavirus S protein.
  • the coronavirus S protein antibody of the present invention specifically binds to an epitope (e.g., linear or conformational epitope) on the coronavirus S protein.
  • the coronavirus S protein antibody of the present invention has one or more of the following characteristics:
  • the neutralizing activity of the antibody of the present invention to inhibit virus infection of Vero E6 cells is significantly better than that of the control antibody, and it protects 50% of cells from 100TCID50 attack.
  • the antibody titer is less than about 0.5 nM, such as about 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM.
  • the coronavirus S protein antibody of the present invention comprises HCDR1 shown in SEQ ID NO: 5 or a variant of HCDR1 shown in SEQ ID NO: 5 with no more than 2 amino acid changes, SEQ ID NO: 6 No more than 2 amino acid variants of HCDR2 or HCDR2 shown in SEQ ID NO: 6 and HCDR3 shown in SEQ ID NO: 7 or HCDR3 shown in SEQ ID NO: 7 not more than 2 A variant of amino acid change; LCDR1 shown in SEQ ID NO: 8 or a variant of LCDR1 shown in SEQ ID NO: 8 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO: 9 or SEQ ID NO: The variant of LCDR2 shown in 9 with no more than 2 amino acid changes, and the LCDR3 shown in SEQ ID NO: 10 or the variant of LCDR3 shown in SEQ ID NO: 10 with no more than 2 amino acid changes, wherein Amino acid changes are additions, deletions or substitutions of amino acids, for example
  • the coronavirus S protein antibody of the present invention comprises HCDR1 shown in SEQ ID NO: 11 or a variant of HCDR1 shown in SEQ ID NO: 11 with no more than 2 amino acid changes, SEQ ID NO: 12 No more than 2 amino acid variants of HCDR2 or HCDR2 shown in SEQ ID NO: 12, and HCDR3 shown in SEQ ID NO: 13 or HCDR3 shown in SEQ ID NO: 13 not more than 2 A variant of amino acid changes; LCDR1 shown in SEQ ID NO: 14 or a variant of LCDR1 shown in SEQ ID NO: 14 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO: 15 or SEQ ID NO: A variant of LCDR2 shown in 15 with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO: 16 or a variant of LCDR3 shown in SEQ ID NO: 16 with no more than 2 amino acid changes, wherein Amino acid changes are additions, deletions or substitutions of amino acids, for example
  • the coronavirus S protein antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 1 or has at least 90% of the sequence of SEQ ID NO:1. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region contains the sequence of SEQ ID NO: 2 or has A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.
  • the amino acid change does not occur in the CDR region.
  • the coronavirus S protein antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3 or has a sequence of at least 90%. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region contains the sequence of SEQ ID NO: 4 or has A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.
  • the amino acid change does not occur in the CDR region.
  • the coronavirus S protein antibody of the present invention comprises an Fc region derived from IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region is derived from human IgG1 or human IgG4.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • Conservative substitution refers to the substitution of an amino acid by another amino acid in the same category, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid replace. Exemplary substitutions are shown in Table B below:
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu, Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val
  • the amino acid changes described in the present invention occur in regions outside the CDR (for example, in the FR). More preferably, the amino acid changes described in the present invention occur in the Fc region.
  • an anti-coronavirus S protein antibody comprising an Fc domain containing one or more mutations that enhance or weaken the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH, for example .
  • the present invention includes a coronavirus S protein antibody containing mutations in the C H Fc domain or 2 C H 3 region, wherein the one or more mutations that improve Fc domain in an acidic environment (e.g.
  • Fc modifications include, for example, positions 250 (for example, E or Q), positions 250 and 428 (for example, L or F), positions 252 (for example, L/Y/F/W or T), 254 Bit (such as S or T) and 256 bit (such as S/R/Q/E/D or T) modification; or 428 bit and/or 433 bit (such as H/L/R/S/P/Q or K ) And/or modification of position 434 (for example, A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modification of position 250 and/or 428; or modification of position 307 or 308 ( For example, 308F, V308F) and the modification of position 434.
  • positions 250 for example, E or Q
  • positions 250 and 428 for example, L or F
  • positions 252 for example, L/Y/F/W or T
  • 254 Bit such as S or T
  • 256 bit such as
  • the modification includes 428L (e.g. M428L) and 434S (e.g. N434S) modifications; 428L, 2591 (e.g. V259I) and 308F (e.g. V308F) modifications; 433K (e.g. H433K) and 434 (e.g. 434Y) modifications; 252, 254, and 256 (e.g. 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g. T250Q and M428L); and 307 and/or 308 modifications (e.g. 308F or 308P).
  • the modification includes 265A (e.g. D265A) and/or 297A (e.g. N297A) modifications.
  • the present invention includes an anti-coronavirus S protein antibody containing an Fc domain containing one or more pairs (groups) of mutations selected from: 250Q and 248L (for example, T250Q and M248L); 252Y , 254T and 256E (such as M252Y, S254T and T256E); 428L and 434S (such as M428L and N434S); 257I and 311I (such as P257I and Q311I); 257I and 434H (such as P257I and N434H); 376V and 434H (such as D376V and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); and 433K and 434F (e.g., H433K and N434F).
  • groups selected from: 250Q and 248L (for example, T250Q and M248L); 252Y , 254
  • the present invention includes an anti-coronavirus S protein antibody containing an Fc domain that includes the S108P mutation in the hinge region of IgG4 to promote dimer stabilization. Any possible combination of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are included within the scope of the present invention.
  • the coronavirus S protein antibodies provided herein are modified to increase or decrease the degree of glycosylation.
  • the addition or deletion of glycosylation sites of the coronavirus S protein antibody can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites.
  • the carbohydrates linked to the Fc region can be changed.
  • modifications to remove unwanted glycosylation sites may be useful, such as removing fucose moieties to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733 ).
  • ADCC antibody-dependent cellular cytotoxicity
  • galactosidation can be modified to modulate complement dependent cytotoxicity (CDC).
  • one or more amino acid modifications can be introduced into the Fc region of the coronavirus S protein antibody provided herein to generate Fc region variants, so as to enhance the prevention of the coronavirus S protein antibody of the present invention, for example. And/or the effectiveness of the treatment of coronavirus infections.
  • the coronavirus S protein antibody of the present invention is in the form of a bispecific or multispecific antibody molecule.
  • the bispecific antibody molecule has a first binding specificity for a first epitope of the coronavirus S protein and a second binding specificity for a second epitope of the coronavirus S protein, wherein the first table The position and the second epitope can be the same, or they can be different and non-overlapping.
  • the bispecific antibody molecule comprises the antibodies P17-A11 Fab and P16-A3 Fab of the invention.
  • the bispecific antibody molecule comprises the antibodies P17-A11 scFv and P16-A3 scFv of the present invention.
  • the present invention relates to an antibody combination comprising the antibody or antigen-binding fragment of the present invention that specifically binds to the epitope of the coronavirus S protein, and/or other antibodies or antigens that specifically bind to the epitope of the coronavirus S protein Combine fragments.
  • the antibody combination is a combination of antibody P17-A11 or an antigen-binding fragment thereof and antibody P16-A3 or an antigen-binding fragment thereof.
  • nucleic acid of the present invention and the host cell containing it
  • the present invention provides a nucleic acid encoding any of the above coronavirus S protein antibodies or antigen-binding fragments or any chain thereof.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector.
  • a host cell comprising the nucleic acid or the vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (such as CHO cells or 293 cells), or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, and 4, or a nucleic acid that encodes an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, and 4.
  • the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 17, 18, 19, 20, or a nucleic acid that encodes an amino acid sequence selected from SEQ ID NO: 17, 18,
  • the amino acid sequence shown in any one of 19 and 20 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity Amino acid sequence of nucleic acid.
  • the present invention also covers nucleic acids that hybridize with the following nucleic acids under stringent conditions, or nucleic acids that encode polypeptide sequences having one or more amino acid substitutions (eg conservative substitutions), deletions, or insertions compared with the following nucleic acids: comprising coding options A nucleic acid derived from the nucleic acid sequence of the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4; or a nucleic acid containing a code selected from any one of SEQ ID NO: 1, 2, 3, 4
  • the amino acid sequence has a nucleic acid sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include but are not limited to viruses, plasmids, cosmids, lambda phage or yeast artificial chromosomes (YAC).
  • the expression vector can be transfected or introduced into a suitable host cell.
  • Various techniques can be used to achieve this goal, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.
  • protoplast fusion the cells are grown in a culture medium and screened for appropriate activity.
  • the methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the methods known in this specification and the prior art, depending on the specific expression vector and Mammalian host cell changes or optimizations.
  • the selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements can include splicing signals, as well as transcription promoters, enhancers, and termination signals.
  • a host cell comprising the polynucleotide of the invention.
  • a host cell comprising an expression vector of the invention is provided.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies. Suitable host cells include prokaryotic microorganisms such as Escherichia coli.
  • the host cell can also be a eukaryotic microorganism such as filamentous fungus or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as hosts.
  • a mammalian cell line modified to be suitable for growth in suspension can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells ( CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver Cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0.
  • COS-7 SV40 transformed monkey kidney CV1 line
  • HEK 293 or 293F cells human embryonic kidney line
  • 293 cells BHK
  • monkey kidney cells CV1
  • African green monkey kidney cells VERO-76
  • HELA human cervical cancer cells
  • MDCK buffalo rat liver cells
  • W138 human lung cells
  • Hep G2 Chinese ham
  • the host cell is a CHO cell or 293 cell.
  • the present invention provides a method for preparing a coronavirus S protein antibody, wherein the method comprises culturing a nucleic acid encoding the coronavirus S protein antibody under conditions suitable for expressing a nucleic acid encoding the coronavirus S protein antibody.
  • the nucleic acid of the antibody or the host cell containing the expression vector of the nucleic acid, and optionally the coronavirus S protein antibody is isolated.
  • the method further includes recovering the coronavirus S protein antibody from the host cell (or host cell culture medium).
  • the coronavirus S protein antibody of the present invention In order to recombinantly produce the coronavirus S protein antibody of the present invention, first isolate the nucleic acid encoding the coronavirus S protein antibody of the present invention, and insert the nucleic acid into a vector for further cloning and/or expression in host cells. Such nucleic acids are easily separated and sequenced using conventional procedures, for example, by using oligonucleotide probes that can specifically bind to the nucleic acid encoding the coronavirus S protein antibody of the present invention.
  • the coronavirus S protein antibody of the present invention prepared as described herein can be purified by known prior art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these are obvious to those skilled in the art.
  • the purity of the coronavirus S protein antibody of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, and the like.
  • the coronavirus S protein antibody provided herein can be identified, screened, or characterized by a variety of assays known in the art, or its physical/chemical properties and/or biological activity.
  • the coronavirus S protein antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot and the like.
  • the binding to the coronavirus S protein can be determined using methods known in the art, and exemplary methods are disclosed herein.
  • SPR or biofilm layer interference is used to determine the binding of the coronavirus S protein antibody of the present invention to the coronavirus S protein.
  • the present invention also provides an assay method for identifying the coronavirus S protein antibody with biological activity.
  • the biological activity may include, for example, blocking the binding of ACE2 on the cell surface.
  • the present invention provides a composition comprising any coronavirus S protein antibody described herein, preferably the composition is a pharmaceutical composition.
  • the composition further comprises pharmaceutical excipients.
  • the composition e.g., pharmaceutical composition
  • the composition comprises the coronavirus S protein antibody of the present invention and a combination of one or more other therapeutic agents (e.g., anti-infective agents, small molecule drugs).
  • the anti-infective agent and small molecule drug are any anti-infective agent or small molecule drug used to treat, prevent or alleviate coronavirus infection in subjects, including but not limited to remdesivir, ribavirin, Oseltamivir, zanamivir, hydroxychloroquine, interferon- ⁇ 2b, analgesics, azithromycin, and corticosteroids.
  • coronavirus infections include infections caused by coronaviruses (including but not limited to 2019-n CoV and SARS-CoV).
  • the pharmaceutical composition or pharmaceutical preparation of the present invention contains suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
  • pharmaceutical carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, etc. that are physiologically compatible.
  • Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. It is also possible to use saline solutions and aqueous dextrose and glycerol solutions as liquid carriers, especially for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skimmed milk, glycerin , Propylene, glycol, water, ethanol, etc.
  • excipients see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.
  • the composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, and saccharin.
  • coronavirus S protein antibody of the present invention can be prepared by mixing the coronavirus S protein antibody of the present invention with the required purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)).
  • the pharmaceutical preparation of the coronavirus S protein antibody described herein is preferably in the form of a lyophilized preparation or an aqueous solution.
  • the pharmaceutical composition or formulation of the present invention may also contain more than one active ingredient that is required for the specific indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other.
  • active ingredients such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators.
  • the active ingredients are suitably present in combination in an amount effective for the intended use.
  • sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the coronavirus S protein antibody of the present invention, which matrices are in the form of shaped articles, such as films or microcapsules.
  • the present invention also provides a combination product, which comprises at least one coronavirus S protein antibody or antigen-binding fragment thereof of the present invention, or further comprises one or more other therapeutic agents (e.g., anti-infective activity). Agents, small molecule drugs or immunomodulators, etc.).
  • two or more ingredients in the combination product may be administered to the subject in combination sequentially, separately, or simultaneously.
  • the present invention also provides a kit containing the coronavirus S protein antibody, pharmaceutical composition, or combination product of the present invention, and optionally a package insert to guide administration.
  • the present invention also provides a pharmaceutical product comprising the coronavirus S protein antibody, pharmaceutical composition, and combination product of the present invention.
  • the pharmaceutical product further includes a package insert for guiding administration.
  • the present invention provides a method for preventing a coronavirus-related disease or disease in a subject, which comprises administering the antibody, antibody combination or multispecific antibody of the present invention to the subject.
  • Subjects at risk of coronavirus-related diseases include subjects who have been in contact with an infected person or subjects who have been exposed to the coronavirus in some other way.
  • the administration of the prophylactic agent may be administered before the symptomatic characteristics of the coronavirus-related disease are manifested in order to prevent the disease, or alternatively delay the progression of the disease.
  • the present invention also provides methods for treating coronavirus-related diseases in patients.
  • the method involves administering an antibody, antibody combination or multispecific antibody of the invention that neutralizes coronavirus to a patient suffering from the disease.
  • a method of treating a coronavirus infection in a patient comprising administering at least one antibody or antigen-binding fragment thereof selected from the group consisting of antibodies P16-A3, P17-A11.
  • two of the antibodies P16-A3, P17-A11 or fragments thereof are administered to the patient together,
  • the antibodies or antigen-binding fragments thereof of the present invention can cross-neutralize human and animal infectious coronavirus isolates.
  • the antibodies of the invention or antigen-binding fragments thereof are administered within the first 24 hours after coronavirus infection.
  • any of the coronavirus S protein antibodies provided herein can be used to detect the presence of coronavirus in a biological sample.
  • detection includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule complexed magnetic beads, ELISA assays Law.
  • a coronavirus S protein antibody for use in a method of diagnosis or detection is provided.
  • a method of detecting the presence of coronavirus in a biological sample includes detecting the presence of coronavirus S protein in a biological sample.
  • the method includes contacting a biological sample with a coronavirus S protein antibody as described herein under conditions that allow the coronavirus S protein antibody to bind to the coronavirus S protein, and detecting the presence of the coronavirus S protein Whether a complex is formed between the antibody and the coronavirus S protein. The formation of the complex indicates the presence of coronavirus.
  • the method can be an in vitro or in vivo method.
  • Exemplary diagnostic assays for coronavirus include, for example, contacting a sample obtained from a patient with the anti-coronavirus S protein of the present invention, where the anti-coronavirus S protein is labeled with a detectable marker or reporter molecule or used as a capture ligand to select sexually isolate the coronavirus from patient samples.
  • the unlabeled anti-coronavirus S protein can be used in diagnostic applications in combination with a second antibody that itself is detectably labeled.
  • the detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125 I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine, or enzymes such as alkali Phosphatase, ⁇ -galactosidase, horseradish peroxidase or luciferase.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • the samples that can be used in the coronavirus diagnostic assay according to the present invention include any biological samples available from patients that contain a detectable amount of coronavirus spike protein or fragments thereof under normal or physiological conditions.
  • the biological sample is blood, serum, throat swabs, lower respiratory tract samples (e.g. tracheal secretions, tracheal aspirates, alveolar lavage fluid) or other samples of biological origin.
  • the level of coronavirus spike protein in a specific sample obtained from healthy patients will be measured to initially establish a baseline or standard coronavirus level. This baseline level of coronavirus can then be compared with the level of coronavirus measured in a sample obtained from an individual suspected of having a coronavirus-related condition or symptoms related to the condition.
  • the antibody specific for the coronavirus spike protein may not contain other labels, or it may contain N-terminal or C-terminal labels.
  • the label is biotin.
  • the position of the label determines the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, the peptide containing the N-terminal biotin will be oriented so that the C-terminal part of the peptide is away from the surface.
  • the following antigens and ACE2 proteins are shared: S protein RBD-His (319Arg-532Asn), S protein S1-huFc (14Gln-685Arg), human ACE2-huFc (18Gln-740Ser), human ACE2-His (18Gln- 740Ser), S protein RBD-mFc (purchased from Sinobio, 40592-V05H), the specific preparation methods of the first four proteins are as follows.
  • Each protein sequence was obtained from NCBI, among which the human ACE2 sequence was obtained from NCBI Gene ID: 59272, and the S protein sequence was obtained from NCBI Gene ID: 43740568.
  • Each protein sequence was obtained according to the position of the above amino acid fragments, and then transformed into gene sequences by GenScript Biotechnology Co., Ltd. conducts gene synthesis of the target fragment.
  • Each target fragment was amplified by PCR, and then constructed into the eukaryotic expression vector pcDNA3.3-TOPO (Invitrogen) by the method of homologous recombination for subsequent expression of the recombinant protein.
  • the constructed recombinant protein expression vectors were transformed into Escherichia coli SS320 and cultured overnight at 37°C. Plasmid extraction was performed using endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for supply Eukaryotic expression use.
  • OEGA endotoxin-free plasmid extraction kit
  • the resulting Fc-tagged protein expression supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100mM sodium acetate (pH3.0), followed by 1M Tris -HCl neutralization; the obtained His tag protein expression supernatant was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with a gradient concentration of imidazole. The eluted proteins were replaced into PBS buffer through an ultrafiltration tube (Millipore, UFC901096). After SDS-PAGE identification and activity identification qualified, frozen at -80°C for later use.
  • S protein S1-huFc also known as Spike-S1-huFc; or S1-huFc
  • S protein RBD-mFc also known as Spike-RBD-mFc; or RBD-mFc
  • a phage display library of antibody genes was constructed, and the recombinant 2019-nCoV coronavirus RBD protein (ie, Spike-RBD-mFc, Sinobio, 40592-V05H) was used as the screening antigen to screen the library, and obtained Multiple antibody molecules that specifically bind to the 2019-nCoV coronavirus RBD protein.
  • the recombinant 2019-nCoV coronavirus RBD protein ie, Spike-RBD-mFc, Sinobio, 40592-V05H
  • Ficoll-Paque density gradient separation solution (purchased from GE Company, catalog number: 17144003S) and slowly add it to a 50 mL centrifuge tube. Tilt the centrifuge tube and slowly add 15 mL of collected normal human blood along the tube wall in batches, so that the Ficoll-Paque density gradient separation solution and normal human blood maintain a clear separation interface. Centrifuge the 50 mL centrifuge tube containing the blood and the separating solution at about 15°C for 20 minutes, where the centrifuge is set to 400 g, the acceleration is 3, and the deceleration is 0 parameters.
  • the entire liquid surface is divided into four layers, the upper layer is plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid.
  • the middle layer is Ficoll-Paque liquid.
  • PBMC PBMC Cell layer.
  • the separated PBMC was first rinsed with PBS twice, then centrifuged at 1500 rpm at 4°C for 10 min, and finally resuspended with 1.5 mL of PBS, and counted by a cell counter (CountStar, CountStar Altair).
  • RNA was extracted from the isolated PBMC cells by conventional methods.
  • Reverse transcription kit purchased from TaKaRa company, catalog number: 6210A
  • degenerate primers were designed at the front end of the V region of the heavy chain and the light chain and the rear end of the first constant region (Li Xiaolin, large-capacity non-immune human Fab phage antibody
  • the construction and preliminary screening of the library the master's thesis of "Peking Union Medical College of China", June 2007), after PCR, the antibody heavy chain variable region gene fragment and light chain variable region gene fragment were obtained.
  • the fragment containing the light and heavy chain variable region of the antibody is amplified by the fusion PCR method, and then the PCR product and the vector for phage display are performed Enzyme digestion, recovery and ligation, and the ligation product is recovered by a recovery kit (Omega, catalog number: D6492-02).
  • a recovery kit (Omega, catalog number: D6492-02).
  • the transformed Escherichia coli SS320 broth was inoculated with antibiotic-free 2YT broth at a volume of 1:50, incubated at 37°C and 220rpm for 1.5-2h until OD600 reached 0.5-0.6, and then taken out to room temperature.
  • First select the counting hole first select the counting hole with the number of clones in 3-20 clones, get the number of rows X, and count the number of clones in the corresponding hole n, the calculation formula is 5 ⁇ 100 ⁇ 10 X ⁇ n, By calculation, an antibody gene library with a storage capacity of 3 ⁇ 10 11 cfu per milliliter of bacterial liquid, which is 3 ⁇ 10 11 antibody genes, was obtained.
  • MOI multiplicity of infection
  • the culture was centrifuged at 10000 rpm for 5 minutes, the supernatant was discarded, and the culture medium was replaced with carbenicillin 50 ⁇ g/mL/kanamycin 40 ⁇ g/mL double-resistant 2-YT medium (hereinafter also referred to as C + /K + 2-YT medium), and continue to culture overnight at 30°C and 220 rpm.
  • C + /K + 2-YT medium carbenicillin 50 ⁇ g/mL/kanamycin 40 ⁇ g/mL double-resistant 2-YT medium
  • the bacterial solution was centrifuged at 13000g for 10 minutes, and the supernatant was collected and 20% PEG/NaCl (prepared from 20% PEG6000 and 2.5M NaCl) was added to make the final concentration of PEG/NaCl 4%. Mix well. And placed on ice for 1 hour, and then centrifuged at 13000g for 10 min. The precipitated phage was rinsed with PBS and stored and used for subsequent phage screening.
  • the magnetic bead method is based on the antigen protein (Spike-RBD-mFc, Sinobio, 40592-V05H) is biotin-labeled, and then combined with streptavidin-coupled magnetic beads. By combining the antigen-bound magnetic beads The panning process of incubation, washing and elution with the antibody gene phage display library usually undergoes 3-4 rounds of panning, so that the specific monoclonal antibodies against the antigen can be enriched in a large amount.
  • the biotin-labeled 2019-nCoV coronavirus RBD protein was used for phage display library screening, and after 3 rounds of panning, a preliminary screening of monoclonal antibodies against the RBD protein was performed.
  • the specific implementation method of antibody screening is as follows:
  • the biotin-labeled 2019-nCoV coronavirus RBD protein (Spike-RBD-mFc, Sinobio, 40592-V05H) is incubated with streptavidin-coupled magnetic beads, so that the biotin-labeled RBD protein binds to the magnetic beads superior.
  • the magnetic beads that bind RBD protein and the constructed phage library were incubated for 2h at room temperature. After washing 6-8 times with PBST, the non-specifically adsorbed phages were removed, Trypsin (Gibco, 25200072) was added and mixed gently and reacted for 20 minutes to elute the specifically bound antibody display phages.
  • the eluted phage was used to infect the log phase SS320 bacteriophage (Lucigen, MC1061F) and let it stand for 30 min, then cultured at 220 rpm for 1 h, then added the VSCM13 helper phage and let it stand for 30 min, and continued at 220 rpm. Cultivate for 1 hour, centrifuge and replace into C + /K + 2-YT medium, and the finally obtained phage will continue to be used in the next round of panning.
  • the purpose of the immunotube method and the magnetic bead method are both to enrich specific antibodies against the antigen, and are two mutually complementary and validated experimental methods.
  • the principle of immune tube screening is to coat the 2019-nCoV coronavirus RBD protein (Spike-RBD-mFc, Sinobio, 40592-V05H) on the surface of an immune tube with high adsorption capacity, and add a phage display antibody library to the immune tube.
  • the panning process of incubation, washing and elution with the antigen protein adsorbed on the surface of the immune tube is carried out. After 2-4 rounds of panning, the specific monoclonal antibodies against the antigen are finally enriched.
  • the phage was precipitated the next day for subsequent 2-4 rounds of screening.
  • the antigen coating concentrations usually used in the second, third and fourth rounds of phage screening gradually decrease, respectively, 30 ⁇ g/mL, 10 ⁇ g/mL and 3 ⁇ g/mL; in addition, the PBS wash strength is gradually increased Large, the PBS elution times are 12 times, 16 times and 20 times in sequence.
  • the phage pool eluted in each round was tested by ELISA to evaluate the effect of enrichment, and 10 clones were randomly selected from the phage pool in each round for sequence analysis. The results are shown in Figure 3A and Figure 3B.
  • the results showed that the antibody sequence was significantly enriched after the third round of screening. Therefore, the clones obtained in the third round were selected for positive clone screening by ELISA.
  • the candidate antibody Fab lysate with gradient dilution (first well 10 ⁇ g/mL, 3-fold dilution) and 0.5 ⁇ g/mL RBD-mFc Mix in equal volume, incubate at 4°C for 1h, then transfer 100 ⁇ l of the mixture to a 96-well plate containing 2.0 ⁇ 10 5 Vero E6 cells/100 ⁇ l (Vero E6 cells obtained from ATCC, CRL-1586), and then incubate for 1h, FACS After rinsing with buffer, 100 ⁇ l of PE-labeled anti-mouse IgG-Fc flow cytometry antibody (Jackson, 115-115-164, 1:200 dilution) was added as the secondary antibody to detect RBD-binding on Vero E6 cells. mFc. The results are shown in Fig. 5, and the results show that the Fabs of the two antibodies (P16-
  • the two Fab antibodies with better blocking activity against ACE2 binding obtained in Example 2 were constructed as human IgG1 type, in which the light chains are all of the kappa type, and the antibody type is a fully human antibody.
  • PCR amplification was used to obtain antibody light and heavy chain variable region fragments, and through homologous recombination, they were constructed into modified eukaryotic expression vector plasmids containing light and heavy chain constant region fragments.
  • pcDNA3.3-TOPO Invitrogen
  • a complete antibody light and heavy chain full-length gene is composed, which encodes the antibody heavy chain and light chain amino acid sequence shown in SEQ ID NO: 17, 18, 19, 20).
  • the constructed vectors containing the full-length antibody light and heavy chain genes were transformed into E. coli SS320, incubated overnight at 37°C, and plasmids were extracted using endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids.
  • OEGA endotoxin-free plasmid extraction kit
  • the antibody light and heavy chain plasmids are used for eukaryotic expression.
  • Candidate antibodies P16-A3, P17-A11 and control antibody CR3022 are expressed by ExpiCHO transient expression system (Thermo Fisher, A29133), the specific method is as follows:
  • ExpiCHO TM Enhancer and ExpiCHO TM Feed to the culture medium. Place the shake flask on a shaker at 32°C and 5% CO 2 to continue culturing. On the 5th day after transfection, add the same Add a volume of ExpiCHO TM Feed slowly while gently mixing the cell suspension. After 7-15 days of transfection, centrifuge the cell culture supernatant expressing the target protein at 15000g for 10 minutes at high speed.
  • SDS-PAGE was used to detect the relative molecular weight and purity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022.
  • IPI is the abbreviation of Ipilimumab, used as a quality control product for physical and chemical properties such as SDS-PAGE, SEC-HPLC, etc.
  • Reducing solution preparation Add 2 ⁇ g of candidate antibody, control antibody and quality control IPI to 5 ⁇ SDS loading buffer and 5mM DTT, heat in a dry bath at 100°C for 10 minutes, cool to room temperature, centrifuge at 12000 rpm for 5 minutes, and take the supernatant.
  • Bis-tris 4-15% gradient gel (purchased from GenScript), constant voltage 110V electrophoresis, when Coomassie Brilliant Blue migrates to the bottom of the gel, stop running, take out the gel piece and place it in Coomassie Brilliant Blue staining solution for 1-2h, Discard the staining solution, add the decolorizing solution, replace the decolorizing solution 2-3 times as needed, and store it in deionized water after decolorizing until the gel background is transparent.
  • the results are shown in Figure 6.
  • the results show that the bands of the candidate antibody and the quality control IPI non-reducing gel are about 150kD, and the bands of the reducing gel are about 55kD and 25kD respectively, which are in line with the expected size, and the purity is greater than 98. %.
  • SEC-HPLC was used to detect the monomer purity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022.
  • Sample preparation Candidate antibody, control antibody and quality control IPI are all diluted to 0.5mg/mL with mobile phase solution.
