CN114656556A - Fully human monoclonal antibody for resisting novel coronavirus and application thereof - Google Patents

Fully human monoclonal antibody for resisting novel coronavirus and application thereof Download PDF

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CN114656556A
CN114656556A CN202210570003.3A CN202210570003A CN114656556A CN 114656556 A CN114656556 A CN 114656556A CN 202210570003 A CN202210570003 A CN 202210570003A CN 114656556 A CN114656556 A CN 114656556A
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monoclonal antibody
antibody
rbd
nucleotide sequence
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CN114656556B (en
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陈磊
高滕森
殷建国
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Yikang Biology Suzhou Co ltd
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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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • 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
    • 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

Abstract

The invention relates to the field of biological medicines, and discloses a fully human monoclonal antibody for resisting novel coronavirus and application thereof. A receptor binding domain RBD of virus S protein is used as a probe, memory B lymphocytes of a novel coronavirus pneumonia rehabilitation patient are screened by flow cytometry, and the affinity constant of the prepared monoclonal antibody and the RBD is 4.48 nM. The three-dimensional structure of the antibody and RBD compound is analyzed by X-ray crystal diffraction, the combination epitope part of the antibody and a host cell receptor (ACE 2) is found to be overlapped, the combination of RBD and ACE2 can be competitively blocked, the virus infection host cell is inhibited, certain genetic mutation of the novel coronavirus can be effectively coped with, the half effective inhibition concentration for blocking the novel coronavirus to infect the host cell can reach 20.93 ng/mL at least, and the compound has the advantages of safety, high efficiency and broad spectrum, and is expected to be used for diagnosing, treating and preventing the novel coronavirus pneumonia.

Description

Fully human monoclonal antibody for resisting novel coronavirus and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a fully human monoclonal antibody for resisting novel coronavirus and application thereof.
Background
The causative agent of the novel coronavirus pneumonia (COVID-19) is a novel coronavirus (SARS-CoV2), which causes acute respiratory diseases. The clinical features of COVID-19 include fever, dry cough and fatigue, respiratory failure, etc. SARS-CoV2 belongs to the family Coronaviridae (Coronaviridae) and genus Coronavir, a single-stranded enveloped positive-strand RNA virus. The virus gene code contains a plurality of structural proteins, such as Spike protein (S protein), Envelope protein (E protein), Membrane protein (M protein) and Nucleocapsid protein (N protein) and the like; among them, the S, M and E proteins are involved in the formation of the envelope of the viral cell, while the N protein is involved in the nucleic acid assembly of the virus. Research shows that the S protein is a key protein for virus to invade host cells, plays a role in binding virus and host cell membrane receptors and fusing membranes, determines the host range and specificity of the virus, and can realize transmission among different hosts through gene recombination or mutation of a Receptor Binding Domain (RBD) of the protein. Like the Sas virus (SARS-CoV), both coronaviruses are combined with angiotensin converting enzyme 2 (ACE 2) on the surface of a host cell through RBD thereof to induce the fusion of a virus envelope and the host cell membrane, so that the infection of the virus to the host cell is realized. The epitope bound by RBD and ACE2 is relatively conserved in genetics, is the weak point of virus, and is an important action site of a broad-spectrum neutralizing antibody and a key target point of antibody drug screening.
There are a number of antibodies present in convalescent patients with new coronavirus pneumonia that neutralize the virus and prevent further infection. But because the plasma source of a rehabilitation patient is limited, the large-scale preparation is difficult, and batch differences and other disease infection risks exist, the therapy cannot be popularized and applied in a large range. The main effective component of the plasma of a rehabilitation patient is a neutralizing antibody, and the fully human monoclonal antibody has single component, strong safety, clear antiviral mechanism and easy large-scale preparation, and is an important direction for developing novel medicaments for treating coronavirus pneumonia.
Disclosure of Invention
In order to develop a high-efficiency fully human monoclonal antibody medicament for treating and preventing COVID-19, the invention designs a screening probe on the basis of observing a novel coronavirus S protein RBD three-dimensional structure, aims to more effectively screen specific memory B lymphocytes from peripheral blood of a rehabilitation patient with novel coronavirus pneumonia, obtains heavy chain and light chain gene sequences of a single B lymphocyte expression antibody by a single cell nested PCR (polymerase chain reaction) technology, then constructs a stable cell line for expressing the antibody by a genetic engineering means, and prepares the monoclonal antibody in large scale in vitro. The three-dimensional structure of the antibody Fab and RBD compound is analyzed by X-ray crystal diffraction, and the antibody and most of the binding epitope of the RBD are highly overlapped with the binding epitope of ACE2, so that the RBD can be competitively blocked from being bound with ACE2, and the virus infection of host cells is inhibited. As the RBD and ACE2 binding epitope is relatively conserved genetically, the antibody provided by the invention can effectively cope with certain genetic mutations generated by novel coronavirus, has good broad spectrum, and verifies the safety, effectiveness and broad spectrum of the monoclonal antibody on 6 different novel coronavirus infection hACE2 humanized cell models including wild type (Wuhan-Hu-1), Alpha (Alpha: B.1.1.7), Kappa (Kappa: B.1.617.1), Delta (Delta: B.1.617.2), Lambda (Lambda: C.37) and Omicrken (Omicron: BA.1). The monoclonal antibody is designated "YK-CoV 2-03" in the present application.