  • Agilent HPLC 1100 chromatographic column (XBridge BEH SEC 3.5 ⁇ m, 7.8mm I.D. ⁇ 30cm, Waters), the flow rate is set to 0.8mL/min, the injection volume is 20 ⁇ L, and the wavelength of the VWD detector is 280nm and 214nm. Inject blank solution, IPI quality control solution and sample solution in sequence.
  • DSF Differential scanning fluorimetry
  • the DSF method was used to detect the T m values of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022.
  • the thermal stability test of the sample adopts ABI 7500FAST RT-PCR instrument, the test type selects the melting curve, the continuous mode is adopted, the scanning temperature range is 25 ⁇ 95°C, the heating rate is 1%, 25°C is equilibrated for 5 minutes, the data is collected during the heating process, and the report is Select “ROX” for the group, select “None” for the quenching group, and a reaction volume of 20 ⁇ L. The temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the melting temperature Tm of the candidate antibody.
  • a 96-well plate was coated with human ACE2-huFc protein, 8 ⁇ g/mL, 30 ⁇ L/well, 4°C overnight. The next day, the 96-well plate was washed 3 times with PBST and then blocked with 5% skimmed milk for 2 hours. Then, the two candidate antibodies P16-A3, P17-A11 or the control antibody CR3022 were diluted gradiently (see Figure 9 for gradient concentration), and premixed with the above-mentioned Spike-RBD-His labeled with biotin for 1.0 h in advance. After washing the plate, transfer it to a 96-well ELISA plate and incubate for 1 hour.
  • IC 50 values calculated (defined as the binding of the viral S protein to ACE2 concentration of antibody required to reduce 50%) as an indication of the effectiveness of blocking.
  • the two candidate antibodies P16-A3 and P17-A11 have IC 50 values of 7.7 nM and 4.2 nM, respectively, indicating that the two candidate antibodies P16-A3 and P17-A11 have excellent blocking and separation of virus S protein.
  • Example 8 Grouping of candidate antibody epitopes based on ELISA assay
  • the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 bind the epitopes of the 2019-nCoV coronavirus S protein into groups.
  • the wells of the 96-well plate were coated with candidate antibodies P16-A3, P17-A11 and control antibody CR3022, respectively, at 2 ⁇ g/mL, 30 ⁇ L/well, overnight at 4°C. The next day, the well plates were washed 3 times with PBST and then blocked with 5% skimmed milk for 2 hours. Then, S protein RBD-His was added, 2 ⁇ g/mL, 30 ⁇ L/well, and incubated for 1h. After washing 3 times with PBST, as shown in Fig. 10, the biotinylated candidate antibody P17-A11 was added in a gradient of 30 ⁇ L/well, and incubated for 1 h.
  • the 96-well plates were coated with S protein RBD-His, 4 ⁇ g/mL, 30 ⁇ L/well, 4°C overnight. The next day, the well plates were washed 3 times with PBST and then blocked with 5% skimmed milk for 2 hours. Then, add the candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 in gradient dilutions (as shown in Figure 11), 30 ⁇ L/well, and incubate for 1 h. Then add biotinylated candidate antibody P17-A11, 30 ⁇ L/well, and incubate for 1h.
  • Example 9 Determination of the binding affinity of candidate antibodies and antigens based on Fortebio
  • the Fortebio BLItz instrument was used to detect the affinity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 with the 2019-nCoV coronavirus S protein RBD-his.
  • Example 10 Grouping of candidate antibody epitopes based on Fortebio
  • the Fortebio BLItz instrument was used to determine the antibody epitope of the candidate antibody and the control antibody.
  • Example 11 Cell-level detection candidate antibody blocks S protein from binding to ACE2
  • the candidate antibody was evaluated based on the FACS method to block the binding activity of the RBD domain of the virus S protein and the receptor ACE2.
  • the Vero E6 cells used in this example belong to the green monkey kidney cell line and naturally express ACE2. Green monkey ACE2 is very conservative with human ACE2, and the sequence homology reaches 95%. Therefore, in this example, Vero E6 was selected to replace the cell line expressing human ACE2 for the experiment.
  • FACS buffer (1X PBS+2% FBS)
  • FACS buffer to dilute the candidate antibody and the control antibody stepwise (see Figure 13 for the dilution ratio)
  • use FACS buffer to dilute RBD-mFc protein to 1 ⁇ g/mL add 100 ⁇ L to the corresponding 96-well plate, gently pipette to mix with a row gun, and place the 96-well plate at 4°C and incubate for 1h.
  • the anti-CD20 antibody Rituxmab (Rituxmab) was used as a negative isotype control (Isotype IgG) to test and evaluate the neutralizing effect of candidate antibodies P16-A3 and P17-A11 on 2019-nCoV coronavirus .
  • the binding of the 2019-nCoV coronavirus S protein to the receptor ACE2 on the cell surface is the first step for the virus to infect host cells.
  • the Vero E6 cells used in this example belong to the green monkey kidney cell line and naturally express ACE2. Because the sequence homology between green monkey ACE2 and human ACE2 reaches 95%, Vero E6 cells are often used for in vitro drug efficacy evaluation of anti-coronavirus drug candidates. Therefore, Vero E6 cells are used for the determination of virus neutralization activity of candidate antibodies.
  • MEM medium Invitrogen, 41500-034, add glutamine (final concentration 2mM), penicillin (final concentration 100U/mL), streptomycin (final concentration 100 ⁇ g/mL), inactivated FBS (final concentration 10 %).
  • MEM medium Invitrogen, 41500-034, add glutamine (final concentration 2mM), penicillin (final concentration 100U/ml), streptomycin (final concentration 100 ⁇ g/mL), inactivated FBS (final concentration 5 %).
  • the initial concentration is 60 ⁇ g/mL, 2-fold serial dilution, a total of 12 dilutions (the prepared antibody concentration is 60, 30, 15, 7.5 , 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029 ⁇ g/mL).
  • the prepared antibody concentration is 60, 30, 15, 7.5 , 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029 ⁇ g/mL.
  • Example 12.1.2 Collect freshly cultured Vero E6 cells, use the virus medium (5% FBS-MEM) prepared in Example 12.1.2 to prepare a cell concentration of 2 ⁇ 10 5 cells/mL, and add 100 ⁇ l to the above-mentioned virus-containing antibody mixture culture Plate and mix well, and place in a 35°C, 5% CO 2 cell incubator.
  • virus medium 5% FBS-MEM
  • CPE classification standard "+” means that less than 25% of cells have CPE; “++” means that more than 25% and less than 50% of cells have CPE; “+++” means that 50%-70% of cells have CPE; “++++” means that more than 75% of the cells have CPE.
  • the antibody group itself has no obvious cytotoxicity, only the normal cell control group with culture medium shows cell growth, and the virus control group with only virus added shows CPE up to ++++.
  • the Karber method is used to calculate the neutralization end point of the antibody against the virus (convert the antibody dilution to a logarithm), that is, the highest dilution concentration of the antibody that can protect 50% of the cells from 100TCID50 attack virus infection is the antibody titer.
  • the experimental results are shown in Table 4.
  • the results show that the candidate antibodies P17-A11 and P16-A3 can significantly inhibit the infection of Vero E6 cells by the 2019-nCoV virus and protect 50% of the cells from 100TCID 50 virus infection. They are 0.015 ⁇ g/mL and 0.029 ⁇ g/mL, namely 0.100nM and 0.193nM.
  • antibodies P16-A3 and P17-A11 were selected, and they were analyzed and sequenced.
  • the variable region of the human antibody sequence is defined, and the sequence of the light chain and heavy chain variable region of the antibody of the present invention (SEQ ID NO: 1-4) is determined.
  • the variable region sequence was analyzed, and AbM was used to define the CDR to determine the complementarity determining region sequence of the antibody heavy chain and light chain (SEQ ID NO: 5-16).
  • the specific sequence information is as follows:

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Abstract

Provided are an antibody or an antigen binding fragment specifically binding to a coronavirus S protein, a multispecific antibody, and an antibody composition. Further provided are a nucleic acid encoding the antibody or the antigen binding fragment, and the multispecific antibody, a host cell comprising the nucleic acid, and a method for preparing the antibody or the antigen binding fragment, and the multispecific antibody. In addition, further provided are prevention, treatment, and/or diagnosis uses of the antibody or the antigen binding fragment, the multispecific antibody, and the antibody composition.

Description

针对冠状病毒的具有中和活性的抗体及其用途Antibody with neutralizing activity against coronavirus and its use 技术领域Technical field
本发明总体上涉及抗体及其用途。更具体地，本发明涉及特异性识别冠状病毒刺突蛋白的抗体和抗原结合片段、其制备方法及其用途。The present invention generally relates to antibodies and their uses. More specifically, the present invention relates to antibodies and antigen-binding fragments that specifically recognize the coronavirus spike protein, preparation methods and uses thereof.
背景技术Background technique
自2002年重症急性呼吸综合征冠状病毒(severe acute respiratory syndromes coronavirus(SARS-CoV))爆发以来，冠状病毒(CoV)成为了一种引起主要公共卫生问题的RNA病毒。目前，全世界都在关注2019新型冠状病毒(2019novel CoV(2019-nCoV)),该病毒最初于2019年12月在中国武汉市被鉴定出，可以在人与人之间传播，患者表现为严重的病毒性肺炎和呼吸系统疾病。从那时起，感染2019-nCoV的病例数量一直在增加。截止2020年3月30日，中国累计确诊的感染该新型冠状病毒的患者有82451例，其中累计死亡3311例，此外其他国家也有近64万例累计确诊的感染该新型冠状病毒的患者，而且确诊病例在急剧攀升，形势非常严峻。Since the outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, coronavirus (CoV) has become an RNA virus that causes major public health problems. At present, the world is paying attention to the 2019 novel coronavirus (2019novel CoV (2019-nCoV)). The virus was originally identified in Wuhan, China in December 2019. It can be transmitted from person to person, and the patient appears to be severe Viral pneumonia and respiratory diseases. Since then, the number of cases infected with 2019-nCoV has been increasing. As of March 30, 2020, China has a total of 82,451 confirmed patients infected with the new coronavirus, of which 3311 have died. In addition, there are also nearly 640,000 confirmed patients infected with the new coronavirus in other countries. The number of cases is rising sharply, and the situation is very serious.
至今为止，针对此类冠状病毒尚无有效的疫苗或治疗剂，临床以对症支持治疗为主。考虑到此类冠状病毒持续危及人类健康，且具有高传播性和致死率等特点，很有可能引起更多的公共卫生问题，因此需要针对此类冠状病毒的致病机制开发出具有抑制或阻断病毒感染的预防和治疗性抗病毒剂，为未来在人群中出现流行或甚至大范围地出现此类冠状病毒感染时提供针对它们的预防和早期治疗。So far, there is no effective vaccine or therapeutic agent for this type of coronavirus, and symptomatic and supportive treatment is the main clinical treatment. Considering that such coronaviruses continue to endanger human health, and have the characteristics of high transmission and lethality, which are likely to cause more public health problems. Therefore, it is necessary to develop an inhibitory or preventive mechanism for the pathogenic mechanism of such coronaviruses. Preventive and therapeutic antiviral agents for virus infections provide preventive and early treatment for such coronavirus infections in the future when such coronavirus infections appear in the population or even appear on a large scale.
研究显示此类冠状病毒通过刺突蛋白(S蛋白)与宿主细胞上的受体血管紧张素转换酶Ⅱ(angiotensin converting enzyme Ⅱ，也称为ACE2)结合，介导病毒进入宿主细胞(Ashour HM等人，Insights into the Recent 2019 Novel Coronavirus(SARS-CoV-2)in Light of Past Human Coronavirus Outbreaks，Pathogens，2020年3月4日；9(3).pii:E186.doi:10.3390/pathogens9030186；Roujian Lu等人，Genomic characterisation and epidemiology of 2019novel coronavirus:implications for virus origins and receptor binding，www.thelancet.com，2020年1月29日网上公开，https://doi.org/10.1016/S0140-6736(20)30251-8)，因此，本领域需要开发出针对冠状病毒S蛋白并且阻断其与宿主细胞上的ACE2受体结合的高亲和力中和抗体，来有效预防和治疗此类冠状病毒(例如，2019-n CoV、SARS-CoV)感染。Studies have shown that this type of coronavirus binds to the receptor angiotensin converting enzyme II (also known as ACE2) on the host cell through the spike protein (S protein), and mediates the virus to enter the host cell (Ashour HM, etc.) Human, Insights into the Recent 2019 Novel Coronavirus (SARS-CoV-2) in Light of Past Human Coronavirus Outbreaks, Pathogens, March 4, 2020; 9(3).pii:E186.doi:10.3390/pathogens9030186; Roujian Lu Et al., Genomic characterisation and epidemiology of 2019novel coronavirus: implications for virus origins and receptor binding, www.thelancet.com, published online on January 29, 2020, https://doi.org/10.1016/S0140-6736(20) 30251-8), therefore, it is necessary in the art to develop high-affinity neutralizing antibodies that target the S protein of coronavirus and block its binding to the ACE2 receptor on host cells to effectively prevent and treat such coronaviruses (for example, 2019 -n CoV, SARS-CoV) infection.
发明概述Summary of the invention
本发明人通过锐意研究，开发了一组以高亲和力特异性结合冠状病毒S蛋白并抑制病毒感染性的人抗体，从而满足了上述需求。本发明的特异性识别冠状病毒S蛋白的抗体能够Through intensive research, the present inventors have developed a set of human antibodies that specifically bind to the coronavirus S protein with high affinity and inhibit viral infectivity, thereby meeting the above-mentioned needs. The antibody that specifically recognizes the S protein of coronavirus of the present invention can
(a)在25℃ELISA测定法中测量，以小于约10nM，例如小于约8nM的IC50阻断冠状病毒S蛋白与分离的ACE2蛋白的结合，优于对照抗体CR3022；(a) Measured in an ELISA assay at 25°C, with an IC50 of less than about 10 nM, for example, less than about 8 nM, blocking the binding of the coronavirus S protein to the isolated ACE2 protein, which is better than the control antibody CR3022;
(b)在25℃ELISA测定法中测量，针对冠状病毒S蛋白的亲和力优于对照抗体CR3022；(b) Measured in the 25°C ELISA assay method, the affinity for the coronavirus S protein is better than the control antibody CR3022;
(c)在25℃生物膜层干涉测定法中测量，以小于约1nM，例如约0.8nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM的结合解离平衡常数K D结合冠状病毒S蛋白； (c) Measured in 25°C biofilm interferometry, with a binding and dissociation equilibrium constant K D of less than about 1 nM, for example, about 0.8 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM Binding to coronavirus S protein;
(d)在FACS测定法中测量，以小于约2nM，例如约1.8nM、1.5nM、1.2nM、0.9nM、0.6nM、0.3nM的IC50阻断冠状病毒S蛋白与Vero E6细胞上表达的天然ACE2蛋白的结合；(d) Measured in the FACS assay, with IC50 less than about 2nM, such as about 1.8nM, 1.5nM, 1.2nM, 0.9nM, 0.6nM, 0.3nM, to block the natural expression of coronavirus S protein and Vero E6 cells Binding of ACE2 protein;
(e)在细胞病变法中测量，本发明的抗体具有抑制病毒侵染Vero E6细胞的中和活性，其保护50％细胞不受100TCID 50攻击病毒液感染的抗体最高稀释浓度(或抗体的滴度)为小于约0.5nM，例如约0.4nM、0.3nM、0.2nM、0.1nM。 (e) Measured in the cytopathic method, the antibody of the present invention has a neutralizing activity to inhibit virus infection of Vero E6 cells, and it protects 50% of cells from 100 TCID 50 attack. Degree) is less than about 0.5 nM, for example, about 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM.
因此，在第一方面，本发明提供了特异性结合冠状病毒S蛋白的分离的抗体或抗原结合片段，其包含Therefore, in the first aspect, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to the S protein of coronavirus, which comprises
(a)SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR；以及与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体；或(a) The 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 and the 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 2; and the same as the above 6 The CDR region has a single or multiple CDR variants that do not exceed 2 amino acid changes in each CDR region; or
(b)SEQ ID NO:3所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:4所示的轻链可变区氨基酸序列中的3个CDR；以及与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体。(b) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 3 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 4; and the same as the above 6 The CDR regions have variants in which single or multiple CDRs do not exceed 2 amino acid changes per CDR region.
在一些实施方案中，本发明提供了特异性结合冠状病毒S蛋白的分离的抗体或抗原结合片段，其包含In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to the S protein of coronavirus, which comprises
(a)SEQ ID NO:5所示的HCDR1或SEQ ID NO:5所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:6所示的HCDR2或SEQ ID NO:6所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:7所示的HCDR3或SEQ ID NO:7所示的HCDR3的不超过2个氨基酸变化的变体；SEQ ID NO:8所示的LCDR1或SEQ ID NO:8所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:9所示的LCDR2或SEQ ID NO:9 所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:10所示的LCDR3或SEQ ID NO:10所示的LCDR3的不超过2个氨基酸变化的变体；或(a) HCDR1 shown in SEQ ID NO: 5 or a variant with no more than 2 amino acid changes of HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6 or SEQ ID NO: 6 HCDR2 not more than 2 amino acid changes, and SEQ ID NO: 7 HCDR3 or SEQ ID NO: 7 HCDR3 variants not more than 2 amino acid changes; SEQ ID NO: 8 The LCDR1 shown or the variant of LCDR1 shown in SEQ ID NO: 8 that does not exceed 2 amino acid changes, the LCDR2 shown in SEQ ID NO: 9 or the LCDR2 shown in SEQ ID NO: 9 does not exceed 2 amino acid changes A variant of and LCDR3 shown in SEQ ID NO: 10 or a variant of LCDR3 shown in SEQ ID NO: 10 with no more than 2 amino acid changes; or
(b)SEQ ID NO:11所示的HCDR1或SEQ ID NO:11所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:12所示的HCDR2或SEQ ID NO:12所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:13所示的HCDR3或SEQ ID NO:13所示的HCDR3的不超过2个氨基酸变化的变体；SEQ ID NO:14所示的LCDR1或SEQ ID NO:14所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:15所示的LCDR2或SEQ ID NO:15所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:16所示的LCDR3或SEQ ID NO:16所示的LCDR3的不超过2个氨基酸变化的变体。(b) HCDR1 shown in SEQ ID NO: 11 or a variant with no more than 2 amino acid changes of HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12 or SEQ ID NO: 12 A variant of HCDR2 with no more than 2 amino acid changes, and a HCDR3 shown in SEQ ID NO: 13 or a variant of HCDR3 shown in SEQ ID NO: 13 with no more than 2 amino acid changes; SEQ ID NO: 14 The LCDR1 shown or the variant of LCDR1 shown in SEQ ID NO: 14 that does not exceed 2 amino acid changes, the LCDR2 shown in SEQ ID NO: 15 or the LCDR2 shown in SEQ ID NO: 15 does not exceed 2 amino acid changes And the LCDR3 shown in SEQ ID NO: 16 or the LCDR3 shown in SEQ ID NO: 16 with no more than 2 amino acid changes.
在一些实施方案中，本发明的分离的抗体或抗原结合片段包含In some embodiments, the isolated antibody or antigen-binding fragment of the invention comprises
(a)SEQ ID NO:5所示的HCDR1、SEQ ID NO:6所示的HCDR2和SEQ ID NO:7所示的HCDR3；SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(a) HCDR1 shown in SEQ ID NO: 5 and HCDR2 shown in SEQ ID NO: 6 and HCDR3 shown in SEQ ID NO: 7; LCDR1 shown in SEQ ID NO: 8 and shown in SEQ ID NO: 9 LCDR2 and LCDR3 shown in SEQ ID NO: 10; or
(b)SEQ ID NO:11所示的HCDR1、SEQ ID NO:12所示的HCDR2和SEQ ID NO:13所示的HCDR3；SEQ ID NO:14所示的LCDR1、SEQ ID NO:15所示的LCDR2和SEQ ID NO:16所示的LCDR3。(b) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12 and HCDR3 shown in SEQ ID NO: 13; LCDR1 shown in SEQ ID NO: 14 and shown in SEQ ID NO: 15 The LCDR2 and the LCDR3 shown in SEQ ID NO: 16.
在一些实施方案中，本发明的分离的抗体或抗原结合片段包含In some embodiments, the isolated antibody or antigen-binding fragment of the invention comprises
(a)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:1的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或(a) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:1 or has at least 90%, 91%, 92%, 93%, 94%, 95% therewith , 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region includes the sequence of SEQ ID NO: 2 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences; or
(b)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。(b) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95% therewith , 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region includes the sequence of SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, Sequences that are 95%, 96%, 97%, 98%, or 99% identical.
在一些实施方案中，本发明的分离的抗体包含In some embodiments, the isolated antibodies of the invention comprise
(a)SEQ ID NO:17或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:18或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；或(a) SEQ ID NO: 17 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it, and SEQ ID NO: 18 or a light chain sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; or
(b)SEQ ID NO:19或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:20或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列。(b) SEQ ID NO: 19 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it, and SEQ ID NO: 20 or a light chain sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it.
在一些实施方案中，本发明的分离的抗体或抗原结合片段是完全人抗体。In some embodiments, the isolated antibodies or antigen-binding fragments of the invention are fully human antibodies.
在一些实施方案中，本发明的分离的抗体或抗原结合片段是IgG1、IgG2、IgG3或IgG4抗体；优选地，是IgG1或IgG4抗体；更优选地，是人IgG1或人IgG4抗体。在一些实施方案中，本发明的抗原结合片段是Fab、Fab’、F(ab’) 2、Fv、单链Fv、单链Fab、双体抗体(diabody)。 In some embodiments, the isolated antibody or antigen-binding fragment of the invention is an IgG1, IgG2, IgG3, or IgG4 antibody; preferably, an IgG1 or IgG4 antibody; more preferably, a human IgG1 or human IgG4 antibody. In some embodiments, the antigen-binding fragments of the present invention are Fab, Fab', F(ab') 2 , Fv, single-chain Fv, single-chain Fab, diabody.
在第二方面，本发明提供了特异性结合冠状病毒S蛋白的多特异性抗体，其包含特异性结合冠状病毒S蛋白表位的上述第一方面所述的抗体或抗原结合片段。In the second aspect, the present invention provides a multispecific antibody that specifically binds to the coronavirus S protein, which comprises the antibody or antigen-binding fragment described in the first aspect that specifically binds to the epitope of the coronavirus S protein.
在第三方面，本发明提供了抗体组合，其包含特异性结合冠状病毒S蛋白第一表位的上述第一方面所述的抗体或抗原结合片段，和/或其他特异性结合冠状病毒S蛋白第二表位的抗体或抗原结合片段，其中所述第一表位和第二表位可以是相同的表位或不同的表位。In a third aspect, the present invention provides an antibody combination comprising the antibody or antigen-binding fragment described in the first aspect that specifically binds to the first epitope of the coronavirus S protein, and/or other specific binding to the coronavirus S protein The antibody or antigen-binding fragment of the second epitope, wherein the first epitope and the second epitope may be the same epitope or different epitopes.
在第四方面，本发明提供了编码上述第一方面所述的抗体或抗原结合片段或编码上述第二方面所述的多特异性抗体的核酸、包含所述核酸的载体(优选地，表达载体)、包含所述核酸或所述载体的宿主细胞。在一些实施方案中，所述宿主细胞是原核的或真核的，例如，选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或抗原结合片段、多特异性抗体的其它细胞。在一些实施方案中，所述宿主细胞是293细胞或CHO细胞。In the fourth aspect, the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment of the above-mentioned first aspect or the multispecific antibody of the above-mentioned second aspect, a vector containing the nucleic acid (preferably, an expression vector ), a host cell containing the nucleic acid or the vector. In some embodiments, the host cell is prokaryotic or eukaryotic, for example, selected from E. coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments, multispecific antibodies. In some embodiments, the host cell is a 293 cell or a CHO cell.
在第五方面，本发明提供了制备本发明的抗体或抗原结合片段、或本发明的多特异性抗体的方法，所述方法包括在适于表达编码本发明的抗体或抗原结合片段或编码本发明的多特异性抗体的核酸的条件下培养本发明的宿主细胞，任选地从所述宿主细胞或从培养基回收本发明的抗体或抗原结合片段、或本发明的多特异性抗体。In the fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment of the present invention, or the multispecific antibody of the present invention. The host cell of the present invention is cultured under the condition of the nucleic acid of the multispecific antibody of the present invention, and the antibody or antigen-binding fragment of the present invention, or the multispecific antibody of the present invention is optionally recovered from the host cell or from the culture medium.
在第六方面，本发明提供了药物组合物，其包含本发明的抗体或抗原结合片段、本发明的多特异性抗体、或本发明的抗体组合，以及可药用载体。In the sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention, or the antibody combination of the present invention, and a pharmaceutically acceptable carrier.
在第七方面，本发明提供了本发明的抗体或抗原结合片段、或本发明的多特异性抗体的用途，用于制备预防和/或治疗冠状病毒感染的药物。在一些实施方案中，所述冠状病毒是2019-nCoV病毒。In the seventh aspect, the present invention provides the use of the antibody or antigen-binding fragment of the present invention, or the multispecific antibody of the present invention, for the preparation of drugs for preventing and/or treating coronavirus infections. In some embodiments, the coronavirus is 2019-nCoV virus.
在第八方面，本发明提供了在受试者中预防和/或治疗冠状病毒感染的方法，包括向受试者施用有效量的本发明的抗体或抗原结合片段、本发明的多特异性抗体、本发明的抗体组合、或本发明的药物组合物。在一些实施方案中，所述冠状病毒是2019-nCoV病毒。In an eighth aspect, the present invention provides a method for preventing and/or treating coronavirus infection in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention , The antibody combination of the present invention, or the pharmaceutical composition of the present invention. In some embodiments, the coronavirus is 2019-nCoV virus.
本发明的抗体以及本发明的抗体组合可有效阻断和/或抑制冠状病毒感染，能够用于冠状病毒的预防和/或治疗。The antibody of the present invention and the antibody combination of the present invention can effectively block and/or inhibit coronavirus infection, and can be used for the prevention and/or treatment of coronavirus.
在第九方面，本发明提供了检测样品中冠状病毒S蛋白的试剂盒，所述试剂盒包含本发明的抗体或抗原结合片段、本发明的多特异性抗体、或本发明的抗体组合，用于实施以下步骤：In a ninth aspect, the present invention provides a kit for detecting coronavirus S protein in a sample, the kit comprising the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention, or the antibody combination of the present invention, with To implement the following steps:
(a)将样品与本发明的抗体或抗原结合片段、本发明的多特异性抗体、或本发明的抗体组合接触；和(a) contacting the sample with the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention, or the antibody combination of the present invention; and
(b)检测本发明的抗体或抗原结合片段、本发明的多特异性抗体、或本发明的抗体组合与冠状病毒S蛋白之间的复合物的形成，(b) detecting the formation of a complex between the antibody or antigen-binding fragment of the present invention, the multispecific antibody of the present invention, or the antibody combination of the present invention and the coronavirus S protein,
由此，判断来自受试者或个体的样品中是否存在冠状病毒感染或者是否自冠状病毒感染后治愈。From this, it is determined whether there is coronavirus infection in the sample from the subject or individual or whether it has been cured after the coronavirus infection.
发明的效果The effect of the invention
本发明提供了针对冠状病毒S蛋白的抗体或抗体组合，至少具有以下的有益技术效果：The present invention provides antibodies or antibody combinations against coronavirus S protein, which have at least the following beneficial technical effects:
第一，S蛋白和受体ACE2结合是冠状病毒侵染宿主的第一步，且S蛋白的S1和S2亚基承载着结合受体、促进病毒包膜和宿主细胞膜融合的功能，S蛋白是开发具有病毒中和活性的抗体的理想靶抗原。First, the binding of S protein to the receptor ACE2 is the first step for coronavirus to infect the host, and the S1 and S2 subunits of the S protein carry the function of binding receptors and promoting the fusion of the virus envelope and the host cell membrane. The S protein is The ideal target antigen for the development of antibodies with virus-neutralizing activity.
第二，S蛋白属于作为冠状病毒(例如，2019-n CoV、SARS-CoV)的宿主(例如，人)的外源蛋白，相对于受体蛋白ACE2而言，针对S蛋白开发的抗体不容易与受试者有组织交叉反应，安全性更高。Second, S protein is a foreign protein that serves as a host (e.g., human) of coronaviruses (e.g., 2019-n CoV, SARS-CoV). Compared with the receptor protein ACE2, it is not easy to develop antibodies against S protein. There is an organized cross-reaction with subjects, and the safety is higher.
第三，相对于小分子药物，抗体药物特异性更高，脱靶带来的毒性效应更低，而且半衰期相对较长，用药频率较低。Third, compared to small molecule drugs, antibody drugs have higher specificity, lower toxic effects caused by off-target, and relatively long half-life, lower frequency of medication.
第四，本发明的抗体为全人抗体，不需要人源化就可以进行临床治疗，免疫原性更低，成药性更好。Fourth, the antibody of the present invention is a fully human antibody, which can be clinically treated without humanization, has lower immunogenicity and better druggability.
在本发明的一个实施方案中，针对冠状病毒(例如，2019-nCoV冠状病毒)S蛋白的抗体是通过筛选人类天然抗体库获得的，其中两个抗体P16-A3、P17-A11在体外经ELISA测定法测定，针对S蛋白的亲和力均优于对照抗体CR3022，且可以更加显著地阻断S蛋白和ACE2的结合；在细胞水平上，所述抗体也可显著阻断S蛋白与Vero E6细胞上ACE2 的结合，进一步的实验还发现所述抗体抑制病毒侵染Vero E6细胞的中和活性显著优于对照抗体，50％细胞不受100TCID 50病毒液感染的最低抗体浓度约为0.100nM。 In one embodiment of the present invention, antibodies against the S protein of coronavirus (for example, 2019-nCoV coronavirus) are obtained by screening human natural antibody libraries, in which two antibodies P16-A3 and P17-A11 are tested in vitro by ELISA As determined by the assay, the affinity for S protein is better than that of the control antibody CR3022, and can more significantly block the binding of S protein and ACE2; at the cellular level, the antibody can also significantly block S protein and Vero E6 cells. In combination with ACE2, further experiments also found that the neutralizing activity of the antibody to inhibit virus infection of Vero E6 cells was significantly better than that of the control antibody, and the minimum antibody concentration at which 50% of the cells were not infected by 100TCID 50 virus solution was about 0.100nM.
本发明的抗体以及抗体组合可有效抑制冠状病毒的感染，极具潜力成为针对此类冠状病毒预防和治疗的有效药物。The antibodies and antibody combinations of the present invention can effectively inhibit the infection of coronaviruses, and have great potential to become effective drugs for the prevention and treatment of such coronaviruses.
附图简述Brief description of the drawings
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。When read together with the following drawings, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the invention, the figure shows a currently preferred embodiment. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
图1显示本发明的靶向2019-nCoV冠状病毒S蛋白的全人抗体的产生方法和活性检测方法。Figure 1 shows the production method and activity detection method of the fully human antibody targeting the 2019-nCoV coronavirus S protein of the present invention.
图2A显示人ACE2-huFc与S蛋白RBD-mFc的结合活性。Figure 2A shows the binding activity of human ACE2-huFc and S protein RBD-mFc.
图2B显示人ACE2-His与S蛋白S1-huFc(也称为Spike S1-huFc)或S蛋白RBD-mFc(也称为Spike RBD-mFc)的结合活性。Figure 2B shows the binding activity of human ACE2-His to S protein S1-huFc (also called Spike S1-huFc) or S protein RBD-mFc (also called Spike RBD-mFc).
图3A显示抗体在第一轮和第二轮淘选中输出(Output)的噬菌体于ELISA测定法中与S蛋白RBD-mFc的结合能力。从该图中可见，每一轮都有更好的富集。使用VSCM13辅助噬菌体作为阴性对照。Figure 3A shows the binding ability of the output phage of the antibody to the S protein RBD-mFc in the ELISA assay in the first and second rounds of panning. It can be seen from this figure that each round has better enrichment. VSCM13 helper phage was used as a negative control.
图3B显示了抗体在第二轮和第三轮淘选中输出的噬菌体于ELISA测定法中与S蛋白RBD-mFc的结合能力。从该图中可见，每一轮都有更好的富集，且富集最好的是3rd-1(即，第三轮淘选中的1号样品)。Figure 3B shows the ability of the antibody to bind the S protein RBD-mFc in the ELISA assay of the phage output in the second and third rounds of panning. It can be seen from this figure that there is better enrichment in each round, and the best enrichment is 3rd-1 (that is, the No. 1 sample selected in the third round of panning).
图4显示在ELISA测定法中候选抗体Fab上清与S蛋白RBD-mFc的结合能力。从该图中可见，候选抗体P17-A11 Fab上清与S蛋白RBD-mFc有较好的结合能力；候选抗体P16-A3 Fab上清与S蛋白RBD-mFc也有一定的结合能力。Figure 4 shows the binding ability of the Fab supernatant of the candidate antibody to the S protein RBD-mFc in the ELISA assay. It can be seen from the figure that the candidate antibody P17-A11 Fab supernatant has good binding ability with S protein RBD-mFc; the candidate antibody P16-A3 Fab supernatant also has a certain binding ability with S protein RBD-mFc.
图5显示细胞水平上候选抗体阻断S蛋白与细胞上表达的ACE2结合的能力。结果显示抗体P16-A3 Fab和P17-A11 Fab均表现出了显著的阻断S蛋白与细胞结合的活性。图中的MFI表示平均荧光强度。Figure 5 shows the ability of candidate antibodies to block the binding of S protein to ACE2 expressed on cells at the cellular level. The results showed that the antibodies P16-A3 Fab and P17-A11 Fab both showed significant activity to block the binding of S protein to cells. The MFI in the figure represents the average fluorescence intensity.
图6显示候选抗体的分子量和纯度鉴定结果。两个候选抗体P16-A3、P17-A11以及对照抗体CR3022的纯度均大于98％。Figure 6 shows the molecular weight and purity identification results of candidate antibodies. The purity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 are all greater than 98%.
图7A显示候选抗体P16-A3的单体纯度鉴定结果，其单体纯度大于98％。Figure 7A shows the monomer purity identification result of the candidate antibody P16-A3, and the monomer purity is greater than 98%.
图7B显示候选抗体P17-A11的单体纯度鉴定结果，其单体纯度大于98％。Figure 7B shows the monomer purity identification results of the candidate antibody P17-A11, and the monomer purity is greater than 98%.
图7C显示对照抗体CR3022的单体纯度鉴定结果，其单体纯度大于98％。Figure 7C shows the monomer purity identification result of the control antibody CR3022, and its monomer purity is greater than 98%.
图8显示基于ELISA测定法的候选抗体特异性结合S蛋白的亲和活性。候选抗体 P16-A3和P17-A11表现出优异的且明显优于对照抗体CR3022的特异性结合S蛋白的活性。图中的阴性抗体是同种型人IgG1抗体。Figure 8 shows the affinity activity of candidate antibodies specifically binding to protein S based on an ELISA assay. Candidate antibodies P16-A3 and P17-A11 showed excellent and significantly better than the control antibody CR3022's specific binding to S protein activity. The negative antibody in the figure is an isotype human IgG1 antibody.
图9显示基于ELISA检测候选抗体的阻断活性。候选抗体P16-A3和P17-A11都表现出优异的阻断病毒S蛋白与分离的ACE2蛋白结合的能力，而对照抗体CR3022无阻断活性。图中的阴性抗体是同种型人IgG1抗体。Figure 9 shows the detection of the blocking activity of candidate antibodies based on ELISA. Candidate antibodies P16-A3 and P17-A11 both show excellent ability to block the binding of viral S protein to isolated ACE2 protein, while the control antibody CR3022 has no blocking activity. The negative antibody in the figure is an isotype human IgG1 antibody.
图10显示利用ELISA的双抗夹心法对候选抗体的表位进行分组。结果显示，在2019-nCoV冠状病毒S蛋白RBD结构域上，抗体P17-A11和抗体CR3022结合不同的表位，而抗体P17-A11和抗体P16-A3结合相同的表位。Figure 10 shows the grouping of epitopes of candidate antibodies using the double antibody sandwich method of ELISA. The results show that on the RBD domain of the 2019-nCoV coronavirus S protein, antibody P17-A11 and antibody CR3022 bind different epitopes, while antibody P17-A11 and antibody P16-A3 bind the same epitope.
图11显示利用ELISA的竞争法对候选抗体的表位进行分组。结果显示，在2019-nCoV冠状病毒S蛋白RBD结构域上，抗体P17-A11和抗体CR3022结合不同的表位，而抗体P17-A11和抗体P16-A3结合相同的表位。Figure 11 shows the grouping of epitopes of candidate antibodies using the competitive method of ELISA. The results show that on the RBD domain of the 2019-nCoV coronavirus S protein, antibody P17-A11 and antibody CR3022 bind different epitopes, while antibody P17-A11 and antibody P16-A3 bind the same epitope.
图12A显示基于Fortebio的候选抗体P16-A3和P17-A11的表位分组结果，抗体P16-A3和P17-A11的表位一致。Figure 12A shows the epitope grouping results of candidate antibodies P16-A3 and P17-A11 based on Fortebio, and the epitopes of antibodies P16-A3 and P17-A11 are the same.
图12B显示基于Fortebio的候选抗体P16-A3和对照抗体CR3022的表位分组结果，抗体P16-A3和对照抗体CR3022的表位不同。Figure 12B shows the epitope grouping results of the candidate antibody P16-A3 and the control antibody CR3022 based on Fortebio. The epitopes of the antibody P16-A3 and the control antibody CR3022 are different.
图12C显示基于Fortebio的候选抗体P17-A11和对照抗体CR3022的表位分组结果，抗体P17-A11和对照抗体CR3022的表位不同。Figure 12C shows the epitope grouping results of the candidate antibody P17-A11 and the control antibody CR3022 based on Fortebio. The epitopes of the antibody P17-A11 and the control antibody CR3022 are different.
图13显示细胞水平检测候选抗体阻断S蛋白在ACE2天然表达细胞上结合的活性。候选抗体P16-A3和P17-A11均显示出剂量依赖的阻断活性，在细胞水平上有优异的阻断效果且显著优于对照抗体CR0322。图中的MFI表示平均荧光强度。Figure 13 shows the cell-level detection of candidate antibodies to block the activity of S protein binding on ACE2 naturally expressing cells. Candidate antibodies P16-A3 and P17-A11 both show dose-dependent blocking activity, have excellent blocking effects at the cellular level and are significantly better than the control antibody CR0322. The MFI in the figure represents the average fluorescence intensity.
发明详述Detailed description of the invention
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be changed. In addition, the terms used herein are only for describing specific embodiments, and are not intended to be limiting.
I.定义I. Definition
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purpose of the present invention, the following terms are defined below.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value means to cover a numerical value within a range having a lower limit 10% smaller than the specified numerical value and an upper limit 10% larger than the specified numerical value.
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。When the term "and/or" is used to connect two or more alternatives, it should be understood to mean any one of the alternatives or any two or more of the alternatives.
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.
术语“冠状病毒(Coronaviruses，CoV)”在本文中是指属于冠状病毒科(Coronaviridae)β冠状病毒属的病毒，病毒颗粒呈球形或椭圆形，直径约60～220nm。病毒为单股正链RNA(+ssRNA)病毒。在几种对人类有致病性的冠状病毒中，大多数与轻度的临床症状有关(Su S,Wong G,Shi W等人，Epidemiology,genetic recombination,and pathogenesis of coronaviruses.Trends Microbiol 2016；24:490–502)，但有两种冠状病毒是值得注意的例外：一种是SARS-CoV，其于2002年11月在中国广东省出现，并在2002年到2003年间在37个国家和地区造成8000多例人类感染和774例死亡(Chan-Yeung M,Xu RH.SARS:epidemiology.Respirology 2003；8(suppl):S9–14)；另一种是引起人类新型冠状病毒疾病(Corona Virus Disease 2019，COVID-19)的2019新型冠状病毒(2019-nCoV)，具有强烈的在人群中传播的能力，大多数感染患者发高烧，有些患有呼吸困难，胸部X光片显示双肺都有浸润性病变(Huang C,Wang Y,Li X等人.Clinical features of patients infected with 2019 novel coronavirus in Wuhan,China.Lancet，2020年1月24日网上公开)。世界卫生组织(WHO)最近将所述2019-nCoV命名为SARS-CoV-2。在本文中，“2019-nCoV”和“SARS-CoV-2”可互换地使用。The term "Coronaviruses (CoV)" herein refers to viruses belonging to the genus Coronaviridae (Coronaviridae) β-coronavirus. The virus particles are spherical or elliptical with a diameter of about 60-220 nm. The virus is a single-stranded positive-strand RNA (+ssRNA) virus. Among several coronaviruses that are pathogenic to humans, most are related to mild clinical symptoms (Su S, Wong G, Shi W, et al., Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol 2016; 24 :490–502), but there are two notable exceptions to the coronavirus: one is SARS-CoV, which appeared in Guangdong Province, China in November 2002, and appeared in 37 countries and regions from 2002 to 2003 Caused more than 8,000 human infections and 774 deaths (Chan-Yeung M, Xu RH.SARS: epidemiology.Respirology 2003; 8(suppl): S9-14); the other is caused by human new coronavirus disease (Corona Virus Disease) 2019, COVID-19), the 2019 novel coronavirus (2019-nCoV), has a strong ability to spread among the population. Most infected patients have high fever, some suffer from breathing difficulties, and chest X-rays show both lungs have infiltrates Sexual disease (Huang C, Wang Y, Li X and others. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet, published online on January 24, 2020). The World Health Organization (WHO) recently named the 2019-nCoV as SARS-CoV-2. In this article, "2019-nCoV" and "SARS-CoV-2" are used interchangeably.
术语“抗体”在本文中以最广意义使用并且包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)，只要它们显示出所需的抗原结合活性即可。抗体可以是任何型和亚型(例如，IgM、IgD、IgG1、IgG2、IgG3、IgG4、IgE、IgA1和IgA2)的完整抗体(例如，具有两个全长的轻链和两个全长的重链)。完整抗体的单体是由二硫键连接两个全长的轻链和两个全长的重链形成的一个四肽链分子，也称为Ig分子的单体。抗体单体是构成抗体的基本结构。The term "antibody" is used in the broadest sense herein and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), as long as they exhibit the desired antigen-binding activity . The antibody can be a complete antibody of any type and subtype (e.g., IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1 and IgA2) (e.g., having two full-length light chains and two full-length heavy chains). chain). The monomer of a complete antibody is a tetrapeptide chain molecule formed by disulfide bonds connecting two full-length light chains and two full-length heavy chains, also known as the monomer of an Ig molecule. Antibody monomer is the basic structure that constitutes an antibody.
本文所用的“分离的抗体”旨在指基本不含其他具有不同抗原特异性的抗体(Ab)的抗体(例如，分离的特异性结合冠状病毒S蛋白的抗体或其抗原结合片段基本不含特异性结合冠状病毒S蛋白以外的抗原的Ab)。在某些实施方案中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)来确定。As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Ab) with different antigen specificities (for example, an isolated antibody that specifically binds to the coronavirus S protein or an antigen-binding fragment thereof is substantially free of specific Sexually binds to antigens other than the Coronavirus S protein). In certain embodiments, the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse Phase HPLC) to determine.
本文所用的“阻断抗体”、“中和性抗体”、“具有中和活性的抗体”或“中和抗体”在本文中可互换地使用，指这样的抗体，其与靶抗原结合或与之相互作用并阻止靶抗原与结合配偶体如受体结合或缔合，因而抑制或阻断本将因靶抗原与结合配偶体如受体的相互作用而产生的生物学反应。在本发明的情况下，指所述抗体与冠状病毒S蛋白的结合导致冠状病毒的至少一种生物活性被抑制。例如，本发明的中和抗体可以阻止或阻断冠状病毒S蛋白与ACE2结合。As used herein, "blocking antibody", "neutralizing antibody", "antibody with neutralizing activity" or "neutralizing antibody" are used interchangeably herein and refer to an antibody that binds to a target antigen or It interacts with it and prevents the target antigen from binding or associating with a binding partner such as a receptor, thereby inhibiting or blocking the biological response that would otherwise occur due to the interaction of the target antigen with a binding partner such as the receptor. In the context of the present invention, it means that the binding of the antibody to the coronavirus S protein results in the inhibition of at least one biological activity of the coronavirus. For example, the neutralizing antibody of the present invention can prevent or block the binding of coronavirus S protein to ACE2.
“表位”或“抗原决定子”指与抗体分子的可变区中称为互补位的特异性抗原结合部位相互作用的抗原决定簇。单个抗原可以具有一个以上表位。因此，不同抗体可以结合抗原上的不同区域，并可以具有不同的生物学效应。表位可以由连续氨基酸或经蛋白质的三级折叠并接的不连续氨基酸形成。由连续氨基酸形成的表位在暴露于变性溶剂时通常保留，而通过三级折叠形成的表位在用变性溶剂处理时通常消失。表位通常在独特空间构象中包括至少3个，并且更通常至少5个、约9个或约8-10个氨基酸。"Epitope" or "antigenic determinant" refers to an antigenic determinant that interacts with a specific antigen-binding site called a paratope in the variable region of an antibody molecule. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be formed by contiguous amino acids or discrete amino acids joined by tertiary folding of the protein. Epitopes formed by consecutive amino acids usually remain when exposed to a denaturing solvent, while epitopes formed by tertiary folding usually disappear when treated with a denaturing solvent. Epitopes usually include at least 3, and more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
术语“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv(scFv)、单链Fab、双体抗体(diabody)、单结构域抗体(sdAb，纳米抗体)、骆驼Ig、Ig NAR、F(ab)' 3片段、双-scFv、(scFv) 2、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(“dsFv”)。所述术语还包括经遗传工程改造的形式，例如嵌合抗体(例如人类化鼠抗体)、杂结合抗体(例如双特异性抗体)和其抗原结合片段。更详细的描述也请参见：皮尔斯目录与手册(Pierce Catalog and Handbook),1994-1995(皮尔斯化学公司(PierceChemical Co.),罗克福德(Rockford),伊利诺伊州(IL))；Kuby,免疫学杂志,第3版,W.H.弗里曼公司(W.H.Freeman&Co.),纽约,1997。 The term "antigen-binding fragment" is a part or section of a complete or complete antibody that has fewer amino acid residues than a complete or complete antibody, which can bind to the antigen or compete with the complete antibody (ie, the complete antibody from which the antigen-binding fragment is derived) Binding antigen. The antigen-binding fragment can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of the intact antibody. Antigen-binding fragments include but are not limited to Fab, Fab', F(ab') 2 , Fv, single-chain Fv (scFv), single-chain Fab, diabody, single domain antibody (sdAb, Nanobody), Camel Ig, Ig NAR, F(ab)' 3 fragment, double-scFv, (scFv) 2 , mini-antibody, bi-functional antibody, tri-functional antibody, tetra-functional antibody, disulfide stabilized Fv protein ("dsFv") . The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), hybrid antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. For a more detailed description, please see: Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Illinois (IL)); Kuby, Journal of Immunology , 3rd edition, WH Freeman & Co., New York, 1997.
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(HC)和两条轻链(LC)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。哺乳动物重链分类为α、δ、ε、γ和μ。哺乳动物轻链分类为λ或κ。包含α、δ、ε、γ和μ重链的免疫球蛋白分类为免疫球蛋白(Ig)A、IgD、IgE、IgG和IgM。完全抗体形成“Y”形状。Y的茎由两条重链的第二和第三恒定区(并且对于IgE和IgM，第四恒定区)结合在一起组成，并且二硫键(链间)在铰链中形成。重链γ、α和δ具有由三个串联(成一行)Ig结构域构成的恒定区，和用于增加柔性的铰链区；重链μ和ε具有由四个免疫球蛋白结构域构成的恒定区。第二和第三恒定区分别称为“CH2结构域”和“CH3结构域”。Y的每个臂 包括结合到单个轻链的可变和恒定区的单个重链的可变区和第一恒定区。轻链和重链的可变区负责抗原结合。The terms "whole antibody", "full-length antibody", "full antibody" and "whole antibody" are used interchangeably herein to refer to at least two heavy chains (HC) and two Light chain (LC) glycoprotein. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three structural domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. Mammalian heavy chains are classified into α, δ, ε, γ, and μ. Mammalian light chains are classified as lambda or kappa. Immunoglobulins containing alpha, delta, epsilon, gamma, and mu heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. The complete antibody forms a "Y" shape. The stem of Y is composed of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchains) are formed in the hinge. The heavy chains γ, α, and δ have a constant region composed of three tandem (in a row) Ig domains, and a hinge region for increasing flexibility; the heavy chains μ and ε have a constant region composed of four immunoglobulin domains Area. The second and third constant regions are called "CH2 domain" and "CH3 domain", respectively. Each arm of Y includes the variable region and the first constant region of a single heavy chain that are bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.
轻链可变区和重链可变区分别包含间插有三个高变区(也称为“互补决定区”或“CDR”)的“构架”区。“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。The light chain variable region and the heavy chain variable region respectively comprise a "framework" region with three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs") intervening. "Complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" (herein can be used interchangeably with hypervariable region "HVR") is an antibody variable domain that is hypervariable in sequence and A structurally defined loop ("hypervariable loop") and/or a region containing antigen contact residues ("antigen contact point") is formed. CDR is mainly responsible for binding to antigen epitopes. The CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr/), and North CDR definition based on affinity propagation clustering using a large number of crystal structures .
然而，应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。例如，对使用Kabat和Chothia编号的CDR区域在不同指派系统定义下的残基范围如下表A所示。However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. For example, the residue ranges of CDR regions using Kabat and Chothia numbering under different assignment systems are shown in Table A below.
表A.不同指派系统定义下的CDR残基范围Table A. CDR residue ranges defined by different assignment systems
Figure PCTCN2020084478-appb-000001
Figure PCTCN2020084478-appb-000001
Figure PCTCN2020084478-appb-000002
Figure PCTCN2020084478-appb-000002
因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
本发明抗体的CDR可以根据本领域的任何方案或其组合人工地评估确定边界。除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。The CDR of the antibody of the present invention can be artificially evaluated and determined according to any scheme in the art or a combination thereof. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.
不同轻链或重链的构架区的序列在物种(例如人类)内具有相对保存性。抗体的构架区(其是成分轻链和重链的组合构架区)用以在三维空间中定位和比对CDR。CDR主要负责结合到抗原的表位。具有不同特异性(即针对不同抗原有不同组合位点)的抗体具有不同CDR。尽管抗体与抗体之间的CDR不同，但CDR内仅有限数目的氨基酸位置直接参与抗原结合。CDR内的这些位置称为特异性决定残基(SDR)。The sequences of the framework regions of different light chains or heavy chains have relative preservation in species (such as humans). The framework region of the antibody (which is the combined framework region of the light chain and the heavy chain) is used to locate and align the CDRs in a three-dimensional space. CDR is mainly responsible for binding to the epitope of the antigen. Antibodies with different specificities (that is, different combination sites for different antigens) have different CDRs. Although the CDRs are different between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDR are called specificity determining residues (SDR).
“单克隆抗体”是由B淋巴细胞的单个克隆或由其中已经转染单个抗体的轻链和重链基因的细胞产生的抗体。单克隆抗体通过本领域的技术人员已知的方法产生，例如通过由骨髓瘤细胞与免疫脾细胞的融合体制备杂交抗体形成细胞。单克隆抗体包括人类化单克隆抗体。A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells in which the light chain and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from a fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
“Fv”是含有完全抗原结合位点的最小抗体片段。在一个实施例中，双链Fv种类由一个重链可变结构域和一个轻链可变结构域呈紧密非共价缔合的二聚体组成。在单链Fv(scFv)种类中，一个重链可变结构域与一个轻链可变结构域可以通过柔性肽连接子共价连接，使得轻链和重链可以按类似于双链Fv种类的“二聚”结构缔合。在这一配置中，每个可变结构域的三个高变区(HVR)相互作用以定义VH-VL二聚体的表面上的抗原结合位点。六个HVR共同地赋予对抗体的抗原结合特异性。然而，即使单个可变结构域(或包含仅三个对抗原具有特异性的HVR的Fv的一半)也具有识别和结合抗原的能力，但亲和力低于完整结合位点。"Fv" is the smallest antibody fragment that contains a complete antigen binding site. In one embodiment, the double-chain Fv species is composed of a dimer of a heavy chain variable domain and a light chain variable domain in a tight non-covalent association. In the single-chain Fv (scFv) category, a heavy-chain variable domain and a light-chain variable domain can be covalently linked through a flexible peptide linker, so that the light chain and the heavy chain can be similar to the double-chain Fv category The "dimeric" structure is associated. In this configuration, the three hypervariable regions (HVR) of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific to the antigen) has the ability to recognize and bind to the antigen, but the affinity is lower than the complete binding site.
Fab片段含有重链可变结构域和轻链可变结构域并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab′片段与Fab片段不同之处在于，重链CH1结构域的羧基末 端增添了几个残基，包括一个或多个来自抗体铰链区的半胱氨酸。Fab′-SH是本文关于Fab′的名称，其中恒定结构域的半胱氨酸残基携有游离硫醇基。F(ab′)2抗体片段最初是作为其间具有铰链半胱氨酸的Fab′片段对产生。还已知抗体片段的其它化学偶合。The Fab fragment contains the variable domain of the heavy chain and the variable domain of the light chain and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The difference between the Fab' fragment and the Fab fragment is that several residues are added to the carboxyl end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of an antibody. Fab'-SH is the name of Fab' herein, in which the cysteine residue of the constant domain carries a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.
当谈及抗原和抗体时使用的术语“特异性结合”或“结合”意指抗体与生理条件下相对稳定的抗原形成复合物。用于确定抗体是否与抗原特异性结合的方法是本领域熟知的并且例如包括表面等离振子共振测定法、MSD测定法(Estep,P.等人,High throughput solution-based measurement of antibody-antigen affinity and epitope binning,MAbs,2013.5(2):p.270-278)、ForteBio亲和力测定法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)等。在一个实施方案中，本发明的“特异性结合”冠状病毒S蛋白抗体如ForteBio亲和力测定法中测量，以至少约10 -8M，优选10 -9M；更优选10 -10M，进一步优选10 -11M，更优选10 -12M的K D与S蛋白结合，由此阻断或抑制冠状病毒S蛋白与其受体ACE2结合以及随后的膜融合。 The term "specifically binds" or "binding" used when talking about antigens and antibodies means that the antibody forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, surface plasmon resonance assays, MSD assays (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2): p.270-278), ForteBio affinity assay (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs, 2013.5(2): p.270-8) and so on. In one embodiment, the "specific binding" coronavirus S protein antibody of the present invention is measured in the ForteBio affinity assay at at least about 10 -8 M, preferably 10 -9 M; more preferably 10 -10 M, and still more preferably A K D of 10 -11 M, more preferably 10 -12 M binds to the S protein, thereby blocking or inhibiting the binding of the coronavirus S protein to its receptor ACE2 and subsequent membrane fusion.
“亲和力”是指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。除非另有说明，在用于本文时，“结合亲和力”指反映结合对的成员(例如抗体与抗原)之间1∶1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可用结合解离平衡常数(K D)来表述。亲和力可通过本领域知道的常用方法来测量，包括现有技术已知以及本文中所描述的那些。 "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X to its partner Y can usually be expressed by the binding and dissociation equilibrium constant (K D ). Affinity can be measured by common methods known in the art, including those known in the prior art and those described herein.
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如S蛋白或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methods in Enzymology 9：242-253)。通常所述测定法涉及使用结合带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。When the term "competition" is used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding protein or neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay: In the assay, the antigen-binding protein to be detected (for example, an antibody or immunological functional fragment thereof) prevents or inhibits (for example, reduces) a reference antigen-binding protein (for example, a ligand or a reference antibody) and a common antigen (for example, S protein or Its fragment) specific binding. Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), Sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253). Generally, the assay involves the use of purified antigen bound to a solid surface or cell with either an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Usually the tested antigen binding protein is present in excess. The antigen binding proteins identified by competition assays (competitive antigen binding proteins) include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding that binds to adjacent epitopes that are sufficiently close to the binding epitope of the reference antigen binding protein Proteins, the two epitopes sterically hinder each other from binding. Additional details on the methods used to determine competitive binding are provided in the examples herein.
如本文所用，术语“变体”是指已经修饰至少一个，例如1、2或3个氨基酸取代、缺失或添加的重链可变区或轻链可变区，其中包含重链或轻链变体的经修饰的抗原结合蛋白基 本上保留修饰前抗原结合蛋白的生物学特征。在一个实施方案中，含有变体重链可变区或轻链可变区序列的抗原结合蛋白保留修饰前抗原结合蛋白的60％、70％、80％、90％、100％生物学特征。应当理解，可以单独或在与另一个重链可变区或轻链可变区组合修饰每个重链可变区或轻链可变区。本公开的抗原结合蛋白包含与本文描述的重链可变区氨基酸序列90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源的重链可变区氨基酸序列。本公开的抗原结合蛋白包括与本文描述的轻链可变区氨基酸序列90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源的轻链可变区氨基酸序列。同源性百分比可以在整个重链可变区和/或整个轻链可变区上，或者百分比同源性可以限于构架区，而对应于CDR的序列与重链可变区和/或轻链可变区内本文中公开的CDR具有100％同一性。如本文所用，术语“CDR变体”是指已经修饰至少一个，例如1、2或3个氨基酸取代、缺失或添加的CDR，其中包含CDR变体的经修饰的抗原结合蛋白基本上保留修饰前抗原结合蛋白的生物学特征。在一个实施方案中，含有变体CDR的抗原结合蛋白保留修饰前抗原结合蛋白的60％、70％、80％、90％、100％生物学特征。应当理解，可以修饰的每个CDR可以单独或与另一个CDR组合修饰。在一个实施方案中，修饰是取代，特别是保守取代。As used herein, the term "variant" refers to a heavy chain variable region or a light chain variable region that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, including heavy or light chain variants. The modified antigen-binding protein of the body basically retains the biological characteristics of the pre-modified antigen-binding protein. In one embodiment, the antigen binding protein containing the sequence of the variable heavy chain variable region or the variable region of the light chain retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein before modification. It should be understood that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region. The antigen binding protein of the present disclosure contains 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the heavy chain variable region described herein The amino acid sequence of the variable region of the heavy chain. The antigen binding protein of the present disclosure includes 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the light chain variable region described herein The amino acid sequence of the variable region of the light chain. The percent homology can be in the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology can be limited to the framework region, and the sequence corresponding to the CDR is the same as the heavy chain variable region and/or light chain. The CDRs disclosed herein have 100% identity within the variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, wherein the modified antigen binding protein comprising the CDR variant substantially retains the pre-modification The biological characteristics of antigen binding proteins. In one embodiment, the antigen binding protein containing the variant CDR retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein before modification. It should be understood that each CDR that can be modified can be modified alone or in combination with another CDR. In one embodiment, the modification is a substitution, especially a conservative substitution.