The first object of the present invention is to provide a fully human monoclonal antibody YK-CoV2-03 against a novel coronavirus,
(I) three CDR regions of the heavy chain variable region of the monoclonal antibody: the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID Nos. 1, 2 and 3, respectively; and is
(II) three CDR regions of the light chain variable region of the monoclonal antibody: the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID Nos. 4, 5 and 6, respectively.
Further, the monoclonal antibody has:
(III), 4 FR regions of the heavy chain of the monoclonal antibody: the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID Nos. 7, 8, 9 and 10, respectively; and is
(IV), 4 FR regions of the light chain of the monoclonal antibody: the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID Nos. 11, 12, 13 and 14, respectively.
Further, the monoclonal antibody has:
(V) the heavy chain has an amino acid sequence shown as SEQ ID No.15, and
(VI), the light chain has an amino acid sequence shown as SEQ ID No. 16.
The fully human monoclonal antibody YK-CoV2-03 is obtained by screening blood of a novel coronavirus pneumonia rehabilitation patient through an S protein RBD specific probe marking-flow sorting-single cell nested PCR technology of SARS-CoV 2. The affinity constant of the antibody to the RBD was 4.48 nM.
The second purpose of the invention is to provide a nucleic acid molecule for coding the monoclonal antibody YK-CoV 2-03.
Further, the nucleic acid molecule encoding the heavy chain has:
(VII) a nucleotide sequence shown as SEQ ID No. 17; or
(VIII) a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID No. 17; or
A nucleotide sequence of (IX) which encodes the same protein as the nucleotide sequence of (VII) or (VIII) but which differs from the nucleotide sequence of (VII) or (VIII) due to the degeneracy of the genetic code.
Further, the nucleic acid molecule encoding the light chain has:
(X) a nucleotide sequence shown as SEQ ID No. 18; or
(XI) the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID No. 18; or
(XII), a nucleotide sequence which encodes the same protein as the nucleotide sequence of (X) or (XI) but which differs from the nucleotide sequence of (X) or (XI) due to the degeneracy of the genetic code.
The third purpose of the invention is to provide an expression vector containing the nucleic acid molecule of the monoclonal antibody YK-CoV 2-03.
The fourth object of the present invention is to provide a host cell containing said nucleic acid molecule, or containing said expression vector.
Further, the host cell is a CHO cell, a HEK293 cell, a yeast cell or a plant cell.
The host cell is used for transient expression of the antibody and construction of a stable cell line, and scientific research and industrial production of the antibody are realized.
The fifth purpose of the invention is to provide the application of the monoclonal antibody YK-CoV2-03 in medicaments for treating and preventing COVID-19.
The sixth purpose of the invention is to provide a novel coronavirus drug, which comprises a safe and effective dose of the monoclonal antibody YK-CoV2-03 and pharmaceutically acceptable auxiliary materials.
The seventh purpose of the invention is to provide a diagnostic kit for diagnosing COVID-19, wherein the diagnostic kit contains the neutralizing antibody YK-CoV 2-03.
The invention discloses a method for carrying out X-ray diffraction on a Fab and RBD protein compound crystal of a monoclonal antibody YK-CoV2-03 and analyzing the protein crystallography of a three-dimensional structure of the compound, and finds that the antibody and RBD binding epitope are overlapped with most of the ACE2 binding epitope, and the RBD and ACE2 binding epitope are relatively conservative genetically, so that the ACE2 binding epitope which is conservative genetically in RBD can be accurately identified, namely the weakness of viruses, and the antibody can effectively deal with genetic variation of novel coronavirus and has good broad-spectrum characteristics.
The invention discloses an antibody neutralization activity detection method based on SARS-CoV2 pseudovirus, aiming at six SARS-CoV2 virus strains, comprising wild type (Wuhan-Hu-1), Alpha (Alpha: B.1.1.7),The neutralizing activity of YK-CoV2-03 on wild-type and 5 different SARS-CoV2 strains was examined by constructing mutant S protein expression plasmids by local deletion mutation of 19 amino acids in the cytoplasmic peptide segment in the S protein sequences of Kappa (Kappa: B.1.617.1), Deltay (Delta: B.1.617.2), Lambda (Lambda: C.37) and Oromkron (Omicron: BA.1), packaging of SARS-CoV2 pseudovirus on 293T cells using a lentivirus packaging system, and performing in vitro neutralization experiments using the pseudovirus. The monoclonal antibody YK-CoV2-03 can play an effective neutralization role against wild type and 5 variant strains, and blocks half of effective Inhibitory Concentration (IC) of pseudovirus infected host cells50) The minimum can reach 20.93 ng/mL, has the advantages of safety, high efficiency and broad spectrum, and is expected to be used for diagnosing, treating and preventing novel coronavirus pneumonia.