“人源化抗体”是指一类工程化抗体，其具有源自非人供体免疫球蛋白的CDR，该人源化抗体的剩余免疫球蛋白部分是来源自一种(或多种)人免疫球蛋白。此外，构架支持残基可以改变以保留结合亲和力(参见例如Queen等,Proc.Natl Acad Sci USA,86:10029-10032(1989),Hodgson等,Bio/Technology,9:421(1991))。合适的人接受抗体可以是通过与供体抗体的核苷酸和氨基酸序列的同源性从常规数据库，例如Los Alamos数据库和Swiss蛋白质数据库选择的抗体。以与供体抗体的构架区的同源性(基于氨基酸)表征的人抗体可以适合于提供用于插入供体CDR的重链恒定区和/或重链可变构架区。可以以类似的方式选择能够提供轻链恒定或可变构架区的合适受体抗体。应当注意的是，受体抗体重链和轻链不需要来源于相同的受体抗体。"Humanized antibody" refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, and the remaining immunoglobulin portion of the humanized antibody is derived from one (or more) humans Immunoglobulin. In addition, framework support residues can be changed to retain binding affinity (see, for example, Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al., Bio/Technology, 9: 421 (1991)). Suitable human accepting antibodies may be antibodies selected from conventional databases such as Los Alamos database and Swiss protein database by homology with the nucleotide and amino acid sequence of the donor antibody. Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody may be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDR. A suitable acceptor antibody capable of providing constant or variable framework regions of the light chain can be selected in a similar manner. It should be noted that the acceptor antibody heavy chain and light chain need not be derived from the same acceptor antibody.
如本领域已知，在本文中可交换使用的“多核苷酸”或“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。As known in the art, "polynucleotide" or "nucleic acid" used interchangeably herein refers to a chain of nucleotides of any length, and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.
如下进行序列之间序列同一性的计算。The calculation of sequence identity between sequences is performed as follows.
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位 置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, the first and second amino acid sequences or nucleic acid sequences may be used for optimal alignment. Gaps can be introduced in one or both or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。Mathematical algorithms can be used to achieve sequence comparison between two sequences and calculation of percent identity. In a preferred embodiment, the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used. Available), use Blossum 62 matrix or PAM250 matrix and gap weight 16, 14, 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6, to determine the difference between two amino acid sequences Percent identity. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 are used. Length weights 1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossom 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。You can also use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), using E. Meyers and W. Miller algorithms that have been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determine the percent identity between two amino acid sequences or nucleotide sequences.
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
如本文所用，“载体(vector)”表示构建体，其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂相关的DNA或RNA表达载体、包囊化于脂质体中的DNA或RNA表达载体以及某些真核细胞，例如生产细胞。As used herein, "vector" refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors related to cationic flocculants, DNA or RNA expression encapsulated in liposomes Vectors and certain eukaryotic cells, such as producer cells.
在本发明中术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且是指已经引入外源性核酸的细胞，包括这些细胞的子代。宿主细胞包括“转化子”和“转化的细胞”，其包括原代转化细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与亲代细胞可能不完全相同，但可能含有突变。本文包括与在初始转化的细胞中筛选或选择的细胞具有相同功能或生物学活性的突变子代。In the present invention, the terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The offspring may not be exactly the same as the parent cell in terms of nucleic acid content, but they may contain mutations. This document includes mutant progeny that have the same function or biological activity as the cells screened or selected in the initially transformed cells.
本发明还涉及一种生产单克隆抗体的方法，所述方法包括培养本发明所述的宿主细胞从而生产上述本发明所述的单克隆抗体。The present invention also relates to a method for producing a monoclonal antibody, the method comprising culturing the host cell of the present invention to produce the above-mentioned monoclonal antibody of the present invention.
在本文中，“受试者”、“个体”或“对象”指需要缓解、预防和/或治疗疾病或病症如病毒感染的动物，优选哺乳动物，更优选是人。哺乳动物还包括但不限于农场动物、竞赛动物、宠物、灵长类、马、犬、猫、小鼠和大鼠。该术语包括具有冠状病毒感染或处于具有冠状病毒感染风险的人受试者。在本发明中，向有此需要的受试者施用本发明所述的抗体或本发明所述的药物组合物或制品是指给予有效量的所述抗体或药物组合物或制品等。As used herein, "subject", "individual" or "subject" refers to animals in need of alleviation, prevention and/or treatment of diseases or conditions such as viral infections, preferably mammals, and more preferably humans. Mammals also include, but are not limited to, farm animals, race animals, pets, primates, horses, dogs, cats, mice, and rats. The term includes human subjects who have coronavirus infection or are at risk of coronavirus infection. In the present invention, administering the antibody of the present invention or the pharmaceutical composition or product of the present invention to a subject in need thereof means administering an effective amount of the antibody or pharmaceutical composition or product or the like.
如本发明所用，术语“有效量”表示引发例如研究者或临床医师所追求的组织、系统、动物或人的生物学或药学响应的药物或药剂的量。此外，术语“治疗有效量”表示，与没有接受该量的相应受试者相比，引起疾病、病症或副作用的改进治疗、治愈、预防或减轻的量，或者使疾病或病况的进展速率降低的量。该术语在其范围内还包括有效增强正常生理功能的量。As used in the present invention, the term "effective amount" means the amount of a drug or agent that elicits a biological or pharmaceutical response of a tissue, system, animal, or human, for example, which is sought by a researcher or clinician. In addition, the term "therapeutically effective amount" refers to an amount that causes an improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or reduces the rate of progression of the disease or condition, compared to a corresponding subject who did not receive the amount.的量。 The amount. The term also includes within its scope an amount effective to enhance normal physiological functions.
II.本发明抗体所针对的冠状病毒、其结构和进入宿主细胞的方式II. Coronavirus targeted by the antibody of the present invention, its structure and the way to enter the host cell
冠状病毒(包括SARS-CoV和新近发现的2019-nCoV)是球形单股正链RNA病毒，其特征是，具有从病毒体表面突出的刺突蛋白(Barcena,M.等人，Cryo-electron tomography of mouse hepatitis virus:Insights into the structure of the coronavirion.Proc.Natl.Acad.Sci.USA2009,106,582–587)。病毒颗粒的球形形态以及刺突突起使得冠状病毒在电子显微镜下看起来像冠冕而被命名为冠状病毒。Coronavirus (including SARS-CoV and the newly discovered 2019-nCoV) is a spherical single-stranded positive-stranded RNA virus, which is characterized by a spike protein protruding from the surface of the virion (Barcena, M. et al., Cryo-electron Tomography of mouse hepatitis virus: Insights into the structure of the coronavirion.Proc.Natl.Acad.Sci.USA2009,106,582–587). The spherical shape of the virus particles and the protrusions of the spikes make the coronavirus look like a crown under the electron microscope and it is named the coronavirus.
冠状病毒是被包膜的病毒(所被包膜衍生自宿主细胞膜的脂质双层)，具有主要由病毒结构蛋白(例如刺突蛋白(Spike，S)，膜蛋白(Membrane，M)，包膜蛋白(Envelope，E)和核衣壳蛋白(Nucleocapsid，N))形成的病毒结构，其中S蛋白、M蛋白和E蛋白均嵌入在病毒包膜中，N蛋白与病毒RNA相互作用，位于病毒颗粒的核心，形成核衣壳(Fehr,A.R.等人，Coronaviruses:An overview of their replication and pathogenesis.Methods Mol.Biol.2015,1282,1–23)。S蛋白是一种高度糖基化的蛋白，可在病毒颗粒表面形成同源三聚体的刺突，并介导病毒进入宿主细胞。Coronavirus is an enveloped virus (the envelope is derived from the lipid bilayer of the host cell membrane), which is mainly composed of viral structural proteins (such as spike protein (Spike, S), membrane protein (Membrane, M), A virus structure formed by membrane protein (Envelope, E) and nucleocapsid protein (Nucleocapsid, N), in which S protein, M protein, and E protein are all embedded in the viral envelope, and N protein interacts with viral RNA and is located in the virus The core of the particle forms the nucleocapsid (Fehr, AR et al., Coronaviruses: An overview of their replication and pathogenesis. Methods Mol. Biol. 2015, 1282, 1-23). S protein is a highly glycosylated protein that can form homotrimeric spikes on the surface of virus particles and mediate the virus into host cells.
2019-nCoV是具有膜结构的、大小为80-120nm的单股正链RNA病毒，基因组长度约为29.9kb，该病毒与同属于冠状病毒科β冠状病毒属的SARS-CoV的基因组序列之间的同源性为80％。病毒基因组的可读框(Open reading frame，ORF)ORF1a和ORF1b占基因组的2/3，表达水解酶以及与复制、转录相关的酶，例如，半胱氨酸蛋白酶(PLpro)和丝氨酸蛋白酶(3CLpro)，RNA依赖RNA聚合酶(RdRp)和解旋酶(Hel)；后面的基因组1/3区域主要负责编码结构蛋白，包括刺突蛋白(S)、包膜蛋白(E)、膜蛋白(M)、核衣壳蛋白(N)等主要结构蛋白，其中N蛋白包裹病毒基因组形成核蛋白复合体，E蛋白和M蛋白主要参与病毒的装配过程，S蛋白则主要通过与宿主细胞受体结合介导病毒的入侵并决定病毒的宿主特异性。经序列比对，发现2019-nCoV病毒和SARS-CoV病毒的S蛋白具有75％的相似度，据报道称多株SARS-CoV冠状病毒分离株中位于S蛋白与ACE2受体(在人体中主要分布于呼吸道上皮细胞、肺脏、心脏、肾脏和消化道等位置)复合物界 面的442、472、479、487和491位点的氨基酸残基是高度保守的。与SARS-CoV的S蛋白相比较，在所述5个位点处，2019-nCoV S蛋白仅第491位氨基酸相同，其它4处氨基酸都发生了突变(Xu X等人，Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission，Sci China Life Sci.,2020年3月；63(3):457-460)。尽管如此，通过蛋白质3D结构模拟预测发现虽然2019-nCOV S蛋白与ACE2受体结合的所述4个关键氨基酸都发生了替换，但是相对于SARS-CoV S蛋白，2019-nCoV S蛋白中的受体结合结构域(receptor binding domain，RBD)的三维结构几乎不变，由此2019-nCoV S蛋白与人体ACE2仍然具有较高的亲和力，最近的文章(Wrapp D等人，Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation，Science,.2020年2月19日，网上公开，pii:eabb2507.doi:10.1126/science.abb2507)以及(Xiaolong Tian等人，Potent binding of 2019novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody,Emerging Microbes & Infections，2020,9:1,p382-385,DOI:10.1080/22221751.2020.1729069)通过Fortebio检测，2019-nCoV的S蛋白结合人类ACE2的亲和力(K D)约为15nM，与SARS-CoV的S蛋白结合人类ACE2的亲和力相当，由此可见，ACE2也是2019-nCoV感染人体进入细胞内部的受体蛋白。预期针对冠状病毒S蛋白并且阻断其与ACE2受体结合的高亲和力中和抗体，能够有效预防和治疗冠状病毒(例如，2019-n CoV、SARS-CoV)感染。 2019-nCoV is a single-stranded positive-stranded RNA virus with a membrane structure and a size of 80-120nm. The genome length is about 29.9kb. This virus is between the genome sequence of SARS-CoV, which belongs to the β-coronavirus genus of the coronavirus family. The homology is 80%. The open reading frame (ORF) of the viral genome, ORF1a and ORF1b account for 2/3 of the genome, expressing hydrolases and enzymes related to replication and transcription, such as cysteine protease (PLpro) and serine protease (3CLpro). ), RNA-dependent RNA polymerase (RdRp) and helicase (Hel); the latter 1/3 region of the genome is mainly responsible for encoding structural proteins, including spike protein (S), envelope protein (E), and membrane protein (M) , Nucleocapsid protein (N) and other major structural proteins. Among them, the N protein wraps the viral genome to form a nucleoprotein complex, the E protein and M protein are mainly involved in the assembly process of the virus, and the S protein is mainly mediated by binding to host cell receptors. The invasion of the virus determines the host specificity of the virus. After sequence comparison, it is found that the S protein of 2019-nCoV virus and SARS-CoV virus has a similarity of 75%. It is reported that the S protein and ACE2 receptor (mainly in human The amino acid residues at positions 442, 472, 479, 487, and 491 at the interface of the complex are highly conserved. Compared with the S protein of SARS-CoV, at the 5 positions, only the 491th amino acid of the 2019-nCoV S protein is the same, and the other 4 amino acids are all mutated (Xu X et al., Evolution of the novel coronavirus) from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission, Sci China Life Sci., March 2020; 63(3):457-460). Nevertheless, the prediction of protein 3D structure simulation found that although the four key amino acids that the 2019-nCOV S protein binds to the ACE2 receptor have all been replaced, compared to the SARS-CoV S protein, the receptors in the 2019-nCoV S protein have been replaced. The three-dimensional structure of the receptor binding domain (RBD) is almost unchanged. Therefore, the 2019-nCoV S protein still has a high affinity with human ACE2. A recent article (Wrapp D et al., Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation, Science,. February 19, 2020, published online, pii:eabb2507.doi:10.1126/science.abb2507) and (Xiaolong Tian et al., Potent binding of 2019novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody, Emerging Microbes & Infections, 2020, 9:1, p382-385, DOI: 10.1080/22221751.2020.1729069) tested by Fortebio, the affinity of 2019-nCoV S protein binding to human ACE2 (K D ) It is about 15nM, which is equivalent to the affinity of SARS-CoV's S protein to human ACE2. It can be seen that ACE2 is also the receptor protein for 2019-nCoV to infect the human body into the cell. It is expected that high-affinity neutralizing antibodies that target the coronavirus S protein and block its binding to the ACE2 receptor can effectively prevent and treat coronavirus (for example, 2019-n CoV, SARS-CoV) infections.
III.本发明的针对冠状病毒S蛋白的抗体III. Antibodies against Coronavirus S protein of the present invention
术语“针对冠状病毒S蛋白的抗体”、“抗冠状病毒S蛋白的抗体”、“抗S蛋白抗体”、“冠状病毒S蛋白抗体”、“S蛋白抗体”或“结合S蛋白的抗体”在本文中可互换地使用，是指这样的本发明的抗体，所述抗体能够以足够的亲和力结合冠状病毒S蛋白(例如，2019-n CoV S蛋白、SARS-CoV S蛋白)，由此所述抗体可以用作靶向冠状病毒S蛋白的诊断剂、预防剂和/或治疗剂。The terms "antibody against coronavirus S protein", "antibody against coronavirus S protein", "anti-S protein antibody", "coronavirus S protein antibody", "S protein antibody" or "antibody binding to S protein" are used in Used interchangeably herein, it refers to the antibody of the present invention that can bind to the coronavirus S protein (for example, 2019-n CoV S protein, SARS-CoV S protein) with sufficient affinity. The antibody can be used as a diagnostic, preventive and/or therapeutic agent targeting the S protein of coronavirus.
本发明的抗体和抗原结合片段以高亲和力特异性结合冠状病毒S蛋白。在一些实施方案中，本发明的抗体是阻断抗体或中和抗体，其中抗体可以结合冠状病毒S蛋白，并阻断冠状病毒S蛋白与ACE2的结合。在一些实施方案中，该阻断抗体或中和抗体可以用于预防冠状病毒感染和/或治疗冠状病毒感染的个体。The antibodies and antigen-binding fragments of the present invention specifically bind to the coronavirus S protein with high affinity. In some embodiments, the antibody of the present invention is a blocking antibody or a neutralizing antibody, wherein the antibody can bind to the coronavirus S protein and block the binding of the coronavirus S protein to ACE2. In some embodiments, the blocking antibody or neutralizing antibody can be used to prevent coronavirus infection and/or treat individuals infected with coronavirus.
在一些实施方案中，本发明的冠状病毒S蛋白抗体特异性结合冠状病毒S蛋白，其包含In some embodiments, the coronavirus S protein antibody of the present invention specifically binds to the coronavirus S protein, which comprises
(a)SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR；以及与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体；或(a) The 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 and the 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 2; and the same as the above 6 The CDR region has a single or multiple CDR variants that do not exceed 2 amino acid changes in each CDR region; or
(b)SEQ ID NO:3所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:4所示的轻链可变区氨基酸序列中的3个CDR；以及与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体。(b) 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 3 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 4; and the same as the above 6 The CDR regions have variants in which single or multiple CDRs do not exceed 2 amino acid changes per CDR region.
其中所述氨基酸变化是氨基酸的添加、缺失或取代，例如，所述氨基酸变化是保守氨基酸取代。Wherein the amino acid change is an addition, deletion or substitution of an amino acid, for example, the amino acid change is a conservative amino acid substitution.
在一些实施方案中，本发明的冠状病毒S蛋白抗体结合哺乳动物冠状病毒S蛋白，例如人冠状病毒S蛋白、猴冠状病毒S蛋白。例如，本发明的冠状病毒S蛋白抗体与冠状病毒S蛋白上的表位(例如，线性或构象表位)特异性结合。In some embodiments, the coronavirus S protein antibody of the present invention binds to mammalian coronavirus S protein, such as human coronavirus S protein, monkey coronavirus S protein. For example, the coronavirus S protein antibody of the present invention specifically binds to an epitope (e.g., linear or conformational epitope) on the coronavirus S protein.
在一些实施方案中，本发明的冠状病毒S蛋白抗体具有以下一个或多个特性：In some embodiments, the coronavirus S protein antibody of the present invention has one or more of the following characteristics:
(a)在25℃ELISA测定法中测量，以小于约10nM，例如小于约8nM的IC50阻断冠状病毒S蛋白与分离的ACE2蛋白的结合，优于对照抗体CR3022；(a) Measured in an ELISA assay at 25°C, with an IC50 of less than about 10 nM, for example, less than about 8 nM, blocking the binding of the coronavirus S protein to the isolated ACE2 protein, which is better than the control antibody CR3022;
(b)在25℃ELISA测定法中测量，针对冠状病毒S蛋白的亲和力优于对照抗体CR3022；(b) Measured in the 25°C ELISA assay method, the affinity for the coronavirus S protein is better than the control antibody CR3022;
(c)在25℃生物膜层干涉测定法中测量，以小于约1nM，例如约0.8nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM的结合解离平衡常数K D结合冠状病毒S蛋白； (c) Measured in 25°C biofilm interferometry, with a binding and dissociation equilibrium constant K D of less than about 1 nM, for example, about 0.8 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM Binding to coronavirus S protein;
(d)在FACS测定法中测量，以小于约2nM，例如约1.8nM、1.5nM、1.2nM、0.9nM、0.6nM、0.3nM的IC50阻断冠状病毒S蛋白与Vero E6细胞上表达的天然ACE2蛋白的结合；(d) Measured in the FACS assay, with IC50 less than about 2nM, such as about 1.8nM, 1.5nM, 1.2nM, 0.9nM, 0.6nM, 0.3nM, to block the natural expression of coronavirus S protein and Vero E6 cells Binding of ACE2 protein;
(e)在细胞病变法中测量，本发明的抗体抑制病毒侵染Vero E6细胞的中和活性显著优于对照抗体，其保护50％细胞不受100TCID50攻击病毒液感染的抗体最高稀释浓度(或抗体的滴度)小于约0.5nM，例如约0.4nM、0.3nM、0.2nM、0.1nM。(e) Measured in the cytopathic method, the neutralizing activity of the antibody of the present invention to inhibit virus infection of Vero E6 cells is significantly better than that of the control antibody, and it protects 50% of cells from 100TCID50 attack. The antibody titer) is less than about 0.5 nM, such as about 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM.
在一些实施方案中，本发明的冠状病毒S蛋白抗体包含SEQ ID NO:5所示的HCDR1或SEQ ID NO:5所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:6所示的HCDR2或SEQ ID NO:6所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:7所示的HCDR3或SEQ ID NO:7所示的HCDR3的不超过2个氨基酸变化的变体；SEQ ID NO:8所示的LCDR1或SEQ ID NO:8所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:9所示的LCDR2或SEQ ID NO:9所示的LCDR2的不超过2个氨基酸变化的 变体、和SEQ ID NO:10所示的LCDR3或SEQ ID NO:10所示的LCDR3的不超过2个氨基酸变化的变体，其中所述氨基酸变化是氨基酸的添加、缺失或取代，例如，所述氨基酸变化是保守氨基酸取代。In some embodiments, the coronavirus S protein antibody of the present invention comprises HCDR1 shown in SEQ ID NO: 5 or a variant of HCDR1 shown in SEQ ID NO: 5 with no more than 2 amino acid changes, SEQ ID NO: 6 No more than 2 amino acid variants of HCDR2 or HCDR2 shown in SEQ ID NO: 6 and HCDR3 shown in SEQ ID NO: 7 or HCDR3 shown in SEQ ID NO: 7 not more than 2 A variant of amino acid change; LCDR1 shown in SEQ ID NO: 8 or a variant of LCDR1 shown in SEQ ID NO: 8 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO: 9 or SEQ ID NO: The variant of LCDR2 shown in 9 with no more than 2 amino acid changes, and the LCDR3 shown in SEQ ID NO: 10 or the variant of LCDR3 shown in SEQ ID NO: 10 with no more than 2 amino acid changes, wherein Amino acid changes are additions, deletions or substitutions of amino acids, for example, the amino acid changes are conservative amino acid substitutions.
在一些实施方案中，本发明的冠状病毒S蛋白抗体包含SEQ ID NO:11所示的HCDR1或SEQ ID NO:11所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:12所示的HCDR2或SEQ ID NO:12所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:13所示的HCDR3或SEQ ID NO:13所示的HCDR3的不超过2个氨基酸变化的变体；SEQ ID NO:14所示的LCDR1或SEQ ID NO:14所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:15所示的LCDR2或SEQ ID NO:15所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:16所示的LCDR3或SEQ ID NO:16所示的LCDR3的不超过2个氨基酸变化的变体，其中所述氨基酸变化是氨基酸的添加、缺失或取代，例如，所述氨基酸变化是保守氨基酸取代。In some embodiments, the coronavirus S protein antibody of the present invention comprises HCDR1 shown in SEQ ID NO: 11 or a variant of HCDR1 shown in SEQ ID NO: 11 with no more than 2 amino acid changes, SEQ ID NO: 12 No more than 2 amino acid variants of HCDR2 or HCDR2 shown in SEQ ID NO: 12, and HCDR3 shown in SEQ ID NO: 13 or HCDR3 shown in SEQ ID NO: 13 not more than 2 A variant of amino acid changes; LCDR1 shown in SEQ ID NO: 14 or a variant of LCDR1 shown in SEQ ID NO: 14 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO: 15 or SEQ ID NO: A variant of LCDR2 shown in 15 with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO: 16 or a variant of LCDR3 shown in SEQ ID NO: 16 with no more than 2 amino acid changes, wherein Amino acid changes are additions, deletions or substitutions of amino acids, for example, the amino acid changes are conservative amino acid substitutions.
在一些实施方案中，本发明的冠状病毒S蛋白抗体或抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:1的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。优选地，所述氨基酸变化不发生在CDR区中。In some embodiments, the coronavirus S protein antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 1 or has at least 90% of the sequence of SEQ ID NO:1. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region contains the sequence of SEQ ID NO: 2 or has A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. Preferably, the amino acid change does not occur in the CDR region.
在一些实施方案中，本发明的冠状病毒S蛋白抗体或抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。优选地，所述氨基酸变化不发生在CDR区中。In some embodiments, the coronavirus S protein antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3 or has a sequence of at least 90%. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region contains the sequence of SEQ ID NO: 4 or has A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. Preferably, the amino acid change does not occur in the CDR region.
在一些实施方案中，本发明的冠状病毒S蛋白抗体包含Fc区，所述Fc区来自IgG，例如IgG1、IgG2、IgG3或IgG4。在一些实施方案中，所述Fc区来自IgG1或IgG4。在一些实施方案中，所述Fc区来自人IgG1或人IgG4。In some embodiments, the coronavirus S protein antibody of the present invention comprises an Fc region derived from IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region is derived from human IgG1 or human IgG4.
在本发明的一些实施方案中，本文所述的氨基酸变化包括氨基酸的取代、插入或缺失。优选的，本文所述的氨基酸变化为氨基酸取代，优选地保守取代。保守取代是指一个氨基酸经相同类别内的另一氨基酸取代，例如一个酸性氨基酸经另一酸性氨基酸取代，一个碱性氨基酸经另一碱性氨基酸取代，或一个中性氨基酸经另一中性氨基酸取代。示例性的取代如下表B所示：In some embodiments of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitution refers to the substitution of an amino acid by another amino acid in the same category, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid replace. Exemplary substitutions are shown in Table B below:
表B.示例的氨基酸取代Table B. Example amino acid substitutions
原始残基Original residue 示例性取代Exemplary substitution 优选的取代Preferred substitution
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Asp、Lys；ArgGln; His; Asp, Lys; Arg GlnGln
Asp(D)Asp(D) Glu；AsnGlu; Asn GluGlu
Cys(C)Cys(C) Ser；AlaSer; Ala SerSer
Gln(Q)Gln(Q) Asn；GluAsn; Glu AsnAsn
Glu(E)Glu(E) Asp；GlnAsp; Gln AspAsp
Gly(G)Gly(G) AlaAla AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile(I) Leu，Val；Met；Ala；Phe；正亮氨酸Leu, Val; Met; Ala; Phe; Norleucine LeuLeu
Leu(L)Leu(L) 正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr TyrTyr
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) Val；SerVal; Ser SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine LeuLeu
在优选的实施方案中，本发明所述的氨基酸变化发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸变化发生在Fc区。在一些实施方案中，提供了包含含有一个或多个突变的Fc结构域的抗冠状病毒S蛋白抗体，该突变增强或减弱抗体例如与中性pH相比在酸性pH下与FcRn受体的结合。例如，本发明包括在Fc结构域的C H2或C H3区中含有突变的抗冠状病毒S蛋白抗体，其中该一个或多个突变提高Fc结构域在酸性环境(例如在pH在约5.5至约6.0范围内的内体中)中与FcRn的亲和力。这种突变可以导致对动物施用时抗体血清半衰期的提高。这类Fc修饰的非限制性实例包括，例如：250位(例如E或Q)、250位和428位(例如L或F)、252位(例如L/Y/F/W或T)、254位(例如S或T)和256位(例如S/R/Q/E/D或T)的修饰；或428位和/或433位(例如H/L/R/S/P/Q或K)和/或434位(例如A、W、H、F或Y[N434A、N434W、N434H、N434F或N434Y])的修饰； 或250位和/或428位的修饰；或307位或308位(例如308F、V308F)和434位的修饰。在一个实施方案中，该修饰包括428L(例如M428L)和434S(例如N434S)修饰；428L、259I(例如V259I)和308F(例如V308F)修饰；433K(例如H433K)和434(例如434Y)修饰；252、254和256(例如252Y、254T和256E)修饰；250Q和428L修饰(例如T250Q和M428L)；及307和/或308修饰(例如308F或308P)。还在另一实施方案中，该修饰包括265A(例如D265A)和/或297A(例如N297A)修饰。 In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDR (for example, in the FR). More preferably, the amino acid changes described in the present invention occur in the Fc region. In some embodiments, there is provided an anti-coronavirus S protein antibody comprising an Fc domain containing one or more mutations that enhance or weaken the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH, for example . For example, the present invention includes a coronavirus S protein antibody containing mutations in the C H Fc domain or 2 C H 3 region, wherein the one or more mutations that improve Fc domain in an acidic environment (e.g. pH about 5.5 Affinity to FcRn in endosomes within the range of about 6.0. This mutation can lead to an increase in antibody serum half-life when administered to animals. Non-limiting examples of such Fc modifications include, for example, positions 250 (for example, E or Q), positions 250 and 428 (for example, L or F), positions 252 (for example, L/Y/F/W or T), 254 Bit (such as S or T) and 256 bit (such as S/R/Q/E/D or T) modification; or 428 bit and/or 433 bit (such as H/L/R/S/P/Q or K ) And/or modification of position 434 (for example, A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modification of position 250 and/or 428; or modification of position 307 or 308 ( For example, 308F, V308F) and the modification of position 434. In one embodiment, the modification includes 428L (e.g. M428L) and 434S (e.g. N434S) modifications; 428L, 2591 (e.g. V259I) and 308F (e.g. V308F) modifications; 433K (e.g. H433K) and 434 (e.g. 434Y) modifications; 252, 254, and 256 (e.g. 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g. T250Q and M428L); and 307 and/or 308 modifications (e.g. 308F or 308P). In yet another embodiment, the modification includes 265A (e.g. D265A) and/or 297A (e.g. N297A) modifications.
例如，本发明包括含有Fc结构域的抗冠状病毒S蛋白抗体，该Fc结构域包含选自以下的一对(组)或多对(组)突变：250Q和248L(例如T250Q和M248L)；252Y、254T和256E(例如M252Y、S254T和T256E)；428L和434S(例如M428L和N434S)；257I和311I(例如P257I和Q311I)；257I和434H(例如P257I和N434H)；376V和434H(例如D376V和N434H)；307A、380A和434A(例如T307A、E380A和N434A)；和433K和434F(例如H433K和N434F)。在一个实施方案中，本发明包括含有Fc结构域的抗冠状病毒S蛋白抗体，该Fc结构域包含IgG4铰链区中的S108P突变以促进二聚体稳定化。前述Fc结构域突变和本文公开的抗体可变结构域内的其他突变的任意可能的组合都包含在本发明的范围之内。For example, the present invention includes an anti-coronavirus S protein antibody containing an Fc domain containing one or more pairs (groups) of mutations selected from: 250Q and 248L (for example, T250Q and M248L); 252Y , 254T and 256E (such as M252Y, S254T and T256E); 428L and 434S (such as M428L and N434S); 257I and 311I (such as P257I and Q311I); 257I and 434H (such as P257I and N434H); 376V and 434H (such as D376V and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present invention includes an anti-coronavirus S protein antibody containing an Fc domain that includes the S108P mutation in the hinge region of IgG4 to promote dimer stabilization. Any possible combination of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are included within the scope of the present invention.
在某些实施方案中，本文中所提供的冠状病毒S蛋白抗体经改变以增加或降低其糖基化的程度。对冠状病毒S蛋白抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一个或多个糖基化位点而方便地实现。当冠状病毒S蛋白抗体包含Fc区时，可以改变与Fc区连接的糖类。在一些应用中，除去不想要的糖基化位点的修饰可以是有用的，例如除去岩藻糖模块以提高抗体依赖性细胞性细胞毒性(ADCC)功能(参见Shield等(2002)JBC277:26733)。在其它应用中，可以进行半乳糖苷化修饰以调节补体依赖性细胞毒性(CDC)。在某些实施方案中，可在本文中所提供冠状病毒S蛋白抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体，以便增强例如本发明的冠状病毒S蛋白抗体预防和/或治疗冠状病毒感染的有效性。In certain embodiments, the coronavirus S protein antibodies provided herein are modified to increase or decrease the degree of glycosylation. The addition or deletion of glycosylation sites of the coronavirus S protein antibody can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites. When the coronavirus S protein antibody contains the Fc region, the carbohydrates linked to the Fc region can be changed. In some applications, modifications to remove unwanted glycosylation sites may be useful, such as removing fucose moieties to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733 ). In other applications, galactosidation can be modified to modulate complement dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the coronavirus S protein antibody provided herein to generate Fc region variants, so as to enhance the prevention of the coronavirus S protein antibody of the present invention, for example. And/or the effectiveness of the treatment of coronavirus infections.
在一些实施方案中，本发明的冠状病毒S蛋白抗体是双特异性或多特异性抗体分子形式。在一个实施方案中，双特异性抗体分子具有针对冠状病毒S蛋白第一表位的第一结合特异性和对冠状病毒S蛋白第二表位的第二结合特异性，其中所述第一表位和第二表位可以是相同的，也可以是不同且非重叠的。在一个实施方案中，双特异性抗体分子包含本发明的抗体P17-A11 Fab和P16-A3 Fab。又在一个实施方案中，双特异性抗体分子包含本发明的抗体P17-A11 scFv和P16-A3 scFv。In some embodiments, the coronavirus S protein antibody of the present invention is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity for a first epitope of the coronavirus S protein and a second binding specificity for a second epitope of the coronavirus S protein, wherein the first table The position and the second epitope can be the same, or they can be different and non-overlapping. In one embodiment, the bispecific antibody molecule comprises the antibodies P17-A11 Fab and P16-A3 Fab of the invention. In yet another embodiment, the bispecific antibody molecule comprises the antibodies P17-A11 scFv and P16-A3 scFv of the present invention.
在一些实施方案中，本发明涉及抗体组合，其包含特异性结合冠状病毒S蛋白表位的本发明的抗体或抗原结合片段，和/或其他特异性结合冠状病毒S蛋白表位的抗体或抗原结合片段。在一个实施方案中，抗体组合是抗体P17-A11或其抗原结合片段和抗体P16-A3或其抗原结合片段的组合。In some embodiments, the present invention relates to an antibody combination comprising the antibody or antigen-binding fragment of the present invention that specifically binds to the epitope of the coronavirus S protein, and/or other antibodies or antigens that specifically bind to the epitope of the coronavirus S protein Combine fragments. In one embodiment, the antibody combination is a combination of antibody P17-A11 or an antigen-binding fragment thereof and antibody P16-A3 or an antigen-binding fragment thereof.
IV.本发明的核酸以及包含其的宿主细胞IV. The nucleic acid of the present invention and the host cell containing it
在一方面，本发明提供了编码以上任何冠状病毒S蛋白抗体或其抗原结合片段或其任一条链的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。In one aspect, the present invention provides a nucleic acid encoding any of the above coronavirus S protein antibodies or antigen-binding fragments or any chain thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (such as CHO cells or 293 cells), or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.
例如，本发明的核酸包含编码选自SEQ ID NO:1、2、3、4中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO:1、2、3、4中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。For example, the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, and 4, or a nucleic acid that encodes an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, and 4. A nucleic acid whose amino acid sequence is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence.
又在一个实施方案中，本发明的核酸包含编码选自SEQ ID NO:17、18、19、20中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO:17、18、19、20中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。In yet another embodiment, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 17, 18, 19, 20, or a nucleic acid that encodes an amino acid sequence selected from SEQ ID NO: 17, 18, The amino acid sequence shown in any one of 19 and 20 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity Amino acid sequence of nucleic acid.
本发明还涵盖与下述核酸在严格性条件下杂交的核酸或与下述核酸相比编码具有一个或多个氨基酸取代(例如保守性取代)、缺失或插入的多肽序列的核酸：包含编码选自SEQ ID NO:1、2、3、4中任一项所示氨基酸序列的核酸序列的核酸；或包含编码与选自SEQ ID NO:1、2、3、4中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸序列的核酸。The present invention also covers nucleic acids that hybridize with the following nucleic acids under stringent conditions, or nucleic acids that encode polypeptide sequences having one or more amino acid substitutions (eg conservative substitutions), deletions, or insertions compared with the following nucleic acids: comprising coding options A nucleic acid derived from the nucleic acid sequence of the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4; or a nucleic acid containing a code selected from any one of SEQ ID NO: 1, 2, 3, 4 The amino acid sequence has a nucleic acid sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
在一个实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include but are not limited to viruses, plasmids, cosmids, lambda phage or yeast artificial chromosomes (YAC).
一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转 染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。Once the expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this goal, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in a culture medium and screened for appropriate activity. The methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the methods known in this specification and the prior art, depending on the specific expression vector and Mammalian host cell changes or optimizations.
另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。标记物可以例如向营养缺陷型宿主提供原养型、杀生物抗性(例如，抗生素)或重金属(如铜)抗性等。可选择标记基因可以与待表达的DNA序列直接连接或通过共转化引入相同的细胞中。也可能需要额外元件以便最佳合成mRNA。这些元件可以包括剪接信号，以及转录启动子、增强子和终止信号。In addition, it is possible to select cells that have stably incorporated DNA into their chromosomes by introducing one or more markers that allow the selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (for example, antibiotics), or heavy metal (for example, copper) resistance, etc., to the auxotrophic host. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements can include splicing signals, as well as transcription promoters, enhancers, and termination signals.
在一个实施方案中，提供了包含本发明多核苷酸的宿主细胞。在一些实施方案中，提供了包含本发明表达载体的宿主细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体的其它细胞。合适的宿主细胞包括原核微生物，如大肠杆菌。宿主细胞还可以是真核微生物如丝状真菌或酵母，或各种真核细胞，例如昆虫细胞等。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK 293或293F细胞)、293细胞、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(Hep G2)、中国仓鼠卵巢细胞(CHO细胞)、CHOS细胞、NSO细胞、骨髓瘤细胞系如Y0、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO细胞或293细胞。In one embodiment, a host cell comprising the polynucleotide of the invention is provided. In some embodiments, a host cell comprising an expression vector of the invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies. Suitable host cells include prokaryotic microorganisms such as Escherichia coli. The host cell can also be a eukaryotic microorganism such as filamentous fungus or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as hosts. For example, a mammalian cell line modified to be suitable for growth in suspension can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells ( CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver Cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (Edited by B.K.C.Lo, Humana Press, Totowa, NJ), pages 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or 293 cell.
V.本发明的冠状病毒S蛋白抗体的生产和纯化V. Production and purification of the coronavirus S protein antibody of the present invention
在一个实施方案中，本发明提供了制备冠状病毒S蛋白抗体的方法，其中所述方法包括在适于表达编码所述冠状病毒S蛋白抗体的核酸的条件下培养包含编码所述冠状病毒S蛋白抗体的核酸或包含所述核酸的表达载体的宿主细胞，以及任选地分离所述冠状病毒S蛋白抗体。在某个实施方案中，所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收冠状病毒S蛋白抗体。In one embodiment, the present invention provides a method for preparing a coronavirus S protein antibody, wherein the method comprises culturing a nucleic acid encoding the coronavirus S protein antibody under conditions suitable for expressing a nucleic acid encoding the coronavirus S protein antibody. The nucleic acid of the antibody or the host cell containing the expression vector of the nucleic acid, and optionally the coronavirus S protein antibody is isolated. In a certain embodiment, the method further includes recovering the coronavirus S protein antibody from the host cell (or host cell culture medium).
为了重组产生本发明的冠状病毒S蛋白抗体，首先分离编码本发明冠状病毒S蛋白抗体的核酸，并将所述核酸插入载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序，例如通过使用能够与编码本发明冠状病毒S蛋白抗体的核酸特异性结合的寡核苷酸探针进行。In order to recombinantly produce the coronavirus S protein antibody of the present invention, first isolate the nucleic acid encoding the coronavirus S protein antibody of the present invention, and insert the nucleic acid into a vector for further cloning and/or expression in host cells. Such nucleic acids are easily separated and sequenced using conventional procedures, for example, by using oligonucleotide probes that can specifically bind to the nucleic acid encoding the coronavirus S protein antibody of the present invention.
如本文所述制备的本发明的冠状病毒S蛋白抗体可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而 易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的冠状病毒S蛋白抗体的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。The coronavirus S protein antibody of the present invention prepared as described herein can be purified by known prior art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these are obvious to those skilled in the art. The purity of the coronavirus S protein antibody of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, and the like.
VI.本发明的冠状病毒S蛋白抗体的活性测定法VI. The method for determining the activity of the coronavirus S protein antibody of the present invention
可以通过本领域中已知的多种测定法对本文中提供的冠状病毒S蛋白抗体鉴定，筛选，或表征其物理/化学特性和/或生物学活性。一方面，对本发明的冠状病毒S蛋白抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹等来进行。可使用本领域已知方法来测定对冠状病毒S蛋白的结合，本文中公开了例示性方法。在一些实施方案中，使用SPR或生物膜层干涉测定本发明的冠状病毒S蛋白抗体对冠状病毒S蛋白的结合。The coronavirus S protein antibody provided herein can be identified, screened, or characterized by a variety of assays known in the art, or its physical/chemical properties and/or biological activity. On the one hand, the coronavirus S protein antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot and the like. The binding to the coronavirus S protein can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, SPR or biofilm layer interference is used to determine the binding of the coronavirus S protein antibody of the present invention to the coronavirus S protein.
本发明还提供了用于鉴定具有生物学活性的冠状病毒S蛋白抗体的测定法。生物学活性可以包括例如阻断对细胞表面ACE2的结合。The present invention also provides an assay method for identifying the coronavirus S protein antibody with biological activity. The biological activity may include, for example, blocking the binding of ACE2 on the cell surface.
VII.药物组合和药物制剂VII. Pharmaceutical combinations and pharmaceutical preparations
在一些实施方案中，本发明提供包含本文所述的任何冠状病毒S蛋白抗体的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物(例如，药物组合物)包含本发明的冠状病毒S蛋白抗体，以及一种或多种其它治疗剂(例如抗感染活性剂、小分子药物)的组合。所述抗感染活性剂、小分子药物是用来治疗、预防或缓解受试者中冠状病毒感染的任何抗感染活性剂、小分子药物，包括但不限于瑞德西韦、利巴韦林、奥司他韦、扎那米韦、羟氯喹、干扰素-α2b、镇痛药、阿奇霉素和皮质类固醇。在本发明的上下中，冠状病毒感染包括由冠状病毒(包括但不限于2019-n CoV、SARS-CoV)引起的感染。In some embodiments, the present invention provides a composition comprising any coronavirus S protein antibody described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises pharmaceutical excipients. In one embodiment, the composition (e.g., pharmaceutical composition) comprises the coronavirus S protein antibody of the present invention and a combination of one or more other therapeutic agents (e.g., anti-infective agents, small molecule drugs). The anti-infective agent and small molecule drug are any anti-infective agent or small molecule drug used to treat, prevent or alleviate coronavirus infection in subjects, including but not limited to remdesivir, ribavirin, Oseltamivir, zanamivir, hydroxychloroquine, interferon-α2b, analgesics, azithromycin, and corticosteroids. In the context of the present invention, coronavirus infections include infections caused by coronaviruses (including but not limited to 2019-n CoV and SARS-CoV).
在一些实施方案中，本发明的药物组合物或药物制剂包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present invention contains suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等 的形式。口服配制剂可以包含标准药用载体和/或赋形剂，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, etc. that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. It is also possible to use saline solutions and aqueous dextrose and glycerol solutions as liquid carriers, especially for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skimmed milk, glycerin , Propylene, glycol, water, ethanol, etc. For the use and use of excipients, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, and saccharin.
可以通过将具有所需纯度的本发明的冠状病毒S蛋白抗体与一种或多种任选的药用辅料(Remington’s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本文所述的冠状病毒S蛋白抗体的药物制剂，优选地以冻干制剂或水溶液的形式。It can be prepared by mixing the coronavirus S protein antibody of the present invention with the required purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). The pharmaceutical preparation of the coronavirus S protein antibody described herein is preferably in the form of a lyophilized preparation or an aqueous solution.
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它抗感染活性成分，例如其它抗体、抗感染活性剂、小分子药物或免疫调节剂等。所述活性成分以对于目的用途有效的量合适地组合存在。The pharmaceutical composition or formulation of the present invention may also contain more than one active ingredient that is required for the specific indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators. The active ingredients are suitably present in combination in an amount effective for the intended use.
可制备持续释放制剂。持续释放制剂的合适实例包括含有本发明的冠状病毒S蛋白抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the coronavirus S protein antibody of the present invention, which matrices are in the form of shaped articles, such as films or microcapsules.
VIII.组合产品或试剂盒VIII. Combination products or kits
在一些实施方案中，本发明还提供了组合产品，其包含至少一种本发明的冠状病毒S蛋白抗体或其抗原结合片段，或者还包含一种或多种其它治疗剂(例如，抗感染活性剂、小分子药物或免疫调节剂等)。In some embodiments, the present invention also provides a combination product, which comprises at least one coronavirus S protein antibody or antigen-binding fragment thereof of the present invention, or further comprises one or more other therapeutic agents (e.g., anti-infective activity). Agents, small molecule drugs or immunomodulators, etc.).
在一些实施方案中，所述组合产品中的两种或多种成分可以依次、分开或同时联合施用给受试者。In some embodiments, two or more ingredients in the combination product may be administered to the subject in combination sequentially, separately, or simultaneously.
在一些实施方案中，本发明还提供了包含本发明的冠状病毒S蛋白抗体、药物组合物或组合产品的试剂盒，以及任选的指导施用的包装插页。In some embodiments, the present invention also provides a kit containing the coronavirus S protein antibody, pharmaceutical composition, or combination product of the present invention, and optionally a package insert to guide administration.
在一些实施方案中，本发明还提供了包含本发明的冠状病毒S蛋白抗体、药物组合物、组合产品的药物制品，任选地，所述药物制品还包括指导施用的包装插页。In some embodiments, the present invention also provides a pharmaceutical product comprising the coronavirus S protein antibody, pharmaceutical composition, and combination product of the present invention. Optionally, the pharmaceutical product further includes a package insert for guiding administration.
IX.本发明的冠状病毒S蛋白抗体的预防和/或治疗用途IX. Preventive and/or therapeutic use of the coronavirus S protein antibody of the present invention
本发明提供了用于预防受试者中冠状病毒相关疾病或病患的方法，其包括向受试者施用本发明的抗体、抗体组合或多特异性抗体。The present invention provides a method for preventing a coronavirus-related disease or disease in a subject, which comprises administering the antibody, antibody combination or multispecific antibody of the present invention to the subject.
具有冠状病毒相关疾病风险的受试者包括与感染者接触的受试者或以一些其他方式暴露于冠状病毒的受试者。预防剂的施用可以在表现出冠状病毒相关疾病的症状特征之前施用，以便阻止疾病，或可选择地延迟疾病的进展。Subjects at risk of coronavirus-related diseases include subjects who have been in contact with an infected person or subjects who have been exposed to the coronavirus in some other way. The administration of the prophylactic agent may be administered before the symptomatic characteristics of the coronavirus-related disease are manifested in order to prevent the disease, or alternatively delay the progression of the disease.
本发明还提供了治疗患者中冠状病毒相关疾病的方法。在一个实施方案中，该方法涉及将中和冠状病毒的本发明的抗体、抗体组合或多特异性抗体施用至患所述疾病的患者。The present invention also provides methods for treating coronavirus-related diseases in patients. In one embodiment, the method involves administering an antibody, antibody combination or multispecific antibody of the invention that neutralizes coronavirus to a patient suffering from the disease.
在一些实施方案中，提供了治疗患者中冠状病毒感染的方法，所述方法包括施用选自由以下组成的组的至少一种抗体或其抗原结合片段：抗体P16-A3、P17-A11。在一个优选的实施方案中，将抗体P16-A3、P17-A11或其片段的两种一起施用给所述患者，In some embodiments, there is provided a method of treating a coronavirus infection in a patient, the method comprising administering at least one antibody or antigen-binding fragment thereof selected from the group consisting of antibodies P16-A3, P17-A11. In a preferred embodiment, two of the antibodies P16-A3, P17-A11 or fragments thereof are administered to the patient together,
在一些实施方案中，本发明的抗体或其抗原结合片段可以交叉中和人和动物传染性冠状病毒分离株。In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can cross-neutralize human and animal infectious coronavirus isolates.
在一些实施方案中，本发明的抗体或其抗原结合片段在冠状病毒感染后的最初24小时内施用。In some embodiments, the antibodies of the invention or antigen-binding fragments thereof are administered within the first 24 hours after coronavirus infection.
X.用于诊断和检测冠状病毒的方法和组合物X. Methods and compositions for diagnosing and detecting coronavirus
在一些实施方案中，本文中提供的任何冠状病毒S蛋白抗体可以用于检测冠状病毒在生物样品中的存在。术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法。In some embodiments, any of the coronavirus S protein antibodies provided herein can be used to detect the presence of coronavirus in a biological sample. When the term "detection" is used herein, it includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule complexed magnetic beads, ELISA assays Law.
在一个实施方案中，提供了用于诊断或检测方法中的冠状病毒S蛋白抗体。在另一个方面中，提供检测冠状病毒在生物样品中的存在的方法。在某些实施方案中，所述方法包括检测冠状病毒S蛋白在生物样品中的存在。在某些实施方案中，所述方法包括将生物样品与如本文所述的冠状病毒S蛋白抗体在允许冠状病毒S蛋白抗体与冠状病毒S蛋白结合的条件下接触，并检测在冠状病毒S蛋白抗体和冠状病毒S蛋白之间是否形成复合物。复合物的形成表示存在冠状病毒。该方法可以是体外或体内方法。In one embodiment, a coronavirus S protein antibody for use in a method of diagnosis or detection is provided. In another aspect, a method of detecting the presence of coronavirus in a biological sample is provided. In certain embodiments, the method includes detecting the presence of coronavirus S protein in a biological sample. In certain embodiments, the method includes contacting a biological sample with a coronavirus S protein antibody as described herein under conditions that allow the coronavirus S protein antibody to bind to the coronavirus S protein, and detecting the presence of the coronavirus S protein Whether a complex is formed between the antibody and the coronavirus S protein. The formation of the complex indicates the presence of coronavirus. The method can be an in vitro or in vivo method.
用于冠状病毒的示例性诊断测定包括例如使用本发明的抗冠状病毒S蛋白接触从患者获得的样品，其中使用可检测的标记或报告分子标记抗冠状病毒S蛋白或用作捕获配体以选择性地从患者样品分离冠状病毒。备选地，未标记的抗冠状病毒S蛋白可与本身被可检测地标记的第二抗体组合在诊断应用中使用。可检测的标记或报告分子可以是放射性同位素、如 3H、 14C、 32P、 35S或 125I；荧光或化学发光的部分如荧光素异硫氰酸酯或罗丹明，或酶如碱性磷酸酶、β-半乳糖苷酶、辣根过氧化物酶或荧光素酶。可用于检测或测量样品中冠状病毒的具体的示例性测定包括酶连接的免疫吸附测定(ELISA)、放射免疫测定(RIA)和荧光活化细胞分选(FACS)。 Exemplary diagnostic assays for coronavirus include, for example, contacting a sample obtained from a patient with the anti-coronavirus S protein of the present invention, where the anti-coronavirus S protein is labeled with a detectable marker or reporter molecule or used as a capture ligand to select Sexually isolate the coronavirus from patient samples. Alternatively, the unlabeled anti-coronavirus S protein can be used in diagnostic applications in combination with a second antibody that itself is detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125 I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine, or enzymes such as alkali Phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure coronavirus in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
可在根据本发明的冠状病毒诊断测定中使用的样品包括从患者可获得的任何生物样品，其包含在正常或生理条件下可检测量的冠状病毒刺突蛋白或其片段。在一些实施方案 中，生物样品是血液、血清、咽拭子、下呼吸道样本(如气管分泌物、气管吸取物、肺泡灌洗液)或生物来源的其他样品。一般而言，将测量从健康患者获得的特定样品中的冠状病毒刺突蛋白水平(例如未受与冠状病毒相关的疾病所扰的患者)以初始性地建立基线或标准冠状病毒水平。该冠状病毒基线水平可随后与从疑似具有冠状病毒相关病况或与该病况相关的症状的个体获得的样品中测量的冠状病毒水平进行比较。The samples that can be used in the coronavirus diagnostic assay according to the present invention include any biological samples available from patients that contain a detectable amount of coronavirus spike protein or fragments thereof under normal or physiological conditions. In some embodiments, the biological sample is blood, serum, throat swabs, lower respiratory tract samples (e.g. tracheal secretions, tracheal aspirates, alveolar lavage fluid) or other samples of biological origin. In general, the level of coronavirus spike protein in a specific sample obtained from healthy patients (for example, patients not affected by coronavirus-related diseases) will be measured to initially establish a baseline or standard coronavirus level. This baseline level of coronavirus can then be compared with the level of coronavirus measured in a sample obtained from an individual suspected of having a coronavirus-related condition or symptoms related to the condition.
特异性针对冠状病毒刺突蛋白的抗体可不包含其他标记物，或其可包含N-末端或C末端的标记物。在一个实施方案中，标记物是生物素。在结合测定中，标记物(如果存在的话)的位置可确定肽相对于所结合的表面的方向。例如，如果表面以抗生物素蛋白包被，包含N末端生物素的肽将被定向，使得肽的C末端部分远离表面。The antibody specific for the coronavirus spike protein may not contain other labels, or it may contain N-terminal or C-terminal labels. In one embodiment, the label is biotin. In a binding assay, the position of the label (if present) determines the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, the peptide containing the N-terminal biotin will be oriented so that the C-terminal part of the peptide is away from the surface.
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。The following examples are described to assist the understanding of the present invention. It is not intended and should not be construed in any way to limit the scope of protection of the present invention.
实施例Example
通过参考以下实施例将更容易地理解本文一般地描述的本发明，这些实施例是以举例说明的方式提供的，并且不旨在限制本发明的范围。这些实施例并不旨在表示下面的实验是全部或仅进行的实验。The present invention generally described herein will be more easily understood by referring to the following examples, which are provided by way of illustration and are not intended to limit the scope of the present invention. These examples are not intended to indicate that the following experiments are all or only experiments performed.
实施例1 冠状病毒S蛋白抗原的制备和检测用ACE2的制备Example 1 Preparation of Coronavirus S Protein Antigen and Preparation of ACE2 for Detection
实施例中共用到如下抗原和ACE2蛋白：S蛋白RBD-His(319Arg-532Asn),S蛋白S1-huFc(14Gln-685Arg)，人ACE2-huFc(18Gln-740Ser)，人ACE2-His(18Gln-740Ser)，S蛋白RBD-mFc(购自Sinobio，40592-V05H)，其中前四种蛋白的具体制备方法如下。In the examples, the following antigens and ACE2 proteins are shared: S protein RBD-His (319Arg-532Asn), S protein S1-huFc (14Gln-685Arg), human ACE2-huFc (18Gln-740Ser), human ACE2-His (18Gln- 740Ser), S protein RBD-mFc (purchased from Sinobio, 40592-V05H), the specific preparation methods of the first four proteins are as follows.
1.1质粒构建1.1 Plasmid construction
从NCBI获取各蛋白序列，其中人ACE2序列获自NCBI Gene ID:59272，S蛋白序列获自NCBI Gene ID:43740568，分别按照上述氨基酸片段位置获得各蛋白序列，转化成基因序列后由金斯瑞生物科技股份有限公司进行目的片段基因合成。PCR扩增各目的片段，然后通过同源重组的方法构建至真核表达载体pcDNA3.3-TOPO(Invitrogen)，用于后续重组蛋白的表达。Each protein sequence was obtained from NCBI, among which the human ACE2 sequence was obtained from NCBI Gene ID: 59272, and the S protein sequence was obtained from NCBI Gene ID: 43740568. Each protein sequence was obtained according to the position of the above amino acid fragments, and then transformed into gene sequences by GenScript Biotechnology Co., Ltd. conducts gene synthesis of the target fragment. Each target fragment was amplified by PCR, and then constructed into the eukaryotic expression vector pcDNA3.3-TOPO (Invitrogen) by the method of homologous recombination for subsequent expression of the recombinant protein.
1.2质粒制备1.2 Plasmid preparation
将构建好的各重组蛋白表达载体分别转化到大肠杆菌SS320中，37℃过夜培养，利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的各质粒以供真核表达使用。The constructed recombinant protein expression vectors were transformed into Escherichia coli SS320 and cultured overnight at 37°C. Plasmid extraction was performed using endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for supply Eukaryotic expression use.
1.3各蛋白的表达纯化1.3 Expression and purification of each protein
S蛋白RBD-His(319Arg-532Asn)，S蛋白S1-huFc(14Gln-685Arg)，人ACE2-huFc(18Gln-740Ser)和人ACE2-His(18Gln-740Ser)均通过Expi293瞬转表达系统(ThermoFisher，A14635)表达，具体方法如下：S protein RBD-His (319Arg-532Asn), S protein S1-huFc (14Gln-685Arg), human ACE2-huFc (18Gln-740Ser) and human ACE2-His (18Gln-740Ser) were all passed through the Expi293 transient expression system (ThermoFisher , A14635) expression, the specific method is as follows:
转染当天，确认细胞密度为每毫升4.5×10 6至5.5×10 6个活细胞左右，细胞存活率>95％，此时用37℃预温的新鲜Expi293表达培养基将细胞调整到终浓度为每毫升3×10 6个细胞。用4℃预冷的Opti-MEM TM稀释目的质粒(1mL Opti-MEM TM中加入1μg质粒)，同时用Opti-MEM TM稀释ExpiFectamine TM293试剂，再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamine TM293试剂/质粒DNA混合液，室温孵育10-20min，缓慢加入到准备好的细胞悬液中，并同时轻轻摇晃，最后置于细胞培养摇床中，在37℃，8％CO 2条件下培养。 On the day of transfection, confirm that the cell density is about 4.5×10 6 to 5.5×10 6 viable cells per milliliter, and the cell survival rate is >95%. At this time, use fresh Expi293 expression medium pre-warmed at 37°C to adjust the cells to the final concentration This is 3×10 6 cells per milliliter. Dilute the target plasmid with Opti-MEM TM pre-cooled at 4°C (1μg plasmid is added to 1mL Opti-MEM TM ), and at the same time dilute ExpiFectamine TM 293 reagent with Opti-MEM TM , then mix the two in equal volumes and gently pipette to mix. Prepare a mixture of ExpiFectamine TM 293 reagent/plasmid DNA, incubate at room temperature for 10-20 minutes, slowly add it to the prepared cell suspension while shaking gently, and finally place it in a cell culture shaker at 37°C, 8% Cultivation under CO 2 conditions.