By the scheme, the invention at least has the following advantages:
in the process of analyzing the physical chemistry, biology, immunology and virology of the monoclonal antibody YK-CoV2-03, the antibody is found to have the following characteristics: (1) high affinity and high neutralizing activity, and the binding affinity constant (KD) of the antibody and the RBD is 4.48 nM as determined by a macromolecular interaction apparatus. (2) The action mechanism is clear: the diffraction result of the crystal structure shows that the monoclonal antibody YK-CoV2-03 is highly specifically combined with RBD, and the RBD combined epitope of the antibody is overlapped with the combined epitope of most ACE2, so that the combination of virus and host cells can be blocked through specific competition, and the antiviral effect is exerted. (3) Good broad spectrum characteristics: the crystal structure shows that the antibody can accurately recognize the ACE2 binding epitope conserved in RBD heredity, and effectively cope with the genetic variation of novel coronavirus. The monoclonal antibody YK-CoV2-03 has high neutralizing activity on six SARS-CoV2 virus strains, can effectively inhibit virus infection of host cells, and has half effective Inhibitory Concentration (IC)50) The lowest can reach 20.93 ng/mL. (4) The stability is good: the heavy chain and light chain genes of the antibody are derived from the same human B lymphocyte and are naturally paired, the half-life period of the IgG1 antibody in a human body is known to be 21-28 days, and theoretically, the disclosed monoclonal antibody has similar half-life period in the human body. (5) Hair brushThe monoclonal antibody is a fully human source, does not need humanized modification in clinical application to reduce human anti-mouse antibody reaction, has high affinity and high neutralization activity, has good drug forming property, and has wide diagnosis, treatment and prevention application prospects aiming at novel coronavirus pneumonia.
The foregoing description is only an overview of the technical solutions and partial results of the present invention, and in order to make the technical means of the present invention more clearly understood and to be implemented according to the content of the description, the following description is made with reference to the preferred embodiments of the present invention and the detailed drawings.
Drawings
FIG. 1, flow sorting memory B cells to which SARS-CoV 2S protein specifically binds; wherein A in FIG. 1 shows the delineation of the human peripheral blood mononuclear cell population, and B in FIG. 1 shows the selection of Aqua Blue from the cells delineated by A in FIG. 1- CD3- CD8a-CD14-D19+ CD20+ IgGhiIgMlo CD27+C in FIG. 1 shows that Ag (+) is further selected from the cells defined in B in FIG. 1+Ag(-)-The cell of (a);
FIG. 2 is a diagram of agarose gel electrophoresis of nucleic acid, which is a PCR amplification product of the light and heavy chain gene of an antibody;
FIG. 3 is a graph showing the output of variable region sequences of YK-CoV2-03 antibody in IMGT search, wherein A in FIG. 3 is a graph showing the output of heavy chain results; b in FIG. 3 is a light chain result output diagram;
FIG. 4, chromatographic purification and SDS-PAGE detection profile of YK-CoV2-03 antibody; wherein, A in figure 4 is Superdex 200 molecular sieve column separation and purification map; b in FIG. 4 is an SDS-PAGE detection profile;
FIG. 5, determination of binding affinity activity of YK-CoV2-03 antibody for RBD using a macromolecular interactor Gator;
FIG. 6, determination of YK-CoV2-03 antibody competitive blocking the binding of RBD of SARS-CoV 2S protein to the receptor ACE2 using a macromolecular interaction apparatus Gator;
FIG. 7, neutralizing ability of YK-CoV2-03 antibody against 6 different SARS-CoV-2 pseudoviruses; wherein A and B in FIG. 7 are YK-CoV2-03 against 6 different pseudovirusesAnd experimental results and half effective inhibitory concentrations IC against different viruses50Comparing;
FIG. 8, the three-dimensional structure of Fab and RBD complex of YK-CoV2-03 is resolved by X-ray crystal diffraction, wherein A in FIG. 8 is the integral three-dimensional structure of YK-CoV2-03 and RBD complex; b in FIG. 8 is a binding mapping diagram of YK-CoV2-03 and the receptor ACE2 on RBD; wherein, HC: a heavy chain binding epitope; LC: a light chain binding epitope; the dotted line includes the binding epitope for the receptor ACE2 on the RBD, the dark grey surface included within the solid line is the binding epitope for the antibody on the RBD;
FIG. 9, heavy and light chain sequences of monoclonal neutralizing antibodies provided by the invention: wherein, A in FIG. 9 is the amino acid sequence of the heavy chain variable region; b in FIG. 9 is the amino acid sequence of the light chain variable region; c in FIG. 9 is the nucleotide sequence of the heavy chain variable region; d in FIG. 9 is the nucleotide sequence of the light chain variable region.
Detailed Description
The invention discloses a fully human monoclonal antibody specifically binding SARS-CoV2 virus and application thereof, and the technical personnel can use the content for reference and can realize the appropriate improvement of the process parameters. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The terms:
unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
An "antibody" refers to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together and are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.
The "variable region" of an antibody heavy or light chain is the N-terminal mature region of the chain. The types of antibodies currently known include kappa and lambda light chains, as well as alpha, gamma (IgG 1, IgG2, IgG3, IgG 4), delta, epsilon and mu heavy chains or other type equivalents thereof. Full-length immunoglobulin "light chains" (about 25kDa or about 214 amino acids) contain a variable region of about 110 amino acids at the NH 2-terminus and a kappa or lambda constant region at the COOH-terminus. The full-length immunoglobulin "heavy chain" (about 50kDa or about 446 amino acids) likewise comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, e.g., gamma (about 330 amino acids).
"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.
"humanized antibody" refers to an antibody that comprises CDR regions derived from a non-human antibody and the remainder of the antibody molecule is derived from a human antibody (or antibodies). Furthermore, to preserve binding affinity, some residues of the backbone (referred to as FR) segment may be modified.
The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.
The medicament contains at least one functional component and also comprises a medicinal carrier. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic salt solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.
As used herein, "CDR region" or "CDR" refers to the hypervariable regions of the heavy and light chains of an immunoglobulin, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is intended to indicate one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope, as the case may be.
The invention provides a fully human monoclonal antibody which aims at an ACE2 binding epitope conserved on the RBD heredity of a novel coronavirus S protein and has high affinity.
The fully human monoclonal antibody is a monoclonal antibody against SARS-CoV-2 screened by SARS-CoV2 related specific probe labeling-flow sorting-single cell nested PCR technology, and the expression, separation and purification of the antibody are shown in figures 1-4. The monoclonal antibody consists of a light chain and a heavy chain:
wherein the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region are respectively shown as SEQ ID No.1, 2 and 3, and the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region of the monoclonal antibody are respectively shown as SEQ ID No.4, 5 and 6. 4 FR regions of the heavy chain: the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID Nos. 7, 8, 9 and 10, respectively; 4 FR regions of the light chain: the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID Nos. 11, 12, 13 and 14, respectively; the heavy chain has an amino acid sequence shown as SEQ ID No.15 and a nucleotide sequence shown as SEQ ID No. 17; the light chain has an amino acid sequence shown as SEQ ID No.16 and a nucleotide sequence shown as SEQ ID No. 18.
The invention also provides a host cell containing the functional element. In a preferred embodiment, the cell is an Expi 293F cell for transient expression of the antibody. In another preferred embodiment, the cell is a CHO cell, and the CHO cell can be used for constructing a stable transformation engineering cell strain to realize industrial production.
The monoclonal antibody provided by the invention shows good neutralization protection effect on SARS-CoV2 pseudovirus infected cells, and has wide application prospect. The monoclonal antibody disclosed by the invention also has the following technical advantages: (1) fully human, in clinical applications, there is no need for humanization to reduce the human anti-mouse antibody response (HAMA response), i.e. low immunogenicity. (2) High affinity activity and high neutralizing activity, the affinity constant KD of the monoclonal antibody and the RBD antigen is 4.48 nM, and the half effective inhibition concentration IC is obtained in the detection based on SARS-CoV2 pseudovirus50The lowest can reach 20.92 ng/mL. (3) The action mechanism is clear: YK-CoV2-03 is highly specifically bound with RBD, and shows that the monoclonal antibody targets a receptor binding region and exerts antiviral effect by specifically blocking the binding of virus and a receptor ACE 2. (4) The stability is good: because the monoclonal antibody genes come from the same cell of a human body and are naturally paired, the half-life period of the IgG1 antibody in the human body is known to be 21-28 days, and theoretically, the disclosed monoclonal antibody has consistent half-life period in the human body. The present application further includes a product for treating and/or preventing and/or diagnosing a disease caused by a novel coronavirus, which comprises the antibody of the present invention as an active ingredient.
The invention also provides a method for carrying out X-ray diffraction on the crystal of the Fab and RBD protein compound of YK-CoV2-03 and analyzing the protein crystallography of the three-dimensional structure of the compound, so that the weak points of the virus can be accurately identified by the antibody, and the antibody has good broad-spectrum characteristics. As shown in fig. 8.
The invention discloses a fully human monoclonal neutralizing antibody aiming at a SARS-CoV2 virus S protein Receptor Binding Domain (RBD). The antibody is obtained by adopting a host cell receptor ACE2 binding epitope specific probe designed on the basis of structural biology and aiming at RBD, and screening blood of a novel coronavirus pneumonia rehabilitation patient by using a flow cytometer to sort B lymphocytes and a single cell PCR technology, and consists of a unique light chain and a unique heavy chain. The amino acid sequence of the heavy chain is shown as SEQ ID No.15, and the nucleotide sequence of the heavy chain is shown as SEQ ID No. 17; the amino acid sequence of the light chain is shown as SEQ ID No.16, and the nucleotide sequence of the light chain is shown as SEQ ID No. 18. The antibody of the invention can specifically recognize and bind to SARS-CoV 2S protein RBD. By analyzing the crystal structure of the antibody and RBD complex, CDRH3 (23 amino acids) of the antibody was found to be able to recognize the genetically conserved host cell receptor ACE2 binding domain (563 a) hidden by the virus2Overlap ratio 75%). The antibody and RBD have affinity constant KD of 4.48 nM, and can block the combination of virus and host cell receptor ACE2 effectively. Half-effective Inhibitory Concentration (IC) of antibody against different mutant pseudoviruses50) Can reach 20.92 ng/mL. The monoclonal antibody disclosed by the invention has high efficiency and broad spectrum on the neutralization capacity of SARS-CoV2 virus, and simultaneously has the characteristics of full humanity and good stability, thereby having good industrialization potential.
The fully human monoclonal antibody specifically binding SARS-CoV2 virus and its application, the used raw material and reagent are all available in market.