在转染后18-22h内添加ExpiFectamine TM293 Transfection Enhancer 1和ExpiFectamine TM293 Transfection Enhancer 2，摇瓶放置于32℃摇床和5％CO 2条件下继续培养，在转染5-7天后，将细胞表达上清于15000g高速离心10min，所得Fc标签蛋白表达上清用MabSelect SuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和；所得His标签蛋白表达上清用Ni Smart Beads 6FF(常州天地人和生物科技有限公司，SA036050)进行亲和纯化，然后用梯度浓度的咪唑洗脱目的蛋白。洗脱下来的各蛋白分别通过超滤浓缩管(Millipore，UFC901096)置换至PBS缓冲液中。经SDS-PAGE鉴定和活性鉴定合格后于－80℃冻存待用。 Add ExpiFectamine TM 293 Transfection Enhancer 1 and ExpiFectamine TM 293 Transfection Enhancer 2 within 18-22 hours after transfection, and place the shaker in a shaker at 32°C and 5% CO 2 to continue culturing. After 5-7 days of transfection, The cell expression supernatant was centrifuged at high speed at 15000g for 10 min. The resulting Fc-tagged protein expression supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100mM sodium acetate (pH3.0), followed by 1M Tris -HCl neutralization; the obtained His tag protein expression supernatant was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with a gradient concentration of imidazole. The eluted proteins were replaced into PBS buffer through an ultrafiltration tube (Millipore, UFC901096). After SDS-PAGE identification and activity identification qualified, frozen at -80°C for later use.
1.4各蛋白的质量检测1.4 Quality detection of each protein
将上述1.3制备的S蛋白S1-huFc(也称为Spike-S1-huFc；或S1-huFc)和S蛋白RBD-mFc(也称为Spike-RBD-mFc；或RBD-mFc)与人ACE2-huFc、人ACE2-His通过ELISA测定法进行相互结合测定。结果显示在图2A和图2B中，人ACE2-huFc和RBD-mFc有良好的结合活性；S蛋白S1-huFc和商品化的S蛋白RBD-mFc分别与人ACE2-his有良好的结合活性，且活性相当。The S protein S1-huFc (also known as Spike-S1-huFc; or S1-huFc) and S protein RBD-mFc (also known as Spike-RBD-mFc; or RBD-mFc) prepared in 1.3 above were combined with human ACE2- huFc and human ACE2-His were tested for mutual binding by ELISA assay. The results show that in Figure 2A and Figure 2B, human ACE2-huFc and RBD-mFc have good binding activity; S protein S1-huFc and commercial S protein RBD-mFc have good binding activity to human ACE2-his, respectively. And the activity is comparable.
实施例2 天然的人抗体噬菌体展示文库的构建和筛选Example 2 Construction and screening of natural human antibody phage display library
在本实施例中，构建了抗体基因噬菌体展示文库，并用重组的2019-nCoV冠状病毒RBD蛋白(即，Spike-RBD-mFc，Sinobio，40592-V05H)为筛选抗原对该文库进行筛选，获得了多个具有特异性结合2019-nCoV冠状病毒RBD蛋白的抗体分子。In this example, a phage display library of antibody genes was constructed, and the recombinant 2019-nCoV coronavirus RBD protein (ie, Spike-RBD-mFc, Sinobio, 40592-V05H) was used as the screening antigen to screen the library, and obtained Multiple antibody molecules that specifically bind to the 2019-nCoV coronavirus RBD protein.
2.1构建人抗体的基因文库2.1 Construction of a gene library of human antibodies
取Ficoll-Paque密度梯度分离液(购自GE公司，目录号：17144003S)15mL缓缓加入50mL离心管中。将离心管倾斜并分批次沿管壁缓慢加入15mL采集的正常人血液，使得Ficoll-Paque密度梯度分离液与正常人血液保持清晰的分离界面。将装有所述血液和分 离液的50mL离心管于15℃左右离心20min，其中将离心机设置为400g，加速度为3，减速为0的参数。离心之后，整个液面分为四层，上层为血浆混合物，下层为红细胞和粒细胞，中层为Ficoll-Paque液体，在上、中层交界处有以PBMC为主的白色云雾层狭窄带，即PBMC细胞层。用无菌巴氏吸管小心地吸去上层的血浆混合物，然后再用新的无菌巴氏吸管吸取PBMC，获得分离的PBMC。将分离的PBMC先用PBS润洗两遍，再在4℃下以1500rpm的转速离心10min，最后用1.5mL的PBS重悬，并通过细胞计数仪(CountStar，CountStar Altair)计数。Take 15 mL of Ficoll-Paque density gradient separation solution (purchased from GE Company, catalog number: 17144003S) and slowly add it to a 50 mL centrifuge tube. Tilt the centrifuge tube and slowly add 15 mL of collected normal human blood along the tube wall in batches, so that the Ficoll-Paque density gradient separation solution and normal human blood maintain a clear separation interface. Centrifuge the 50 mL centrifuge tube containing the blood and the separating solution at about 15°C for 20 minutes, where the centrifuge is set to 400 g, the acceleration is 3, and the deceleration is 0 parameters. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid. At the junction of the upper and middle layers, there is a narrow zone of white cloud layer dominated by PBMC, namely PBMC Cell layer. Use a sterile Pasteur pipette to carefully aspirate the upper plasma mixture, and then use a new sterile Pasteur pipette to aspirate PBMC to obtain a separated PBMC. The separated PBMC was first rinsed with PBS twice, then centrifuged at 1500 rpm at 4°C for 10 min, and finally resuspended with 1.5 mL of PBS, and counted by a cell counter (CountStar, CountStar Altair).
通过常规方法自分离的PBMC细胞提取总RNA。使用反转录试剂盒(购自TaKaRa公司,目录号：6210A)将提取的总RNA反转录成cDNA。基于重链和轻链种系基因的序列相似度，分别在重链和轻链的V区前端和第一个恒定区后端设计简并引物(李晓琳，大容量非免疫人源性Fab噬菌体抗体库的构建及初步筛选，《中国协和医科大学》硕士学位论文，2007年6月)，PCR后得到抗体的重链可变区基因片段和轻链可变区基因片段。回收抗体的重链可变区基因片段和轻链可变区基因片段后，通过融合PCR方法扩增得到含有抗体的轻重链可变区的片段，接着，对该PCR产物和噬菌体展示用载体进行酶切、回收和连接，连接产物通过回收试剂盒(Omega，目录号：D6492-02)回收，具体材料和方法参见上文李晓琳的论文。最后，通过电转仪(Bio-Rad，MicroPulser)转化至感受态大肠杆菌SS320(Lucigen，MC1061F)中，并将经转化的大肠杆菌SS320菌液涂布于具有氨苄青霉素抗性的2-YT固体平板(固体平板由1.5％的胰蛋白胨，1％的酵母提取物，0.5％的NaCl，1.5％的琼脂，按质量体积g/mL配制而成)。The total RNA was extracted from the isolated PBMC cells by conventional methods. Reverse transcription kit (purchased from TaKaRa company, catalog number: 6210A) was used to reverse transcribe the extracted total RNA into cDNA. Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the V region of the heavy chain and the light chain and the rear end of the first constant region (Li Xiaolin, large-capacity non-immune human Fab phage antibody The construction and preliminary screening of the library, the master's thesis of "Peking Union Medical College of China", June 2007), after PCR, the antibody heavy chain variable region gene fragment and light chain variable region gene fragment were obtained. After the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody are recovered, the fragment containing the light and heavy chain variable region of the antibody is amplified by the fusion PCR method, and then the PCR product and the vector for phage display are performed Enzyme digestion, recovery and ligation, and the ligation product is recovered by a recovery kit (Omega, catalog number: D6492-02). For specific materials and methods, please refer to the paper by Li Xiaolin above. Finally, transform into competent Escherichia coli SS320 (Lucigen, MC1061F) by electroporator (Bio-Rad, MicroPulser), and spread the transformed Escherichia coli SS320 bacterial solution on a 2-YT solid plate with ampicillin resistance (The solid plate is made up of 1.5% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar, according to mass volume g/mL).
2.2抗体基因库容的计算2.2 Calculation of antibody gene storage capacity
取经转化的大肠杆菌SS320菌液用无抗生素的2YT培养液以1:50的体积进行接菌，37℃，220rpm培养1.5-2h至OD600达到0.5-0.6后取出至室温。将菌液按照90μL/孔添加到96孔圆底稀释板中，每个菌液样品进行10倍梯度稀释，共12个稀释梯度。将稀释好的样品使用8道10μL量程移液器，吸取2μL的液体按照稀释梯度从低到高的顺序加到羧苄青霉素和四环素浓度分别为50μg/mL和50μg/mL的2YT(下文中也简称为C +/T +2YT)平板上，正置5min后倒置放在37℃培养过夜。第二天观察克隆生长的情况，并计算库容。库容的计算方法如下，从A行开始，依次标记为1,2,3,4,5,6,7,8到X行。首先选择计数孔，先将克隆数目在3-20个克隆的计数孔选出，得出行数X,并数出对应孔中的克隆数n，计算公式为5×100×10 X×n，经计算，获得了每毫升菌液库容大小为3×10 11cfu，即3×10 11个抗体基因的抗体基因库。 The transformed Escherichia coli SS320 broth was inoculated with antibiotic-free 2YT broth at a volume of 1:50, incubated at 37°C and 220rpm for 1.5-2h until OD600 reached 0.5-0.6, and then taken out to room temperature. Add 90 μL/well of the bacterial solution to a 96-well round-bottomed dilution plate, and each bacterial solution sample was diluted by a 10-fold gradient for a total of 12 dilution gradients. Use 8 channels of 10μL volume pipettes for the diluted sample, and add 2μL of liquid to 2YT with carbenicillin and tetracycline concentrations of 50μg/mL and 50μg/mL according to the dilution gradient from low to high (also below) (Referred to as C + /T + 2YT) on the plate, put it upright for 5 minutes and then invert it at 37°C for overnight incubation. On the second day, observe the growth of the clones and calculate the storage capacity. The calculation method of storage capacity is as follows, starting from line A, and marking them as lines 1, 2, 3, 4, 5, 6, 7, 8 to X. First select the counting hole, first select the counting hole with the number of clones in 3-20 clones, get the number of rows X, and count the number of clones in the corresponding hole n, the calculation formula is 5×100×10 X ×n, By calculation, an antibody gene library with a storage capacity of 3×10 11 cfu per milliliter of bacterial liquid, which is 3×10 11 antibody genes, was obtained.
2.3抗体基因噬菌体展示文库的制备2.3 Preparation of antibody gene phage display library
基于抗体基因库容容量，吸取50个OD(1个OD为5×10 8cfu)的全人抗体基因库的菌液加入到新鲜的2-YT液体培养基中，使得初始OD值为0.1。将所得物置于37℃，220rpm的摇床中培养至对数生长期(OD600＝0.6左右)，再以50倍于细菌数的数量(即，感染复数(MOI)为大约50)加入VSCM13辅助噬菌体(购自Stratagene)，充分混匀，静置30min后在220rpm的摇床中继续培养1小时。随后，将培养物以10000rpm的转速离心5min后，弃上清，将培养液替换为羧苄青霉素50μg/mL/卡那霉素40μg/mL双抗性的2-YT培养基(下文也称为C +/K +2-YT培养基)，并于30℃，220rpm继续培养过夜。次日，菌液于13000g离心10min，收集上清后加入20％PEG/NaCl(由体积浓度为20％的PEG6000和2.5M NaCl配制而成)使得PEG/NaCl最终浓度为4％，混匀，并置于冰上1小时，再以13000g的转速离心10min，将沉淀的噬菌体用PBS润洗后保存并用于后续噬菌体筛选。 Based on the capacity of the antibody gene library, aspirate 50 OD (1 OD is 5×10 8 cfu) of the bacterial liquid of the fully human antibody gene library and add it to the fresh 2-YT liquid medium, so that the initial OD value is 0.1. The resultant was cultured in a shaker at 37°C and 220 rpm to the logarithmic growth phase (OD600 = about 0.6), and then the VSCM13 helper phage was added in an amount 50 times the number of bacteria (ie, the multiplicity of infection (MOI) was about 50) (Purchased from Stratagene), mix well, let stand for 30 minutes and continue to incubate in a shaker at 220 rpm for 1 hour. Subsequently, the culture was centrifuged at 10000 rpm for 5 minutes, the supernatant was discarded, and the culture medium was replaced with carbenicillin 50μg/mL/kanamycin 40μg/mL double-resistant 2-YT medium (hereinafter also referred to as C + /K + 2-YT medium), and continue to culture overnight at 30°C and 220 rpm. The next day, the bacterial solution was centrifuged at 13000g for 10 minutes, and the supernatant was collected and 20% PEG/NaCl (prepared from 20% PEG6000 and 2.5M NaCl) was added to make the final concentration of PEG/NaCl 4%. Mix well. And placed on ice for 1 hour, and then centrifuged at 13000g for 10 min. The precipitated phage was rinsed with PBS and stored and used for subsequent phage screening.
2.4抗体基因噬菌体展示文库的筛选2.4 Screening of antibody gene phage display library
2.4.1磁珠法筛选抗体基因噬菌体展示文库2.4.1 Screening of antibody gene phage display library by magnetic bead method
磁珠法筛选是基于将抗原蛋白(Spike-RBD-mFc，Sinobio，40592-V05H)进行生物素标记后，再与偶联有链霉亲和素的磁珠结合，通过将结合抗原的磁珠和抗体基因噬菌体展示文库进行孵育、洗涤和洗脱的淘选过程，通常经历3-4轮的淘选，由此针对抗原的特异性单克隆抗体可以大量富集。本实施例中，将生物素标记的2019-nCoV冠状病毒RBD蛋白用于噬菌体展示文库筛选，经过3轮淘选后进行针对RBD蛋白的单克隆抗体初筛。The magnetic bead method is based on the antigen protein (Spike-RBD-mFc, Sinobio, 40592-V05H) is biotin-labeled, and then combined with streptavidin-coupled magnetic beads. By combining the antigen-bound magnetic beads The panning process of incubation, washing and elution with the antibody gene phage display library usually undergoes 3-4 rounds of panning, so that the specific monoclonal antibodies against the antigen can be enriched in a large amount. In this example, the biotin-labeled 2019-nCoV coronavirus RBD protein was used for phage display library screening, and after 3 rounds of panning, a preliminary screening of monoclonal antibodies against the RBD protein was performed.
抗体筛选的具体实施方法如下：The specific implementation method of antibody screening is as follows:
首先用生物素标记的2019-nCoV冠状病毒RBD蛋白(Spike-RBD-mFc，Sinobio，40592-V05H)与链霉亲和素偶联的磁珠孵育，使得生物素标记的RBD蛋白结合到磁珠上。将结合RBD蛋白的磁珠和构建的噬菌体库室温下孵育2h。经PBST洗涤6-8次后，去除非特异性吸附的噬菌体，加入Trypsin(Gibco，25200072)轻轻混匀并反应20min，以洗脱特异性结合的抗体展示噬菌体。随后，用洗脱下来的噬菌体侵染对数期的SS320菌体(Lucigen,MC1061F)并静置30min，然后在220rpm条件下培养1h，再加入VSCM13辅助噬菌体并静置30min，继续在220rpm条件下培养1h，离心并置换至C +/K +2-YT培养基中，最终得到的噬菌体继续用于下一轮的淘选。 First, the biotin-labeled 2019-nCoV coronavirus RBD protein (Spike-RBD-mFc, Sinobio, 40592-V05H) is incubated with streptavidin-coupled magnetic beads, so that the biotin-labeled RBD protein binds to the magnetic beads superior. The magnetic beads that bind RBD protein and the constructed phage library were incubated for 2h at room temperature. After washing 6-8 times with PBST, the non-specifically adsorbed phages were removed, Trypsin (Gibco, 25200072) was added and mixed gently and reacted for 20 minutes to elute the specifically bound antibody display phages. Subsequently, the eluted phage was used to infect the log phase SS320 bacteriophage (Lucigen, MC1061F) and let it stand for 30 min, then cultured at 220 rpm for 1 h, then added the VSCM13 helper phage and let it stand for 30 min, and continued at 220 rpm. Cultivate for 1 hour, centrifuge and replace into C + /K + 2-YT medium, and the finally obtained phage will continue to be used in the next round of panning.
2.4.2免疫管法筛选抗体基因噬菌体展示文库2.4.2 Screening antibody gene phage display library by immunotube method
免疫管法和磁珠法的目的均为富集针对抗原的特异性抗体，为两个相互补充和验证的实验方法。The purpose of the immunotube method and the magnetic bead method are both to enrich specific antibodies against the antigen, and are two mutually complementary and validated experimental methods.
免疫管法筛选的原理是将2019-nCoV冠状病毒RBD蛋白(Spike-RBD-mFc，Sinobio，40592-V05H)包被在具有高吸附力的免疫管表面，通过将噬菌体展示抗体文库加入免疫管中并和吸附于免疫管表面的抗原蛋白进行孵育、洗涤和洗脱的淘选过程，经历2-4轮淘选，最终将针对抗原的特异性单克隆抗体富集下来。The principle of immune tube screening is to coat the 2019-nCoV coronavirus RBD protein (Spike-RBD-mFc, Sinobio, 40592-V05H) on the surface of an immune tube with high adsorption capacity, and add a phage display antibody library to the immune tube. The panning process of incubation, washing and elution with the antigen protein adsorbed on the surface of the immune tube is carried out. After 2-4 rounds of panning, the specific monoclonal antibodies against the antigen are finally enriched.
具体实施方法如下：The specific implementation method is as follows:
第一轮筛选时，在免疫管中加入1mL 100μg/mL的RBD-mFc，4℃包被过夜，第二天弃去包被液，加入5％牛奶的PBS封闭2h，PBS润洗两次后加入构建的总量为10 14个全人抗体基因的噬菌体库，孵育2h，用PBS润洗8遍，然后用PBST润洗2遍，以去除非特异性结合的噬菌体，然后向免疫管中加入0.8mL 0.05％EDTA胰酶消化液，用于洗脱特异性结合目标抗原的噬菌体，接着将其侵染对数期的SS320菌体(Lucigen，60512-1)，37℃静置30min，然后220rpm条件下培养1h，再加入VSCM13辅助噬菌体，静置30min，继续在220rpm条件下培养1h，离心并置换至C +/K +2-YT培养基中，并于30℃，220rpm环境下继续培养过夜。第二天沉淀噬菌体，用于后续2-4轮的筛选。通常用于第二轮、第三轮和第四轮噬菌体筛选的抗原包被浓度依次递减，分别为30μg/mL，10μg/mL和3μg/mL；除此之外，PBS润洗强度也逐渐加大，PBS洗脱次数依次为12次，16次和20次。 In the first round of screening, add 1mL of 100μg/mL RBD-mFc to the immune tube, coat it overnight at 4°C, discard the coating solution the next day, add 5% milk in PBS for blocking for 2h, and rinse twice with PBS Add the constructed phage library with a total of 10 14 fully human antibody genes, incubate for 2 hours, rinse with PBS 8 times, and then rinse with PBST 2 times to remove non-specifically bound phage, and then add 0.8 to the immune tube mL of 0.05% EDTA trypsin digestion solution, used to elute the phage that specifically binds to the target antigen, and then infect the logarithmic phase of SS320 bacteria (Lucigen, 60512-1), stand at 37°C for 30 minutes, and then at 220rpm Incubate for 1 h, then add VSCM13 helper phage, let stand for 30 min, continue to culture at 220 rpm for 1 h, centrifuge and replace in C + /K + 2-YT medium, and continue to culture overnight at 30°C and 220 rpm. The phage was precipitated the next day for subsequent 2-4 rounds of screening. The antigen coating concentrations usually used in the second, third and fourth rounds of phage screening gradually decrease, respectively, 30μg/mL, 10μg/mL and 3μg/mL; in addition, the PBS wash strength is gradually increased Large, the PBS elution times are 12 times, 16 times and 20 times in sequence.
对每轮洗脱下来的噬菌体池进行ELISA检测来评价富集的效果，并从每轮筛选的噬菌体池中随机挑选10个克隆进行序列分析，结果显示在图3A和图3B中。The phage pool eluted in each round was tested by ELISA to evaluate the effect of enrichment, and 10 clones were randomly selected from the phage pool in each round for sequence analysis. The results are shown in Figure 3A and Figure 3B.
结果表明，第三轮筛选后抗体序列富集明显，因此，选择第三轮所得的克隆进行ELISA的阳性克隆筛选。The results showed that the antibody sequence was significantly enriched after the third round of screening. Therefore, the clones obtained in the third round were selected for positive clone screening by ELISA.
2.5单克隆的挑选2.5 Monoclonal selection
在共四轮筛选后，选择第三轮所得的克隆进行ELISA的阳性克隆ELISA筛选。最终，在2304个克隆中共筛选到88个能够与RBD蛋白结合的阳性克隆，经测序分析、ELISA结合和Fab水平的FACS阻断检测后，最终选取了2个克隆的序列构建全长抗体以进行进一步的实验。具体实施方法如下。After a total of four rounds of screening, the clones obtained in the third round were selected for ELISA screening of positive clones. Finally, a total of 88 positive clones that can bind to the RBD protein were screened out of 2304 clones. After sequencing analysis, ELISA binding and FACS blocking detection at the Fab level, the sequences of 2 clones were finally selected to construct full-length antibodies. Further experiments. The specific implementation method is as follows.
2.5.1阳性克隆测序与分析2.5.1 Sequencing and analysis of positive clones
在完成初筛工作后，对88个能够与RBD蛋白结合的阳性克隆进行编号，吸取2μL菌液到2mL的2YT培养基中，37℃，220rpm培养过夜，提取质粒进行二代测序。测序结果通过SeqMan将原始的AB1文件整合、比对、去掉非抗体基因序列，生成抗体基因整合版的fasta文件。随后将DNA序列通过MEGA6翻译为氨基酸序列，并通过氨基酸序列找出含有终止子、非常规序列等，导出氨基酸序列的fasta文件。After completing the preliminary screening, number 88 positive clones that can bind to the RBD protein, pipet 2 μL of bacterial solution into 2 mL of 2YT medium, culture overnight at 37°C, 220 rpm, and extract plasmids for second-generation sequencing. Sequencing results use SeqMan to integrate the original AB1 files, compare them, and remove non-antibody gene sequences to generate an integrated version of fasta files for antibody genes. Subsequently, the DNA sequence was translated into amino acid sequence by MEGA6, and the amino acid sequence was used to find out the terminator, unconventional sequence, etc., to derive the fasta file of the amino acid sequence.
2.5.2 ELISA测定法检测Fab形式抗体的亲和效果2.5.2 ELISA assay method to detect the affinity effect of Fab antibody
首先挑取第三轮筛选的克隆于含有300μL 2-YT培养基的96孔深孔板中，37℃培养过夜，上清即含有表达的Fab，取上清，梯度稀释后加入到包被2μg/mL RBD-mFc的ELISA板中，然后用HRP标记的山羊抗人Fab作为第二抗体(Goat anti-Human Fab-HRP,ThermoFisher,31482,1:6000稀释)进行检测，信号值越高，表明亲和力越强，结果显示在图4中，结果表明，在ELISA测定法中，2个抗体(分别命名为P16-A3抗体、P17-A11抗体)的Fab均表现出了较好的亲和活性。First, pick the clones from the third round of screening in a 96-well deep-well plate containing 300 μL 2-YT medium, and incubate overnight at 37°C. The supernatant contains the expressed Fab. Take the supernatant and add it to the coating 2 μg after serial dilution. /mL RBD-mFc ELISA plate, and then use HRP-labeled goat anti-human Fab as the secondary antibody (Goat anti-Human Fab-HRP, ThermoFisher, 31482, 1:6000 dilution) for detection, the higher the signal value, it indicates The stronger the affinity, the results are shown in Figure 4, and the results show that in the ELISA assay, the Fabs of the two antibodies (named P16-A3 antibody and P17-A11 antibody, respectively) showed better affinity activity.
2.5.3裂解液的制备2.5.3 Preparation of Lysis Solution
将所有的阳性克隆进行测序分析，将序列唯一的克隆接种至50mL 2-YT培养基中，37℃过夜培养，离心得到的菌体沉淀中加入500μL含有浓度为25U/mL Benzonase核酸酶(Merck，70746-3)的裂解液，置于冰上1h，13000rpm离心5min后获得裂解液并进行定量，裂解液将用于细胞水平的阻断效果检测。All positive clones were sequenced and analyzed. The clones with unique sequences were inoculated into 50mL 2-YT medium, cultured overnight at 37°C, and 500 μL of the bacterial pellet obtained by centrifugation was added with a concentration of 25U/mL Benzonase Nuclease (Merck, The lysate of 70746-3), placed on ice for 1 hour, centrifuged at 13000 rpm for 5 minutes to obtain and quantify the lysate, the lysate will be used for cell-level blocking effect detection.
2.5.4细胞水平检测Fab形式抗体的阻断效果2.5.4 Detection of the blocking effect of Fab form antibodies at the cell level
流式细胞水平上用于筛选具有S蛋白阻断活性的候选抗体，方法如下：将梯度稀释(首孔10μg/mL，3倍梯度稀释)的候选抗体Fab裂解液和0.5μg/mL RBD-mFc等体积混合，在4℃孵育1h，然后将100μl混合物转移至含有2.0×10 5个Vero E6细胞/100μl(Vero E6细胞获自ATCC,CRL-1586)的96孔板中，接着孵育1h，FACS缓冲液润洗后，加入100μl PE标记的抗小鼠IgG-Fc流式抗体(Jackson,115-115-164，1:200稀释)作为第二抗体，用于检测Vero E6细胞上结合的RBD-mFc。结果显示在图5中，结果表明，2个抗体(P16-A3抗体、P17-A11抗体)的Fab均表现出较好的细胞水平的阻断活性。 At the level of flow cytometry, it is used to screen candidate antibodies with S protein blocking activity. The method is as follows: the candidate antibody Fab lysate with gradient dilution (first well 10μg/mL, 3-fold dilution) and 0.5μg/mL RBD-mFc Mix in equal volume, incubate at 4°C for 1h, then transfer 100μl of the mixture to a 96-well plate containing 2.0×10 5 Vero E6 cells/100μl (Vero E6 cells obtained from ATCC, CRL-1586), and then incubate for 1h, FACS After rinsing with buffer, 100μl of PE-labeled anti-mouse IgG-Fc flow cytometry antibody (Jackson, 115-115-164, 1:200 dilution) was added as the secondary antibody to detect RBD-binding on Vero E6 cells. mFc. The results are shown in Fig. 5, and the results show that the Fabs of the two antibodies (P16-A3 antibody and P17-A11 antibody) all showed better blocking activity at the cellular level.
本实施例中，发现序列独特的2个Fab形式的抗体(P16-A3抗体、P17-A11抗体)表现出较好的细胞水平的阻断结合活性，因此将所述2个Fab形式的抗体分别构建至人IgG1 Fc中获得全长抗体并进行进一步的验证。In this example, it was found that two Fab forms of antibodies (P16-A3 antibody and P17-A11 antibody) with unique sequences showed better blocking and binding activity at the cellular level. Therefore, the two Fab forms of antibodies were separated Constructed into human IgG1 Fc to obtain a full-length antibody and further verification.
实施例3 全长抗体的构建、表达和纯化Example 3 Construction, expression and purification of full-length antibodies
在本实施例中，将实施例2中获得的2个对结合ACE2阻断活性较好的Fab抗体构建为人IgG1型，其中轻链均为κ型，抗体类型为全人抗体。In this example, the two Fab antibodies with better blocking activity against ACE2 binding obtained in Example 2 were constructed as human IgG1 type, in which the light chains are all of the kappa type, and the antibody type is a fully human antibody.
3.1质粒构建3.1 Plasmid construction
从筛选获得的含有Fab抗体菌株中，PCR扩增获取抗体轻、重链可变区片段，通过同源重组方法，分别构建至经过改造的含有轻、重链恒定区片段的真核表达载体质粒pcDNA3.3-TOPO(Invitrogen)上，组成完整的抗体轻、重链全长基因，编码SEQ ID NO：17、18、19、20所示的抗体重链和轻链氨基酸序列)。From the selected strains containing Fab antibodies, PCR amplification was used to obtain antibody light and heavy chain variable region fragments, and through homologous recombination, they were constructed into modified eukaryotic expression vector plasmids containing light and heavy chain constant region fragments. On pcDNA3.3-TOPO (Invitrogen), a complete antibody light and heavy chain full-length gene is composed, which encodes the antibody heavy chain and light chain amino acid sequence shown in SEQ ID NO: 17, 18, 19, 20).