The invention is further illustrated by the following examples:
example 1: screening and preparation of human anti-SARS-CoV 2 monoclonal antibody
1.1 labeling of probes
The desired SARS-CoV2 related specificity positive and negative probes are subjected to fluorescent dye coupling labeling by the following process:
1.1.1 Biotin labelling of the probes was performed using the AviTag fusion protein biotinylation kit (Avidity, BirA 500). The reaction was prepared according to the procedure described in the specification and incubated at 30 ℃ for 30 min.
1.1.2 excess biotin was removed by exchanging the reaction product with 1 XPBS (pH 7.4) using a 30 kDa ultrafiltration tube.
1.1.3 Add 1/5 molar equivalents of streptavidin-phycoerythrin (SA-PE) or streptavidin-allophycocyanin (SA-APC) to the biotinylated probe stepwise at 20 min intervals until the molar ratio of SA-PE or SA-APC to biotinylated probe reaches 1: 1, incubation is carried out by gentle shaking at 4 ℃, and positive and negative probes with fluorescein labels are respectively prepared: ag (-) -SA-APC and Ag (+) -SA-PE.
1.2 flow cytometry sorting antigen-specific Single B lymphocytes
PBMC of a convalescent patient is separated by a Ficoll-Hypaque density gradient (1.077 g/L) centrifugation method, and the specific steps are as follows:
1.2.1 fresh venous blood (heparin anticoagulated) was diluted with an equal volume of 1 XPBS.
1.2.2 according to the volume ratio of peripheral blood to Ficoll of 10: 3, adding the Ficoll to the bottom of the centrifuge tube gently, and flatly paving the diluted blood sample above the liquid level of the separation liquid to keep the interface of the two liquid levels clear.
1.2.3 at room temperature, centrifuge for 30 min with a horizontal rotor at 900 Xg, acceleration of 4, and deceleration of 0.
1.2.4 after centrifugation, the middle tunica albuginea layer was aspirated and placed in a new centrifuge tube, and 10 volumes of 1 × PBS was added, centrifuged at 600 × g for 10 min at room temperature, the supernatant was discarded, and the washing was repeated.
1.2.5 resuspend 1 XPBS in RPMI1640 medium and count viable cells.
The flow cytometry sorting process is adopted to sort out antigen specific single B lymphocyte from PBMC, and the specific steps are as follows:
1.2.6 washing cells with 1 × PBS, resuspending to a cell density of 1 × 106 cells/mL, adding Aqua Blue dye at a ratio of 1 μ L/mL, incubating at 4 ℃ in the dark for 30 min, centrifuging at 600 × g for 10 min, discarding supernatant, and washing repeatedly.
1.2.7 flow antibody staining of PBMC: anti-CD3-PE-Cy7, anti-CD8a-PE-Cy7, anti-CD14-PE-Cy7, anti-CD20-PerCP-Cy5.5, anti-CD19-FITC, anti-IgG-APC-H7, anti-IgM-BV421, anti-CD27-PE-CF594, Ag (-) -SA-APC and Ag (+) -SA-PE, each 5X 106Adding 2 μ L of the above antibody to each PBMC, incubating at 4 deg.C in the dark for 40 min, washing twice with PBS containing 2% BSA, performing flow sorting with BD FACSAria III Cell Sorter, and using a Cell surface marker specific to B lymphocytes (Aqua Blue)-CD3-CD8a-CD14-D19+CD20+IgGhiIgMloCD27+Ag(+)+Ag(-)-) Sorting B lymphocytes, sorting individual B lymphocytes directly into 96-well plates, each well being prepended with 1 XPBS containing 20U RNase inhibitor and 2. mu.L, frozen at-80 ℃ for use. The results of flow sorting are shown in FIG. 1, where A in FIG. 1 shows the delineation of mononuclear lymphocyte populations; b in FIG. 1 shows selection of Aqua Blue from cells circled by A in FIG. 1-CD3- CD8a-CD14-D19+CD20+IgGhiIgMloCD27+B lymphocytes of (a); c in FIG. 1 shows further selection of Ag (+) -from the cells delineated in B in FIG. 1+Ag(-)-The B lymphocyte of (1).
1.3 amplification of fully human monoclonal antibody variable region Gene by Single cell-PCR
1.3.1 reverse transcription PCR, according to the instructions (TaKaRa, 6210A), the procedure is briefly described as follows: obtaining 38B lymphocytes by a flow cytometer, adding 4. mu.L of 5 Xbuffer solution, 1.1. mu.L of dNTP, 1. mu.L of reverse transcriptase and 1. mu.L of Oligo dT primer into each reaction system at the same time, and supplementing to 20 mL with water, wherein the conditions of the reverse transcription PCR reaction are as follows: after incubation at 65 ℃ for 5min, the cells were rapidly cooled on ice, followed by reverse transcription at 42 ℃ for 60 min, followed by pre-denaturation at 95 ℃ for 5min, and cooling on ice.
1.3.2 nested PCR
Taking 4uL of the reverse transcription product as a template, and carrying out PCR reaction to amplify the heavy chain variable region, the kappa light chain variable region and the lambda light chain variable region, wherein relevant primers are shown in Table 1.
TABLE 1 nested PCR primers
Figure 414791DEST_PATH_IMAGE001
Figure 760322DEST_PATH_IMAGE002
The PCR reaction system comprises: mu.L of 2 XDNA polymerase buffer, 0.5 pM each of the primers shown in Table 1, 2. mu.L of the template (reverse transcription product as the template in the first round of PCR, and PCR product in the first round as the template in the second round of PCR) were supplemented with water to 50. mu.L, and the PCR reaction conditions were: pre-denaturation at 94 ℃ for 4 min, followed by denaturation at 94 ℃ for 30 s, annealing at 58 ℃ (H, lambda chain) or 60 ℃ (kappa chain) for 30 s, extension at 72 ℃ for 45 s, 50 cycles, and final extension at 72 ℃ for 10 min.
1.3.3 agarose gel electrophoresis and sequence analysis
The clone with successfully amplified heavy chain and light chain genes in single B lymphocyte is considered to be a monoclonal antibody with successfully matched pair, 5 μ L of second round PCR amplification product is detected by 2% agarose gel electrophoresis, and the result is shown in FIG. 2, wherein lanes 1 to 5 are PCR products of heavy chain variable region VH, the amplification fragment is about 450 bp, lanes 6 to 8 are PCR products of light chain variable region, and the amplification fragment is about 405 bp. The paired positive clones were ligated to a T-Vector for sequencing, and the antibody variable region sequence obtained by sequencing was analyzed using Vector NTI software and logged in the IMGT website, with the results shown in fig. 3, in which a in fig. 3 is the heavy chain gene analysis result and B in fig. 3 is the light chain gene analysis result.
1.4 construction of eukaryotic expression vector of fully human monoclonal antibody against SARS-CoV2 Virus
1.4.1 the above light/heavy chain genes were inserted into the monoclonal cleavage sites of pcDNA3.1(+/-) expression vectors (purchased from Invitrogen;) respectively, to construct expression vectors of fully human monoclonal antibodies against SARS-CoV2 virus.
1.4.2 DH 5. alpha. competent bacteria were transformed with the above ligation products and cultured overnight at 37 ℃ on a plate containing ampicillin.
1.4.3 selecting 10 single colonies to carry out PCR by using specific primers, wherein the reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, 28 cycles, and final extension at 72 ℃ for 5 min.
1.4.4 taking 5. mu.L of the PCR product and running it on a 1% agarose gel, transformants containing the antibody heavy or light chain gene were identified among the positive transformants. The result shows that the recombinant expression vector of the heavy chain/light chain of the novel coronavirus monoclonal antibody constructed by the invention has correct sequence.
1.5 transient expression and purification of monoclonal antibodies
1.5.1 expression of monoclonal antibodies using Expi293 expression System (Thermo Fisher Scientific, A14635), cell transfection procedure was performed as described in the instructions, cell status was monitored continuously after transfection, cell culture supernatant was harvested when cell viability dropped to 70%, centrifuged at 5000 rpm for 40 min and collected by filtration using a 0.22 μm bottleneck filter.
1.5.2 using 15 mL volume of the existing Protein A affinity chromatography column for affinity chromatography purification, before the sample loading using 8 column volume of 1 x PBS buffer solution for balance, after the conductance display to baseline sample introduction, after the sample loading, using 8 column volume of 1 x PBS buffer solution to wash the chromatographic column until the baseline is stable, using elution buffer solution to elute the target Protein, after OD to separate elution of target Protein280After the baseline approach, the collection was stopped, 1 mL of 1M Tris (pH 9.0) was added to each final eluate, and the eluted protein was concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cut-off of 30 kDa, and the concentration thereof was measured by Nanodrop.
1.5.3 the antibody obtained by ultrafiltration is further purified by a Superdex 200 molecular sieve column, the collected peaks of molecular sieve purification are combined, concentrated by an ultrafiltration tube with a molecular weight cut-off of 30 kDa, and the concentration is detected by Nanodrop.
1.5.4 the purified monoclonal antibody is detected by SDS-PAGE, the result is shown in FIG. 4, wherein A in FIG. 4 is the result of purifying the antibody by gel filtration chromatography, B in FIG. 4 is the result of detecting SDS-PAGE, wherein lane 1 is the result of detecting reduced SDS-PAGE, and lane 2 is the result of detecting non-reduced SDS-PAGE. In the reductive electrophoresis, the molecular weights of the heavy and light chains were expected to be 50kDa and 25kDa, respectively, and lane M is a molecular weight marker. In non-reducing electrophoresis, the molecular weight of the whole monoclonal antibody is expected to be 150 kDa, which is expected.
Example 2: analysis of binding Activity of monoclonal antibody YK-CoV2-03 with RBD protein
Analysis of binding Activity between antibody and antigen Using Gator non-labeled biomolecule Analyzer
2.1. RBD expression by GNTi cells, followed by nickel column purification and molecular sieve purification in this order, was biotinylated according to the procedures described in the DSB-X-protein labeling kit (Invitrogen, D20655).
2.2 biotinylated RBD was diluted 600-fold, 3. mu.L of RBD was added to 2000. mu.L of HBS-EP buffer and mixed, and the diluted RBD was loaded in an amount of 200. mu.L per well into a 96-well sample plate.
2.3 the monoclonal antibody is cut into Fab fragments by HRV 3C protease, the obtained antibody Fab is purified by affinity chromatography and molecular sieve, the antibody Fab is diluted by 1000 times, and then the reaction is continued according to the proportion of 1: 3 gradient dilutions were made for 4 dilutions (36.2 nM, 12.1 nM, 4.02 nM and 1.34 nM), and 200. mu.L of HBSEP buffer was taken for assay at each dilution, with 200. mu.L of HBSEP buffer as a blank and one column of HBS-EP buffer added on either side of the sample well column as a baseline.