3.2质粒准备3.2 Plasmid preparation
将构建好的含抗体轻重链全长基因的载体分别转化到大肠杆菌SS320中，37℃过夜培养，利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的抗体轻重链质粒以供真核表达使用。The constructed vectors containing the full-length antibody light and heavy chain genes were transformed into E. coli SS320, incubated overnight at 37°C, and plasmids were extracted using endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids. The antibody light and heavy chain plasmids are used for eukaryotic expression.
3.3抗体的表达纯化3.3 Expression and purification of antibodies
候选抗体P16-A3、P17-A11以及对照抗体CR3022(参见US10066238)是通过ExpiCHO瞬转表达系统(Thermo Fisher，A29133)表达的，具体方法如下：Candidate antibodies P16-A3, P17-A11 and control antibody CR3022 (see US10066238) are expressed by ExpiCHO transient expression system (Thermo Fisher, A29133), the specific method is as follows:
转染当天，确认细胞密度为7×10 6至1×10 7个活细胞/mL左右，细胞存活率>98％，此时，用37℃预温的新鲜ExpiCHO表达培养基将细胞调整到终浓度为6×10 6个细胞/mL。用4℃预冷的OptiPRO TM SFM稀释目的质粒(向1mL所述培养基中加入1μg质粒)，同时，用OptiPRO TMSFM稀释ExpiFectamine TMCHO，再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamine TMCHO/质粒DNA混合液，室温孵育1-5min，缓慢加入到准备好的细胞悬液中，并同时轻轻摇晃，最后置于细胞培养摇床中，在37℃，8％CO 2条件下培养。 On the day of transfection, confirm that the cell density is about 7×10 6 to 1×10 7 viable cells/mL, and the cell survival rate is >98%. At this time, use fresh ExpiCHO expression medium pre-warmed at 37°C to adjust the cells to the end The concentration is 6×10 6 cells/mL. Dilute the target plasmid with OptiPRO TM SFM pre-chilled at 4°C (add 1 μg plasmid to 1 mL of the medium). At the same time, dilute ExpiFectamine TM CHO with OptiPRO TM SFM, then mix the two in equal volumes and gently pipette to mix. Make ExpiFectamine TM CHO/plasmid DNA mixture, incubate for 1-5 min at room temperature, slowly add to the prepared cell suspension, and shake gently at the same time, and finally place it in a cell culture shaker at 37°C, 8% CO 2 Cultivate under the conditions.
在转染后18-22h，向培养液中添加ExpiCHO TMEnhancer和ExpiCHO TMFeed，摇瓶放置于32℃摇床和5％CO 2条件下继续培养，在转染后的第5天，添加相同体积的ExpiCHO TMFeed，缓慢加入的同时轻轻混匀细胞混悬液，在转染7-15天后，将表达有目的蛋白的细胞培养上清于15000g高速离心10min，所得上清用MabSelect SuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得蛋白置换至PBS缓冲液中。 18-22h after transfection, add ExpiCHO TM Enhancer and ExpiCHO TM Feed to the culture medium. Place the shake flask on a shaker at 32°C and 5% CO 2 to continue culturing. On the 5th day after transfection, add the same Add a volume of ExpiCHO TM Feed slowly while gently mixing the cell suspension. After 7-15 days of transfection, centrifuge the cell culture supernatant expressing the target protein at 15000g for 10 minutes at high speed. Use MabSelect SuRe LX for the supernatant (GE, 17547403) for affinity purification, and then eluted the target protein with 100mM sodium acetate (pH3.0), then neutralized with 1M Tris-HCl, and finally replaced the obtained protein with an ultrafiltration tube (Millipore, UFC901096) PBS buffer.
3.4抗体的浓度测定3.4 Determination of antibody concentration
将经纯化的抗体蛋白用经验证过的超微量分光光度计(杭州奥盛仪器有限公司，Nano-300)进行浓度测定，将经测定的A280数值除以抗体理论消光系数后的数值作为后续研究的抗体浓度值，质检合格后，分装并保存于－80℃。Determine the concentration of the purified antibody protein with a validated ultra-micro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and divide the measured A280 value by the theoretical extinction coefficient of the antibody as the follow-up study After passing the quality inspection, the concentration value of the antibody should be aliquoted and stored at -80°C.
实施例4 候选抗体的分子量和纯度鉴定Example 4 Identification of molecular weight and purity of candidate antibodies
在本实施例中，用SDS-PAGE检测两个候选抗体P16-A3、P17-A11以及对照抗体CR3022的相对分子量和纯度。In this example, SDS-PAGE was used to detect the relative molecular weight and purity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022.
4.1样品溶液制备4.1 Sample solution preparation
非还原溶液制备：候选抗体，对照抗体以及质控品IPI(所述IPI是伊匹木单抗(Ipilimumab)的缩写，用作每次SDS-PAGE、SEC-HPLC等理化性质的质控品)1μg加入5×SDS上样缓冲液，和40mM碘代乙酰胺，75℃干浴加热10min，冷却到室温后，12000rpm离心5min，取上清。Preparation of non-reducing solution: candidate antibody, control antibody and quality control product IPI (the IPI is the abbreviation of Ipilimumab, used as a quality control product for physical and chemical properties such as SDS-PAGE, SEC-HPLC, etc.) Add 1μg of 5×SDS loading buffer and 40mM iodoacetamide, heat in a dry bath at 75°C for 10min, cool to room temperature, centrifuge at 12000rpm for 5min, and take the supernatant.
还原溶液制备：候选抗体，对照抗体以及质控品IPI 2μg加入5×SDS上样缓冲液和5mM DTT，100℃干浴加热10min，冷却到室温后，12000rpm离心5min，取上清。Reducing solution preparation: Add 2μg of candidate antibody, control antibody and quality control IPI to 5×SDS loading buffer and 5mM DTT, heat in a dry bath at 100°C for 10 minutes, cool to room temperature, centrifuge at 12000 rpm for 5 minutes, and take the supernatant.
4.2实验流程4.2 Experimental procedure
Bis-tris 4-15％梯度胶(购于金斯瑞)，恒压110V电泳，当考马斯亮蓝迁移到凝胶底部，停止运行，取出凝胶片置考马斯亮蓝染色液中1-2h，弃去染色液，加入脱色液，根据需要更换2-3次脱色液，脱色至凝胶背景透明后保存在去离子水中。Bis-tris 4-15% gradient gel (purchased from GenScript), constant voltage 110V electrophoresis, when Coomassie Brilliant Blue migrates to the bottom of the gel, stop running, take out the gel piece and place it in Coomassie Brilliant Blue staining solution for 1-2h, Discard the staining solution, add the decolorizing solution, replace the decolorizing solution 2-3 times as needed, and store it in deionized water after decolorizing until the gel background is transparent.
4.3实验结果4.3 Experimental results
结果显示在图6中，结果表明，候选抗体和质控品IPI非还原胶的条带在为150kD左右，还原胶的条带分别是55kD左右和25kD左右，符合预期大小，且纯度均大于98％。The results are shown in Figure 6. The results show that the bands of the candidate antibody and the quality control IPI non-reducing gel are about 150kD, and the bands of the reducing gel are about 55kD and 25kD respectively, which are in line with the expected size, and the purity is greater than 98. %.
实施例5 候选抗体单体纯度鉴定Example 5 Identification of the purity of candidate antibody monomers
在本实施例中，用SEC-HPLC检测两个候选抗体P16-A3、P17-A11以及对照抗体CR3022的单体纯度。In this example, SEC-HPLC was used to detect the monomer purity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022.
5.1材料准备5.1 Material preparation
流动相：150mmol/L磷酸缓冲液，pH 7.4。Mobile phase: 150mmol/L phosphate buffer, pH 7.4.
样品制备：候选抗体、对照抗体以及质控品IPI均用流动相溶液稀释到0.5mg/mL。Sample preparation: Candidate antibody, control antibody and quality control IPI are all diluted to 0.5mg/mL with mobile phase solution.
5.2实验流程5.2 Experimental procedure
Agilent HPLC 1100色谱柱(XBridge BEH SEC 3.5μm,7.8mm I.D.×30cm,Waters)流速设为0.8mL/min，进样体积20μL，VWD检测器波长为280nm和214nm。依次进样空白溶液、IPI质控品溶液和样品溶液。Agilent HPLC 1100 chromatographic column (XBridge BEH SEC 3.5μm, 7.8mm I.D.×30cm, Waters), the flow rate is set to 0.8mL/min, the injection volume is 20μL, and the wavelength of the VWD detector is 280nm and 214nm. Inject blank solution, IPI quality control solution and sample solution in sequence.
5.3实验结果5.3 Experimental results
按照面积归一法计算样品中高分子聚合物，抗体单体和低分子物质百分比，结果显示在图7A-图7C，结果表明，两个候选抗体P16-A3、P17-A11以及对照抗体CR3022的单体纯度均大于98％。The percentages of high molecular weight polymers, antibody monomers and low molecular weight substances in the samples were calculated according to the area normalization method. The results are shown in Figures 7A-7C. The results show that the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 The body purity is greater than 98%.
实施例6 候选抗体热稳定性检测Example 6 Thermal stability test of candidate antibodies
差示扫描荧光法(differential scanning fluorimetry；DSF)能够根据蛋白质图谱中的荧光变化过程提供有关蛋白质结构稳定性的信息，检测蛋白的构型变化，获得蛋白质的熔解温度(T m)。 Differential scanning fluorimetry (DSF) can provide information about the stability of the protein structure according to the fluorescence change process in the protein map, detect the configuration change of the protein, and obtain the melting temperature (T m ) of the protein.
在本实施例中，采用DSF法检测了两个候选抗体P16-A3、P17-A11以及对照抗体CR3022的T m值。 In this example, the DSF method was used to detect the T m values of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022.
6.1实验流程6.1 Experimental procedure
制备抗体溶液，0.2mg/mL，19μL/孔，每个供试品在96孔板(Nunc)中设置三个平行孔，并以PBS和IPI(Ipilimlumab)作为参比，然后在每个孔中加入1μL浓度为100×的SYPRO orange染料，用移液枪吹打混匀，准备上机。样品热稳定测试采用ABI 7500FAST RT-PCR仪器，试验类型选择熔解曲线，采用连续模式，扫描温度范围为25～95℃，升温速率为1％，25℃平衡5min，在升温过程中采集数据，报告基团选择“ROX”，淬灭基团选择“None”，反应体积20μL，以熔解曲线一阶导数的第一个峰谷对应的温度确定为候选抗体的熔解温度Tm。Prepare antibody solution, 0.2mg/mL, 19μL/well, set three parallel wells in a 96-well plate (Nunc) for each test substance, and use PBS and IPI (Ipilimlumab) as a reference, and then place them in each well Add 1μL of SYPRO orange dye with a concentration of 100×, pipette and mix well, and prepare for machine. The thermal stability test of the sample adopts ABI 7500FAST RT-PCR instrument, the test type selects the melting curve, the continuous mode is adopted, the scanning temperature range is 25～95℃, the heating rate is 1%, 25℃ is equilibrated for 5 minutes, the data is collected during the heating process, and the report is Select “ROX” for the group, select “None” for the quenching group, and a reaction volume of 20 μL. The temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the melting temperature Tm of the candidate antibody.
6.2实验结果6.2 Experimental results
实验结果显示在表1中，结果表明，抗体P16-A3、抗体P17-A11与对照抗体CR3022的热稳定性相当，都在70℃左右，具有较好的热稳定性。The experimental results are shown in Table 1. The results show that the thermal stability of antibody P16-A3, antibody P17-A11 and the control antibody CR3022 are equivalent, both at about 70°C, and have good thermal stability.
表1 候选抗体和对照抗体的熔解温度Table 1 Melting temperature of candidate antibody and control antibody
抗体名称Antibody name Tm(℃)Tm(℃)
CR3022CR3022 70.170.1
P16-A3P16-A3 69.369.3
P17-A11P17-A11 70.570.5
实施例7 基于ELISA检测候选抗体的亲和活性和阻断活性Example 7 Detecting the affinity activity and blocking activity of candidate antibodies based on ELISA
在本实施例中，检测了两个候选抗体P16-A3、P17-A11对2019-nCoV冠状病毒S蛋白的亲和活性和阻断S蛋白与分离的ACE2蛋白结合的能力。In this example, the affinity activity of the two candidate antibodies P16-A3 and P17-A11 to the 2019-nCoV coronavirus S protein and the ability to block the binding of the S protein to the isolated ACE2 protein were tested.
7.1基于ELISA检测候选抗体的亲和活性7.1 Detect the affinity activity of candidate antibodies based on ELISA
在96孔ELISA板上，包被重组的2019-nCoV冠状病毒Spike-RBD-mFc，2μg/mL，30μL/孔，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入梯度稀释(梯度浓度见图8)的两个候选抗体P16-A3、P17-A11或对照抗体CR3022，并孵育1h。之后，用PBST清洗3次后加入100μL/孔1:5000稀释的HRP标记的抗人Fc第二抗体(Abcam，ab98624(山羊抗人IgG Fc(HRP)预吸附抗体))并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M的HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板，结果显示在图8中，结果表明，两个候选抗体P16-A3、P17-A11表现出显著优于对照抗体CR3022的S蛋白亲和活性。On a 96-well ELISA plate, coat the recombinant 2019-nCoV coronavirus Spike-RBD-mFc, 2 μg/mL, 30 μL/well, overnight at 4°C. The next day, wash the well plate with PBST 3 times and block it with 5% skimmed milk for 2 hours. After washing the plate 3 times with PBST, add two candidate antibodies P16-A3 and P17-A11 with gradient dilutions (see Figure 8 for gradient concentration). Or control antibody CR3022, and incubate for 1h. After washing 3 times with PBST, 100 μL/well of 1:5000 diluted HRP-labeled secondary anti-human Fc antibody (Abcam, ab98624 (goat anti-human IgG Fc (HRP) pre-adsorbed antibody)) was added and incubated for 1 h. After the incubation, the plate was washed with PBST six times, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, the reaction was terminated by adding 2M HCl, and the plate was read under OD450 with a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in Figure 8. The results show that the two candidate antibodies P16-A3 and P17-A11 It showed significantly better S protein affinity activity than the control antibody CR3022.
7.2基于ELISA检测候选抗体的阻断活性7.2 Detect the blocking activity of candidate antibodies based on ELISA
用人ACE2-huFc蛋白，8μg/mL，30μL/孔，4℃过夜包被96孔板。次日，将96孔板用PBST洗3次后用5％脱脂牛奶封闭2h。然后分别将两个候选抗体P16-A3、P17-A11或对照抗体CR3022梯度稀释(梯度浓度见图9)，并与生物素标记的上述Spike-RBD-His提前预混1.0h，在封闭完成并洗板结束后转移至96孔ELISA板中，孵育1h。用PBST清洗3次后加入100μL/孔1:5000稀释的第二抗体NeutrAvidin-HRP(Thermofisher，31001)并孵育1h。孵育完成后，PBST洗板六次，加入TMB(SurModics，TMBS-1000-01)显色，根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板，结果显示在图9中。用Prism TM软件(GraphPad)内的S形剂量-反应模型进行数据分析。用计算的IC 50值(定义为使病毒S蛋白与ACE2的结合减少50％所需的抗体浓度)作 为阻断效力的指示。经计算，两个候选抗体P16-A3、P17-A11分别具有7.7nM和4.2nM的IC 50值，表明了这两个候选抗体P16-A3、P17-A11具有优异的阻断病毒S蛋白与分离的ACE2蛋白结合的能力。 A 96-well plate was coated with human ACE2-huFc protein, 8μg/mL, 30μL/well, 4°C overnight. The next day, the 96-well plate was washed 3 times with PBST and then blocked with 5% skimmed milk for 2 hours. Then, the two candidate antibodies P16-A3, P17-A11 or the control antibody CR3022 were diluted gradiently (see Figure 9 for gradient concentration), and premixed with the above-mentioned Spike-RBD-His labeled with biotin for 1.0 h in advance. After washing the plate, transfer it to a 96-well ELISA plate and incubate for 1 hour. After washing 3 times with PBST, 100 μL/well of the secondary antibody NeutrAvidin-HRP (Thermofisher, 31001) diluted 1:5000 was added and incubated for 1 h. After the incubation is completed, PBST washes the plate six times, adds TMB (SurModics, TMBS-1000-01) for color development, and according to the color development results, adds 2M HCl to stop the reaction, and reads under OD450 with a microplate reader (Molecular Devices, SpecterMax 190) The results are shown in Figure 9. Data analysis was performed using the S-shaped dose-response model in Prism TM software (GraphPad). IC 50 values calculated (defined as the binding of the viral S protein to ACE2 concentration of antibody required to reduce 50%) as an indication of the effectiveness of blocking. After calculation, the two candidate antibodies P16-A3 and P17-A11 have IC 50 values of 7.7 nM and 4.2 nM, respectively, indicating that the two candidate antibodies P16-A3 and P17-A11 have excellent blocking and separation of virus S protein. The ability of ACE2 protein to bind.
实施例8 基于ELISA测定法的候选抗体表位分组Example 8 Grouping of candidate antibody epitopes based on ELISA assay
在本实施例中，对两个候选抗体P16-A3、P17-A11和对照抗体CR3022结合2019-nCoV冠状病毒S蛋白的表位进行了分组。In this example, the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 bind the epitopes of the 2019-nCoV coronavirus S protein into groups.
8.1双抗夹心法对候选抗体的表位进行分组8.1 Double antibody sandwich method to group epitopes of candidate antibodies
将96孔板的孔分别用候选抗体P16-A3、P17-A11和对照抗体CR3022包被，2μg/mL，30μL/孔，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h。然后，加入S蛋白RBD-His，2μg/mL，30μL/孔，孵育1h。之后，用PBST清洗3次后，如图10加入梯度稀释的生物素化的候选抗体P17-A11，30μL/孔，孵育1h。之后，用PBST清洗3次后加入100μL/孔1:5000稀释的第二抗体NeutrAvidin-HRP(Thermofisher，31001)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色，根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板，结果表明，P17-A11和CR3022结合在2019-nCoV冠状病毒S蛋白RBD结构域不同的表位，而P17-A11和P16-A3结合在2019-nCoV冠状病毒S蛋白RBD结构域相同的表位。The wells of the 96-well plate were coated with candidate antibodies P16-A3, P17-A11 and control antibody CR3022, respectively, at 2 μg/mL, 30 μL/well, overnight at 4°C. The next day, the well plates were washed 3 times with PBST and then blocked with 5% skimmed milk for 2 hours. Then, S protein RBD-His was added, 2μg/mL, 30μL/well, and incubated for 1h. After washing 3 times with PBST, as shown in Fig. 10, the biotinylated candidate antibody P17-A11 was added in a gradient of 30 μL/well, and incubated for 1 h. After washing 3 times with PBST, 100 μL/well of the secondary antibody NeutrAvidin-HRP (Thermofisher, 31001) diluted 1:5000 was added and incubated for 1 h. After the incubation is completed, wash the plate with PBST six times, add TMB (SurModics, TMBS-1000-01) for color development, add 2M HCl to stop the reaction according to the color development results, and read under OD450 with a microplate reader (Molecular Devices, SpecterMax 190) Panel, the results show that P17-A11 and CR3022 bind to different epitopes of the 2019-nCoV coronavirus S protein RBD domain, while P17-A11 and P16-A3 bind to the same table of 2019-nCoV coronavirus S protein RBD domain Bit.
8.2竞争法对候选抗体的表位进行分组8.2 Competition method to group the epitopes of candidate antibodies
将96孔板分别用S蛋白RBD-His进行包板，4μg/mL，30μL/孔，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h。然后，加入梯度稀释(如图11)的候选抗体P16-A3、P17-A11和对照抗体CR3022，30μL/孔，孵育1h。之后加入生物素化的候选抗体P17-A11，30μL/孔，孵育1h。之后，用PBST清洗3次后加入100μL/孔1:5000稀释的第二抗体NeutrAvidin-HRP(Therofisher，31001)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色，根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板，结果显示在图11中，结果表明，P17-A11和CR3022结合在2019-nCoV冠状病毒S蛋白RBD结构域不同的表位，而P17-A11和P16-A3结合在2019-nCoV冠状病毒S蛋白RBD结构域相同的表位，竞争法的结果与上述8.1所述双抗夹心法的结果相符合。The 96-well plates were coated with S protein RBD-His, 4μg/mL, 30μL/well, 4°C overnight. The next day, the well plates were washed 3 times with PBST and then blocked with 5% skimmed milk for 2 hours. Then, add the candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 in gradient dilutions (as shown in Figure 11), 30 μL/well, and incubate for 1 h. Then add biotinylated candidate antibody P17-A11, 30μL/well, and incubate for 1h. After washing 3 times with PBST, 100 μL/well of the secondary antibody NeutrAvidin-HRP (Therofisher, 31001) diluted 1:5000 was added and incubated for 1 h. After the incubation is completed, wash the plate with PBST six times, add TMB (SurModics, TMBS-1000-01) for color development, add 2M HCl to stop the reaction according to the color development results, and read under OD450 with a microplate reader (Molecular Devices, SpecterMax 190) The results are shown in Figure 11. The results show that P17-A11 and CR3022 bind to different epitopes in the RBD domain of the 2019-nCoV coronavirus S protein, while P17-A11 and P16-A3 bind to the 2019-nCoV coronavirus S For the same epitope in the RBD domain of the protein, the result of the competition method is consistent with the result of the double antibody sandwich method described in 8.1 above.
实施例9 基于Fortebio的候选抗体和抗原结合亲和力的测定Example 9 Determination of the binding affinity of candidate antibodies and antigens based on Fortebio
在本实施例中，采用Fortebio BLItz仪器，检测了两个候选抗体P16-A3、P17-A11和对照抗体CR3022与2019-nCoV冠状病毒S蛋白RBD-his的亲和力。In this example, the Fortebio BLItz instrument was used to detect the affinity of the two candidate antibodies P16-A3, P17-A11 and the control antibody CR3022 with the 2019-nCoV coronavirus S protein RBD-his.
9.1材料准备9.1 Material preparation
称取10g的BSA，量取5mL的Tween 20，加入到1000mL的10×PBS，混匀，制成10×KB缓冲液。过滤后分装保存。吸取0.1mL 0.1M且pH＝2.0的甘氨酸溶液加入至0.9mL的超纯水中，混匀，制成传感器再生缓冲液。作为抗原的S蛋白RBD-His以10×KB稀释成10μg/mL，抗体以10×KB稀释成系列浓度梯度，依次为200、66.6、22.2、7.41、0nM。Weigh 10 g of BSA, measure 5 mL of Tween 20, add to 1000 mL of 10×PBS, mix well, and make a 10×KB buffer. After filtering, pack and save. Pipette 0.1mL 0.1M glycine solution with pH=2.0 into 0.9mL ultrapure water, mix well, and make the sensor regeneration buffer. As the antigen, the S protein RBD-His was diluted with 10×KB to 10μg/mL, and the antibody was diluted with 10×KB into a series of concentration gradients, which were 200, 66.6, 22.2, 7.41, 0nM in sequence.
9.2实验流程9.2 Experimental procedure
避光条件下，采用10×KB缓冲液预湿传感器(Anti-Penta-HIS，HIS1K,Fortebio,CA)，至少10min后开始测试样品板(GreinierBio，PN655209)，测试无误后按预设程序进行。首先采用抗原S蛋白RBD-His进行结合，300s,在10×KB缓冲液中平衡30s后，将结合有抗原的传感器转移至不同浓度抗体稀释液中结合300s，待信号稳定后，再转移到10×KB缓冲液中，解离时间为900s，最后通过不同浓度抗体的结合解离数据拟合得到K D、Kon和Koff。 Under dark conditions, use a 10×KB buffer solution to pre-wet the sensor (Anti-Penta-HIS, HIS1K, Fortebio, CA), start testing the sample plate (GreinierBio, PN655209) after at least 10 minutes, and proceed according to the preset program after the test is correct. Firstly, use antigen S protein RBD-His for binding, 300s, after equilibrating in 10×KB buffer for 30s, transfer the antigen-bound sensor to different concentrations of antibody diluents for binding for 300s, and then transfer to 10 In ×KB buffer, the dissociation time is 900s, and finally K D , Kon and Koff are obtained by fitting the binding and dissociation data of different concentrations of antibodies.
9.3结果分析9.3 Analysis of results
结果显示在表2中，候选抗体P16-A3、P17-A11的亲和力分别为0.415nM、0.311nM，对照抗体CR3022亲和力为2.42nM。该实验结果与ELISA测定法中的亲和阻断数据一致(详见实施例7)，候选抗体结合2019-nCoV冠状病毒S蛋白的能力显著优于对照抗体CR3022。The results are shown in Table 2. The affinity of candidate antibodies P16-A3 and P17-A11 are 0.415 nM and 0.311 nM, respectively, and the affinity of control antibody CR3022 is 2.42 nM. The results of this experiment are consistent with the affinity blocking data in the ELISA assay (see Example 7 for details), and the candidate antibody is significantly better than the control antibody CR3022 in binding to the 2019-nCoV coronavirus S protein.
表2 BLI测定抗体对2019-nCoV S蛋白RBD-his的亲和力Table 2 BLI determination of the affinity of antibodies to 2019-nCoV S protein RBD-his
抗体名称Antibody name K D(M) K D (M) k on(1/Ms) k on (1/Ms) k off(1/s) k off (1/s)
CR3022CR3022 2.42E-92.42E-9 1.15E51.15E5 2.78E-42.78E-4
P16-A3P16-A3 4.15E-104.15E-10 1.73E51.73E5 7.17E-57.17E-5
P17-A11P17-A11 3.11E-103.11E-10 2.16E52.16E5 6.71E-56.71E-5
实施例10 基于Fortebio的候选抗体表位分组Example 10 Grouping of candidate antibody epitopes based on Fortebio
在本实施例中，为了考察候选抗体以及对照抗体的结合表位是否一致，采用Fortebio BLItz仪器对候选抗体和对照抗体进行了抗体表位的判定。In this example, in order to investigate whether the binding epitopes of the candidate antibody and the control antibody are consistent, the Fortebio BLItz instrument was used to determine the antibody epitope of the candidate antibody and the control antibody.
10.1材料准备10.1 Material preparation
同实施例9Same as Example 9
10.2实验流程10.2 Experimental procedure
避光条件下，采用10×KB缓冲液预湿传感器(Anti-Penta-HIS，HIS1K,Fortebio,CA),至少10min后开始测试样品板(GreinierBio，PN655209)，测试无误后按预设程序进行。固化抗原S蛋白RBD-His，300s，在10×KB缓冲液中平衡30s后和200nM浓度的第一抗体 结合300s，待信号稳定后，记录其信号强度，再转移到200nM的第二抗体溶液中，并观察其结合强度，此时如果结合信号值在第一抗体基础上明显增强，说明了两个抗体的结合表位不一样，如果结合信号值相较于第一抗体没有增强，说明了两个抗体的结合表位相同。Under dark conditions, use 10×KB buffer to pre-wet the sensor (Anti-Penta-HIS, HIS1K, Fortebio, CA), start testing the sample plate (GreinierBio, PN655209) after at least 10 minutes, and proceed according to the preset program after the test is correct. The solidified antigen S protein RBD-His, 300s, equilibrated in 10×KB buffer for 30s and combined with 200nM of the first antibody for 300s. After the signal is stable, record its signal intensity, and then transfer to 200nM of the second antibody solution , And observe its binding strength. At this time, if the binding signal value is significantly enhanced on the basis of the first antibody, it indicates that the binding epitopes of the two antibodies are not the same. If the binding signal value is not enhanced compared to the first antibody, it indicates that the two The binding epitopes of each antibody are the same.
10.3结果分析10.3 Result analysis
实验结果显示在图12A-12C中，结果表明，候选抗体P16-A3和P17-A11的表位一致，且两者与对照抗体CR3022的表位不同。结果和实施例8中基于ELISA的表位分组结果一致。The experimental results are shown in Figures 12A-12C. The results show that the epitopes of the candidate antibodies P16-A3 and P17-A11 are the same, and they are different from the epitopes of the control antibody CR3022. The results are consistent with the results of ELISA-based epitope grouping in Example 8.
实施例11 细胞水平检测候选抗体阻断S蛋白结合ACE2Example 11 Cell-level detection candidate antibody blocks S protein from binding to ACE2
本实施例中，基于FACS方法评价候选抗体阻断病毒S蛋白RBD结构域和受体ACE2的结合活性。本实施例中使用的Vero E6细胞属于绿猴肾细胞系，天然表达ACE2。绿猴ACE2与人ACE2非常保守，序列同源性达到95％，因此本实施例选择用Vero E6代替表达人ACE2的细胞系进行实验。In this example, the candidate antibody was evaluated based on the FACS method to block the binding activity of the RBD domain of the virus S protein and the receptor ACE2. The Vero E6 cells used in this example belong to the green monkey kidney cell line and naturally express ACE2. Green monkey ACE2 is very conservative with human ACE2, and the sequence homology reaches 95%. Therefore, in this example, Vero E6 was selected to replace the cell line expressing human ACE2 for the experiment.