2.4 assay procedure, each experiment was first baseline equilibrated in HBS-EP buffer on probe card for 120 s, then run for 200 s in cycles, run for 120 s at the front baseline of the sample column, run for 600 s in the sample as binding, run for 400 s in HBSEP in the latter column of the sample column as dissociation, and the fitting procedure used 1: 1 model fitting.
2.5 analysis and results: the affinity constant of YK-CoV2-03 for RBD was calculated by software fitting, see FIG. 5 and Table 2. The YK-CoV2-03 monoclonal antibody has high affinity with RBD.
TABLE 2 result of affinity detection of monoclonal antibody YK-CoV2-03 and RBD antigen
Figure 645101DEST_PATH_IMAGE003
Example 3: analysis of capability of monoclonal antibody YK-CoV2-03 for competitively blocking SARS-CoV2 virus RBD from binding with receptor ACE2
Analysis of the binding capacity of antibody blocking SARS-CoV2 virus RBD and receptor ACE2 by using Gator non-labeled biomolecule analyzer
3.1 expression of ACE2 by Expi-CHO cells, subsequent nickel column purification and molecular sieve purification, biotinylation of ACE2 according to the procedures described in the specification of the DSB-X-channel biotin protein labeling kit (Invitrogen;, D20655), and dilution of biotinylated ACE2 to 40. mu.g/mL at a molar concentration of 570 nM.
3.2 RBD diluted to 30. mu.g/mL, molarity 1000 nM, antibody diluted to 0.2 mg/mL, in a molar ratio of 1: 1, and preparing a mixture with the molar quantity of the antibody being more than that of ACE 2.
3.3 in the test procedure, each experiment was first baseline-equilibrated in HBS-EP buffer of probe card for 120 s, then circulated for 200 s, and run for 120 s at the previous baseline of the sample column, and 20 s in 40. mu.g/mL biotin-ACE2 could reach the maximum binding level of about 1.2 nM response, and in 30. mu.g/mL RBD, bind for 400 s, wherein about 20 s reached the plateau phase of binding, the maximum response was about 0.12 nM, and dissociate for 600 s in HBS-EP buffer, and the dissociation was almost complete.
3.4 analysis and results: the affinity constant of the receptor ACE2 for RBD was calculated by software fitting, see fig. 6. The YK-CoV2-03 monoclonal antibody can completely block the combination of RBD and a receptor ACE 2.
Example 4: method for detecting neutralizing capacity and broad spectrum analysis of monoclonal antibody YK-CoV2-03 against SARS-CoV2 pseudovirus by micro-neutralization method
4.1 SARS-CoV2 pseudovirus preparation:
to test the broad spectrum of neutralizing effect of monoclonal antibody YK-CoV2-03 against SARS-CoV2, wild-type and mutant pseudoviruses were prepared.
The gene encoding the S protein (GenBank ID: QHD 43416.1) was inserted into the pCMV3-CF vector to obtain the wild-type SARS-CoV2 pseudovirus plasmid pCMV-S-CF-WT. On the basis of wild-type pseudoviruses, different mutant pseudovirus plasmids were obtained by mutating the plasmid pCMV-S-CF-WT according to the S protein sequences of the SARS-CoV2 mutant strain Alpha (Alpha: B.1.1.7), Kappa (Kappa: B.1.617.1), Delta (Delta: B.1.617.2), Lambda (Lambda: C.37) and Onckrom (Omicron: BA.1)). 293T cells were seeded at a density of 1.5E +7 cells/mL into a T175 cell culture flask, cultured in a cell incubator at 37 ℃ with 5% CO2, after 24h, transfection was started after a cell density of about 90%, different strains of S protein expression plasmids were transfected into 293T cells with psPAX2 and pCDH-EF 1-CMV-copeGFP-Luc, respectively, using Lipofecta mine 3000 (Invitrogen;, L3000001), the medium was changed after 6 h, the supernatant was collected 48h after transfection, centrifuged at 2000 rpm at 4 ℃ for 10 min to remove cell debris, and then the supernatant was filtered through a 0.45 μm filter.
Filtered viral supernatant with Lenti-XTMConcentrator (Clontech, 631231) as per 3: 1, incubating at 4 deg.C overnight, centrifuging at 1500 Xg for 45 min at 4 deg.C, discarding the supernatant, adding 1/10-1/100 amount of DMEM or PBS, resuspending and precipitating, packaging the concentrated virus into frozen tubes, and storing at-80 deg.C.
4.2 dilution of antibody: the antibodies were diluted with Opti-MEM to the desired concentration, starting at 20. mu.g/mL, and diluted in 5-fold gradients, 25. mu.L of diluted antibody was added to each well, and three replicates per dilution gradient were used.
4.3 dilution of virus: pseudoviruses were diluted to the desired titer with Opti-MEM, an equal amount of 25. mu.L of virus solution was added to each well to which the antibody was added, and mixed well while setting a control group (CC: 50. mu.L DMEM; VC: 25. mu.L of virus + 25. mu.L DMEM), and then the cell culture plate was incubated at 37 ℃ for 1 hour.