11.1实验流程11.1 Experimental procedure
配制FACS缓冲液(1X PBS+2％FBS)，使用FACS缓冲液将候选抗体和对照抗体进行梯度稀释(稀释比例见图13)，将抗体稀释液按100μL每孔加入96孔圆底板中。同样使用FACS缓冲液将RBD-mFc蛋白稀释至1μg/mL，向对应96孔板中加入100μL，用排枪轻轻吹打混匀并将96孔板放置于4℃孵育1h。Prepare FACS buffer (1X PBS+2% FBS), use FACS buffer to dilute the candidate antibody and the control antibody stepwise (see Figure 13 for the dilution ratio), and add 100 μL of antibody diluent to each well of the 96-well circular bottom plate. Similarly, use FACS buffer to dilute RBD-mFc protein to 1μg/mL, add 100μL to the corresponding 96-well plate, gently pipette to mix with a row gun, and place the 96-well plate at 4°C and incubate for 1h.
将传代2-4次且生长状态良好的Vero E6细胞用于实验，胰酶消化后重悬细胞，4℃、300g离心去除上清，随后将细胞用FACS缓冲液重悬，计数后将细胞密度调整为2×10 6个细胞/mL，以每孔100μL加至新的96孔圆底板中，4℃、300g离心并去除上清。向对应96孔板对应位置的细胞中加入预先孵育的抗体/RBD-mFc混合液，每孔180μL，用排枪轻轻吹打混匀，并于4℃孵育30min。 Use Vero E6 cells that have been passaged 2-4 times and grow well in the experiment. Resuspend the cells after trypsinization, remove the supernatant by centrifugation at 4°C and 300g, and then resuspend the cells in FACS buffer. After counting, the cell density is determined. Adjust to 2×10 6 cells/mL, add 100 μL per well to a new 96-well round bottom plate, centrifuge at 4° C., 300 g, and remove the supernatant. Add the pre-incubated antibody/RBD-mFc mixture to the cells corresponding to the corresponding position in the 96-well plate, 180μL per well, gently pipette to mix with a row gun, and incubate at 4°C for 30min.
将孵育后的细胞混合液于4℃、300g离心并去除上清，随后向对应孔中各加入200μL的FACS缓冲液并重悬细胞，4℃、300g离心去上清；重复此步骤2次。使用FACS缓冲液将PE标记的抗小鼠IgG-Fc流式抗体(Jackson,115-115-164)以1:200配制成第二抗体稀释液，使用排枪向对应细胞中加入200μL/孔的该第二抗体稀释液并轻轻吹打重悬细胞，随后将细胞放置于4℃避光孵育30min，孵育结束后将细胞于4℃、300g离心去除上清，加入FACS缓冲液重悬细胞，重复该步骤两次。最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测。Centrifuge the incubated cell mixture at 4°C and 300g to remove the supernatant, then add 200μL of FACS buffer to the corresponding wells and resuspend the cells, centrifuge at 4°C and 300g to remove the supernatant; repeat this step twice. Use FACS buffer to prepare PE-labeled anti-mouse IgG-Fc flow cytometry antibody (Jackson, 115-115-164) as a second antibody dilution at 1:200, and add 200 μL/well of this to the corresponding cells using a row gun. Dilute the secondary antibody and gently pipette to resuspend the cells, then place the cells in the dark at 4°C and incubate for 30 minutes. After the incubation, the cells are centrifuged at 4°C and 300g to remove the supernatant, and FACS buffer is added to resuspend the cells. Repeat the procedure Step twice. Finally, it was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).
11.2实验结果11.2 Experimental results
实验结果显示在表3和图13中，结果表明，两个候选抗体显示出了剂量依赖的受体结合阻断活性，在细胞水平有优异的阻断效果且显著优于对照抗体CR0322。The experimental results are shown in Table 3 and Figure 13, and the results show that the two candidate antibodies show a dose-dependent receptor binding blocking activity, have excellent blocking effects at the cellular level and are significantly better than the control antibody CR0322.
表3 两个候选抗体和对照抗体的IC50Table 3 IC50 of two candidate antibodies and control antibody
抗体名称Antibody name IC50(nM)IC50(nM)
P16-A3P16-A3 0.890.89
P17-A11P17-A11 0.890.89
CR3022CR3022 无明显阻断活性No obvious blocking activity
实施例12 2019-nCoV冠状病毒中和实验Example 12 2019-nCoV coronavirus neutralization experiment
本实施例中，以抗CD20抗体利妥昔单抗(Rituxmab)作为阴性同种型对照(Isotype IgG)，测试评价了候选抗体P16-A3、P17-A11对2019-nCoV冠状病毒的中和效果。2019-nCoV冠状病毒S蛋白结合细胞表面上的受体ACE2是病毒感染宿主细胞的第一步。本实施例中使用的Vero E6细胞属于绿猴肾细胞系，天然表达ACE2。因为绿猴ACE2与人ACE2的序列同源性达到95％，Vero E6细胞常用于抗冠状病毒候选药物的体外药效评价，因此使用Vero E6细胞用于候选抗体的病毒中和活性测定。In this example, the anti-CD20 antibody Rituxmab (Rituxmab) was used as a negative isotype control (Isotype IgG) to test and evaluate the neutralizing effect of candidate antibodies P16-A3 and P17-A11 on 2019-nCoV coronavirus . The binding of the 2019-nCoV coronavirus S protein to the receptor ACE2 on the cell surface is the first step for the virus to infect host cells. The Vero E6 cells used in this example belong to the green monkey kidney cell line and naturally express ACE2. Because the sequence homology between green monkey ACE2 and human ACE2 reaches 95%, Vero E6 cells are often used for in vitro drug efficacy evaluation of anti-coronavirus drug candidates. Therefore, Vero E6 cells are used for the determination of virus neutralization activity of candidate antibodies.
12.1材料准备12.1 Material preparation
12.1.1配制细胞培养基12.1.1 Preparation of cell culture medium
在MEM培养基(Invitrogen,41500-034)中，添加谷氨酰胺(终浓度2mM)、青霉素(终浓度100U/mL)、链霉素(终浓度100μg/mL)、灭活FBS(终浓度10％)。In MEM medium (Invitrogen, 41500-034), add glutamine (final concentration 2mM), penicillin (final concentration 100U/mL), streptomycin (final concentration 100μg/mL), inactivated FBS (final concentration 10 %).
12.1.2配制病毒培养基12.1.2 Preparation of virus culture medium
在MEM培养基(Invitrogen,41500-034)中，添加谷氨酰胺(终浓度2mM)、青霉素(终浓度100U/ml)、链霉素(终浓度100μg/mL)、灭活FBS(终浓度5％)。In MEM medium (Invitrogen, 41500-034), add glutamine (final concentration 2mM), penicillin (final concentration 100U/ml), streptomycin (final concentration 100μg/mL), inactivated FBS (final concentration 5 %).
12.1.3配制抗体稀释液12.1.3 Preparation of antibody diluent
使用无血清培养基稀释测试抗体和阴性同种型对照(Isotype IgG)，初始浓度为60μg/mL，2倍梯度稀释，共稀释12个稀释度(配制的抗体浓度为60、30、15、7.5、3.75、1.875、0.938、0.469、0.234、0.117、0.059、0.029μg/mL)。每个抗体浓度各取50μL至96孔细胞培养板中，并设置3个复孔。Use serum-free medium to dilute the test antibody and negative isotype control (Isotype IgG), the initial concentration is 60μg/mL, 2-fold serial dilution, a total of 12 dilutions (the prepared antibody concentration is 60, 30, 15, 7.5 , 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029 μg/mL). Take 50 μL of each antibody concentration into a 96-well cell culture plate, and set up 3 replicate wells.
12.2实验流程12.2 Experimental procedure
12.2.1病毒和抗体混合孵育12.2.1 Mixed incubation of virus and antibody
获得2019-nCoV病毒(hCoV-19/Hangzhou/ZJU-05/2020，全球共享流感数据倡议组织(GISAID)登录号EPI_ISL_415711)，稀释于无血清MEM中，感染Vero E6细胞。感染6天后，用Karber法计算50％组织培养物(在本实施例中为细胞)感染剂量(TCID 50)。 Obtained 2019-nCoV virus (hCoV-19/Hangzhou/ZJU-05/2020, Global Influenza Data Sharing Initiative (GISAID) accession number EPI_ISL_415711), diluted in serum-free MEM, and infected Vero E6 cells. After 6 days of infection, the Karber method was used to calculate the 50% tissue culture (cells in this example) infectious dose (TCID 50 ).
向各抗体稀释液中加入等体积50μL含有100TCID 50的所述2019-nCoV病毒(hCoV-19/Hangzhou/ZJU-05/2020，稀释在无血清MEM中)，混匀，并置于室温中孵育60 min。此时，抗体终浓度为：30、15、7.5、3.75、1.875、0.938、0.469、0.234、0.117、0.059、0.029、0.015μg/mL。 Add an equal volume of 50 μL of the 2019-nCoV virus (hCoV-19/Hangzhou/ZJU-05/2020, diluted in serum-free MEM) containing 100 TCID 50 to each antibody dilution, mix well, and incubate at room temperature 60 min. At this time, the final antibody concentration is: 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029, 0.015 μg/mL.
12.2.2病毒和抗体混合物感染细胞12.2.2 A mixture of virus and antibody infects cells
收集新鲜培养的Vero E6细胞，用实施例12.1.2配制的病毒培养基(5％FBS-MEM)配制细胞浓度为2×10 5个细胞/mL，取100μl加入至上述含病毒抗体混合物的培养板中并混匀，放置于35℃、5％CO 2细胞培养箱中。 Collect freshly cultured Vero E6 cells, use the virus medium (5% FBS-MEM) prepared in Example 12.1.2 to prepare a cell concentration of 2×10 5 cells/mL, and add 100 μl to the above-mentioned virus-containing antibody mixture culture Plate and mix well, and place in a 35°C, 5% CO 2 cell incubator.
12.2.3观察细胞病变和计算抗体的中和效应12.2.3 Observe the cell pathological changes and calculate the neutralizing effect of antibodies
接种后的第2天起显微镜下观察细胞生长情况，第4天观察细胞病变效应(cytopathic effect(CPE))情况，并记录。第6天最终判定结果。From the second day after inoculation, the cell growth was observed under the microscope, and the cytopathic effect (CPE) was observed and recorded on the fourth day. The final decision on the 6th day.
CPE分级标准：“+”为少于25％的细胞出现CPE；“++”为大于25％、少于50％细胞出现CPE；“+++”为50％－70％的细胞出现CPE；“++++”为大于75％的细胞出现CPE。CPE classification standard: "+" means that less than 25% of cells have CPE; "++" means that more than 25% and less than 50% of cells have CPE; "+++" means that 50%-70% of cells have CPE; "++++" means that more than 75% of the cells have CPE.
判定标准：抗体组本身无明显细胞毒性，仅培养基的正常细胞对照组显示为细胞生长，仅加入病毒的病毒对照组显示为CPE达++++。Judgment criteria: the antibody group itself has no obvious cytotoxicity, only the normal cell control group with culture medium shows cell growth, and the virus control group with only virus added shows CPE up to ++++.
用Karber法计算抗体对病毒的中和终点(将抗体稀释度转化为对数)，即能够保护50％细胞不受100TCID50攻击病毒液感染的抗体最高稀释浓度就是该抗体的滴度。The Karber method is used to calculate the neutralization end point of the antibody against the virus (convert the antibody dilution to a logarithm), that is, the highest dilution concentration of the antibody that can protect 50% of the cells from 100TCID50 attack virus infection is the antibody titer.
12.3实验结果12.3 Experimental results
实验结果显示在表4中，结果表明，候选抗体P17-A11和P16-A3能够显著抑制2019-nCoV病毒对Vero E6细胞的感染，其保护50％细胞不受100TCID 50病毒液感染的抗体滴度分别为0.015μg/mL和0.029μg/mL，即0.100nM和0.193nM。 The experimental results are shown in Table 4. The results show that the candidate antibodies P17-A11 and P16-A3 can significantly inhibit the infection of Vero E6 cells by the 2019-nCoV virus and protect 50% of the cells from 100TCID 50 virus infection. They are 0.015μg/mL and 0.029μg/mL, namely 0.100nM and 0.193nM.
表4.抗体对2019-nCoV冠状病毒的中和作用Table 4. Neutralization of antibodies to 2019-nCoV coronavirus
抗体名称Antibody name 抗病毒滴度(μg/mL)Antiviral titer (μg/mL)
P17-A11P17-A11 0.0150.015
P16-A3P16-A3 0.0290.029
CR3022CR3022 无中和活性No neutralizing activity
同种型IgGIsotype IgG 无中和活性No neutralizing activity
实施例13Example 13
基于上述实施例选定了抗体P16-A3、P17-A11，对它们进行了分析和测序。基于IMGT数据库(http://www.imgt.org/)对人抗体序列可变区进行定义，确定本发明抗体轻链和重链可变区的序列(SEQ ID NO:1-4)，针对可变区序列进行分析，采用AbM定义CDR的方式，确定了抗体重链和轻链的互补决定区序列(SEQ ID NO:5-16)。具体序列信息如下：Based on the above examples, antibodies P16-A3 and P17-A11 were selected, and they were analyzed and sequenced. Based on the IMGT database (http://www.imgt.org/), the variable region of the human antibody sequence is defined, and the sequence of the light chain and heavy chain variable region of the antibody of the present invention (SEQ ID NO: 1-4) is determined. The variable region sequence was analyzed, and AbM was used to define the CDR to determine the complementarity determining region sequence of the antibody heavy chain and light chain (SEQ ID NO: 5-16). The specific sequence information is as follows:
表5 抗体的VH和VL序列Table 5 VH and VL sequences of antibodies
Figure PCTCN2020084478-appb-000003
Figure PCTCN2020084478-appb-000003
表6.抗体的重链可变区CDR序列Table 6. CDR sequences of antibody heavy chain variable region
Figure PCTCN2020084478-appb-000004
Figure PCTCN2020084478-appb-000004
表7.抗体的轻链可变区CDR序列Table 7. CDR sequences of light chain variable region of antibodies
Figure PCTCN2020084478-appb-000005
Figure PCTCN2020084478-appb-000005
Figure PCTCN2020084478-appb-000006
Figure PCTCN2020084478-appb-000006
表8.全长抗体的氨基酸序列Table 8. Amino acid sequences of full-length antibodies
Figure PCTCN2020084478-appb-000007
Figure PCTCN2020084478-appb-000007
Figure PCTCN2020084478-appb-000008
Figure PCTCN2020084478-appb-000008
表9.全长抗体的核苷酸序列Table 9. Nucleotide sequences of full-length antibodies
Figure PCTCN2020084478-appb-000009
Figure PCTCN2020084478-appb-000009
Figure PCTCN2020084478-appb-000010
Figure PCTCN2020084478-appb-000010
Figure PCTCN2020084478-appb-000011
Figure PCTCN2020084478-appb-000011

Claims (16)

  1. 特异性结合冠状病毒S蛋白的分离的抗体或抗原结合片段，其包含An isolated antibody or antigen-binding fragment that specifically binds to the S protein of coronavirus, which comprises
    (a)SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR；或者与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体；或(a) The 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 and the 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 2; or the same as the above 6 The CDR region has a single or multiple CDR variants that do not exceed 2 amino acid changes in each CDR region; or
    (b)SEQ ID NO:3所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:4所示的轻链可变区氨基酸序列中的3个CDR；或者与上述6个CDR区具有单个或者多个CDR不超过每个CDR区2个氨基酸变化的变体。(b) The 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 3 and the 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 4; or the same as the above 6 The CDR regions have variants in which single or multiple CDRs do not exceed 2 amino acid changes per CDR region.
  2. 特异性结合冠状病毒S蛋白的分离的抗体或抗原结合片段，其包含An isolated antibody or antigen-binding fragment that specifically binds to the S protein of coronavirus, which comprises
    (a)SEQ ID NO:5所示的HCDR1或SEQ ID NO:5所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:6所示的HCDR2或SEQ ID NO:6所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:7所示的HCDR3或SEQ ID NO:7所示的HCDR3的不超过2个氨基酸变化的变体；SEQ ID NO:8所示的LCDR1或SEQ ID NO:8所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:9所示的LCDR2或SEQ ID NO:9所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:10所示的LCDR3或SEQ ID NO:10所示的LCDR3的不超过2个氨基酸变化的变体；或(a) HCDR1 shown in SEQ ID NO: 5 or a variant with no more than 2 amino acid changes of HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6 or SEQ ID NO: 6 HCDR2 not more than 2 amino acid changes, and SEQ ID NO: 7 HCDR3 or SEQ ID NO: 7 HCDR3 variants not more than 2 amino acid changes; SEQ ID NO: 8 The LCDR1 shown or the variant of LCDR1 shown in SEQ ID NO: 8 that does not exceed 2 amino acid changes, the LCDR2 shown in SEQ ID NO: 9 or the LCDR2 shown in SEQ ID NO: 9 does not exceed 2 amino acid changes A variant of and LCDR3 shown in SEQ ID NO: 10 or a variant of LCDR3 shown in SEQ ID NO: 10 with no more than 2 amino acid changes; or
    (b)SEQ ID NO:11所示的HCDR1或SEQ ID NO:11所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:12所示的HCDR2或SEQ ID NO:12所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:13所示的HCDR3或SEQ ID NO:13所示的HCDR3的不超过2个氨基酸变化的变体；SEQ ID NO:14所示的LCDR1或SEQ ID NO:14所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:15所示的LCDR2或SEQ ID NO:15所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:16所示的LCDR3或SEQ ID NO:16所示的LCDR3的不超过2个氨基酸变化的变体；(b) HCDR1 shown in SEQ ID NO: 11 or a variant with no more than 2 amino acid changes of HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12 or SEQ ID NO: 12 A variant of HCDR2 with no more than 2 amino acid changes, and a HCDR3 shown in SEQ ID NO: 13 or a variant of HCDR3 shown in SEQ ID NO: 13 with no more than 2 amino acid changes; SEQ ID NO: 14 The LCDR1 shown or the variant of LCDR1 shown in SEQ ID NO: 14 that does not exceed 2 amino acid changes, the LCDR2 shown in SEQ ID NO: 15 or the LCDR2 shown in SEQ ID NO: 15 does not exceed 2 amino acid changes A variant of and LCDR3 shown in SEQ ID NO: 16 or a variant of LCDR3 shown in SEQ ID NO: 16 with no more than 2 amino acid changes;
    优选地，特异性结合冠状病毒S蛋白的分离的抗体或抗原结合片段包含Preferably, the isolated antibody or antigen-binding fragment that specifically binds to the S protein of coronavirus comprises
    (a)SEQ ID NO:5所示的HCDR1、SEQ ID NO:6所示的HCDR2和SEQ ID NO:7所示的HCDR3；SEQ ID NO:8所示的LCDR1、SEQ ID NO:9所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(a) HCDR1 shown in SEQ ID NO: 5 and HCDR2 shown in SEQ ID NO: 6 and HCDR3 shown in SEQ ID NO: 7; LCDR1 shown in SEQ ID NO: 8 and shown in SEQ ID NO: 9 LCDR2 and LCDR3 shown in SEQ ID NO: 10; or
    (b)SEQ ID NO:11所示的HCDR1、SEQ ID NO:12所示的HCDR2和SEQ ID NO:13所示的HCDR3；SEQ ID NO:14所示的LCDR1、SEQ ID NO:15所示的LCDR2和SEQ ID NO:16所示的LCDR3。(b) HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12 and HCDR3 shown in SEQ ID NO: 13; LCDR1 shown in SEQ ID NO: 14 and shown in SEQ ID NO: 15 The LCDR2 and the LCDR3 shown in SEQ ID NO: 16.
  3. 根据权利要求1或2所述的分离的抗体或抗原结合片段，其包含The isolated antibody or antigen-binding fragment of claim 1 or 2, which comprises
    (a)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:1的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或(a) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:1 or has at least 90%, 91%, 92%, 93%, 94%, 95% therewith , 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region includes the sequence of SEQ ID NO: 2 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences; or
    (b)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；(b) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95% therewith , 96%, 97%, 98%, or 99% identity sequence, and the light chain variable region includes the sequence of SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences;
    优选地，所述分离的抗体包含Preferably, the isolated antibody comprises
    (a)SEQ ID NO:17或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:18或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；或(a) SEQ ID NO: 17 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it, and SEQ ID NO: 18 or a light chain sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; or
    (b)SEQ ID NO:19或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:20或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；(b) SEQ ID NO: 19 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it, and SEQ ID NO: 20 or a light chain sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it;
    优选地，所述分离的抗体或抗原结合片段为完全人抗体。Preferably, the isolated antibody or antigen-binding fragment is a fully human antibody.
  4. 根据权利要求1至3中任一项所述的分离的抗体或抗原结合片段，其是IgG1、IgG2、IgG3或IgG4抗体；优选地，其是IgG1或IgG4抗体；更优选地，其是人IgG1或人IgG4抗体。The isolated antibody or antigen-binding fragment according to any one of claims 1 to 3, which is an IgG1, IgG2, IgG3 or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is a human IgG1 Or human IgG4 antibody.
  5. 根据权利要求1至4中任一项所述的分离的抗体或抗原结合片段，其中所述抗原结合片段是Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab、双体抗体(diabody)。The isolated antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab, Diabody.
  6. 根据权利要求1至5中任一项所述的分离的抗体或抗原结合片段，其具有一种或多种以下特性：The isolated antibody or antigen-binding fragment according to any one of claims 1 to 5, which has one or more of the following characteristics:
    (a)在25℃ ELISA测定法中测量，以小于约10nM，例如小于约8nM的IC50阻断冠状病毒S蛋白与分离的ACE2蛋白的结合；(a) Measured in an ELISA assay at 25°C, with an IC50 of less than about 10 nM, for example, less than about 8 nM, to block the binding of the coronavirus S protein to the isolated ACE2 protein;
    (b)在25℃生物膜层干涉测定法中测量，以小于约1nM，例如约0.8nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM的结合解离平衡常数K D结合冠状病毒S蛋白； (b) Measured in 25°C biofilm interferometry, with a binding and dissociation equilibrium constant K D of less than about 1 nM, such as about 0.8 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM Binding to coronavirus S protein;
    (c)在FACS测定法中测量，以小于约2nM，例如约1.8nM、1.5nM、1.2nM、0.9nM、0.6nM、0.3nM的IC50阻断冠状病毒S蛋白与细胞上表达的天然ACE2蛋白的结合；(c) Measured in the FACS assay, with an IC50 of less than about 2nM, such as about 1.8nM, 1.5nM, 1.2nM, 0.9nM, 0.6nM, 0.3nM, to block the coronavirus S protein and the natural ACE2 protein expressed on the cell The combination
    (d)在细胞病变法中测量，以小于约0.5nM，例如约0.4nM、0.3nM、0.2nM、0.1nM的抗体滴度保护50％细胞不受100TCID 50攻击病毒液感染； (d) Measured in the cytopathic method, with an antibody titre of less than about 0.5 nM, for example, about 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, to protect 50% of cells from 100 TCID 50 attack virus infection;
    优选地，所述冠状病毒是2019-nCoV病毒。Preferably, the coronavirus is 2019-nCoV virus.
  7. 多特异性抗体，其包含特异性结合冠状病毒S蛋白表位的权利要求1至6中任一项所述的抗体或抗原结合片段，优选地，所述多特异性抗体是双特异性抗体。A multispecific antibody comprising the antibody or antigen-binding fragment of any one of claims 1 to 6 that specifically binds to the epitope of the coronavirus S protein. Preferably, the multispecific antibody is a bispecific antibody.
  8. 抗体组合，其包含特异性结合冠状病毒S蛋白表位的权利要求1至6中任一项所述的抗体或抗原结合片段，和/或其他特异性结合冠状病毒S蛋白表位的抗体或抗原结合片段。An antibody combination comprising the antibody or antigen-binding fragment of any one of claims 1 to 6 that specifically binds to the epitope of the coronavirus S protein, and/or other antibodies or antigens that specifically bind to the epitope of the coronavirus S protein Combine fragments.
  9. 分离的核酸，其编码权利要求1至6中任一项所述的抗体或抗原结合片段或编码权利要求7所述的多特异性抗体。An isolated nucleic acid that encodes the antibody or antigen-binding fragment of any one of claims 1 to 6 or encodes the multispecific antibody of claim 7.
  10. 包含权利要求9的核酸的载体，优选地所述载体是表达载体，例如ExpiCHO载体。A vector comprising the nucleic acid of claim 9, preferably the vector is an expression vector, such as ExpiCHO vector.
  11. 包含权利要求9的核酸或权利要求10的载体的宿主细胞，优选地，所述宿主细胞是原核的或真核的，更优选的选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或抗原结合片段、多特异性抗体的其它细胞，最优选地，所述宿主细胞是293细胞或CHO细胞。A host cell comprising the nucleic acid of claim 9 or the vector of claim 10, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from E. coli cells, yeast cells, mammalian cells or suitable for preparation Antibodies or antigen-binding fragments, other cells for multispecific antibodies, most preferably, the host cell is 293 cells or CHO cells.
  12. 制备权利要求1至6中任一项所述的抗体或抗原结合片段、或权利要求7所述的多特异性抗体的方法，所述方法包括在适于表达编码权利要求1至6中任一项所述的抗体或抗原结合片段或编码权利要求7所述的多特异性抗体的核酸的条件下培养权利要求11的宿主细胞，任选地从所述宿主细胞或从培养基回收权利要求1至6中任一项所述的抗体或抗原结合片段、或权利要求7所述的多特异性抗体。A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 6, or the multispecific antibody according to claim 7, said method comprising: The host cell of claim 11 is cultured under the conditions of the antibody or antigen-binding fragment or the nucleic acid encoding the multispecific antibody of claim 7, and the host cell of claim 1 is optionally recovered from the host cell or from the culture medium. The antibody or antigen-binding fragment of any one of to 6 or the multispecific antibody of claim 7.
  13. 药物组合物，其包含权利要求1至6中任一项所述的抗体或抗原结合片段、权利要求7所述的多特异性抗体、或权利要求8所述的抗体组合，以及可药用载体。A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 6, the multispecific antibody of claim 7, or the antibody combination of claim 8, and a pharmaceutically acceptable carrier .
  14. 根据权利要求1至6中任一项所述的抗体或抗原结合片段、或权利要求7所述的多特异性抗体的用途，用于制备预防和/或治疗冠状病毒感染的药物，例如，所述冠状病毒是2019-nCoV病毒。The use of the antibody or antigen-binding fragment according to any one of claims 1 to 6, or the use of the multispecific antibody according to claim 7, for the preparation of drugs for the prevention and/or treatment of coronavirus infections, for example, Said coronavirus is 2019-nCoV virus.
  15. 在受试者中预防和/或治疗冠状病毒感染的方法，包括向受试者施用有效量的权利要求1至6中任一项所述的抗体或抗原结合片段、权利要求7所述的多特异性抗体、权利要求8所述的抗体组合、或权利要求13的药物组合物，例如，所述冠状病毒是2019-nCoV病毒。A method for preventing and/or treating coronavirus infection in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment according to any one of claims 1 to 6, or the polyclonal according to claim 7 Specific antibodies, the antibody combination of claim 8, or the pharmaceutical composition of claim 13, for example, the coronavirus is 2019-nCoV virus.
  16. 检测样品中冠状病毒S蛋白的试剂盒，所述试剂盒包含权利要求1至6中任一项所述的抗体或抗原结合片段、权利要求7所述的多特异性抗体、或权利要求8所述的抗体组合，用于实施以下步骤：A kit for detecting coronavirus S protein in a sample, the kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 6, the multispecific antibody of claim 7, or the multispecific antibody of claim 8 The antibody combination described above is used to implement the following steps:
    (a)将样品与权利要求1至6中任一项所述的抗体或抗原结合片段、权利要求7所述的多特异性抗体、或权利要求8所述的抗体组合接触；和(a) contacting the sample with the antibody or antigen-binding fragment of any one of claims 1 to 6, the multispecific antibody of claim 7, or the antibody combination of claim 8; and
    (b)检测权利要求1至6中任一项所述的抗体或抗原结合片段、权利要求7所述的多特异性抗体、或权利要求8所述的抗体组合与冠状病毒S蛋白之间的复合物的形成，(b) Detect the relationship between the antibody or antigen-binding fragment of any one of claims 1 to 6, the multispecific antibody of claim 7, or the antibody combination of claim 8 and the coronavirus S protein The formation of complexes,
    例如，所述冠状病毒是2019-nCoV病毒。For example, the coronavirus is 2019-nCoV virus.
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