4.4 dilution of 293T-hACE2 cells to 2X 105And adding 50 mu L of cell suspension and the antibody-virus mixed solution into each well, mixing the mixture in each well, culturing the mixture for 24 hours in a cell culture box at 37 ℃ and 5% CO2, supplementing 50 mu L of DMEM containing 10% FBS, and placing the mixture in the culture box for continuous culture for 2-4 days.
And 4.52-4 days later, taking out the cell plate, balancing the temperature to room temperature, adding a luciferase reporter gene detection reagent into each hole, reacting for 2min, and then putting into an enzyme labeling instrument to read the RLU value.
4.5 statistical analysis: inhibition =100- (sample signal-blank control signal)/(virus control signal-blank control signal) × 100%, statistical analysis was performed using GraphPad Prism.
4.6 results: YK-CoV2-03 can effectively inhibit six SARS-CoV2 pseudoviruses including wild type (Wuhan-Hu-1), Alpha (Alpha: B.1.1.7), Kappa (Kappa: B.1.617.1), Delta (Delta: B.1.617.2), Lambda (Lambda: C.37) and Omicrken (Omicron: BA.1), wherein Lambda is inhibited best, IC is inhibited best50Can reach 20.92 ng/mL. Still can exert effective neutralization on Omicron, but the inhibition effect is lower than that of other virus strains, IC50319.8ng/mL was reached. See fig. 7.
Example 5: x-ray diffraction crystal structure analysis of monoclonal antibody YK-CoV2-03 and RBD complex
5.1 preparation of antibody Fab and RBD complexes
5.1.1 the antibody Fab to be detected is enzymatically cleaved into Fab fragments by HRV 3C protease, and the obtained antibody Fab is purified by affinity chromatography and molecular sieve.
5.1.2 expression of RBD using GNTi cells. The resulting RBD was purified using nickel column purification and molecular sieve.
5.1.3 RBD and YK-CoV2-03 monoclonal antibody Fab were expressed according to a molecular weight of 1: mixing the raw materials in a proportion of 1.2, incubating the raw materials on ice for 1H to enable the raw materials to be combined with each other to form an antibody Fab and RBD compound, preparing an enzyme digestion system of Endo H endonuclease of the compound, incubating the compound at room temperature for 24H, removing glycosylation modification on the surface of the obtained compound by using the Endo H endonuclease, sequentially passing the obtained enzyme digestion system through a Con A column purification system and a molecular sieve column purification system, purifying the compound without glycosylation modification, and adjusting the concentration of the compound to 5 mg/mL by using an ultrafiltration concentration method.
5.2 preparation of antibody Fab and RBD composite crystals by hanging drop gas phase diffusion method
5.2.1 screening out the optimal conditions for preparing the compound crystal from 384 crystal growth conditions according to the related steps described in the specification of the JCSG crystal preparation series kit, dripping 0.2 mu L of concentrated protein compound on the dot crystal, dripping 0.2 mu L of protein dot crystal pool liquid on the protein compound liquid drop, adding 50 mu L of corresponding dot crystal liquid and 3.5 mu L of 5M NaCl in the liquid pool, placing the crystal culture plate in a thermostat at 22 ℃ for culturing for 10-20 d, and observing the growth condition of the protein crystal, wherein the dot crystal liquid of the selected optimal growth conditions of the crystal is 20% PEG3350 and 0.18M ammonium citrate pH 6.5 solution.
5.2.2 dropwise adding 0.5 mu L of concentrated protein compound onto the spot wafer, simultaneously dropwise adding 0.5 mu L of protein spot crystal pool liquid onto the protein compound liquid drop, reversely buckling the spot wafer onto a crystal culture plate mixed with 500 mu L of protein spot crystal pool liquid and 35 mu L of 5M NaCl, sealing, placing the crystal culture plate in a thermostat at 22 ℃ for culturing for 10-20 days, and taking out the crystal and placing the crystal in liquid nitrogen for storage when the crystal grows to the optimal state. The frozen stock solution was 26% PEG3350, 0.23M ammonium citrate pH 6.5, 40% glycerol solution.
5.2.3 performing crystal diffraction on the obtained Fab and RBD compound crystal at the Shanghai synchrotron radiation light source No.17 line station (SSRF BL 17U) and collecting data, and performing protein three-dimensional structure analysis and optimization correction on the obtained data by using software such as CCP4, Phenix, COOT, PyMol and the like, wherein the structure of the compound is shown in FIG. 8, wherein A in FIG. 8 is the integral three-dimensional structure of the antibody Fab and RBD compound; FIG. 8B is a diagram showing the binding epitope of antibody and the receptor ACE2 on RBD; the results show that the overlapping rate of the binding epitope of the antibody and RBD and the ACE2 binding site is as high as 77%, which indicates that the antibody can effectively cope with the possible genetic variation of the novel coronavirus.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Yikang organism (Suzhou) Co., Ltd
<120> fully human monoclonal antibody against novel coronavirus and application thereof
<160> 61
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> (Artificial sequence)
<400> 1
Gly Leu Thr Val Ser Arg Asn Tyr
1 5
<210> 2
<211> 7
<212> PRT
<213> (Artificial sequence)
<400> 2
Ile Tyr Ala Gly Gly Ser Thr
1 5
<210> 3
<211> 13
<212> PRT
<213> (Artificial sequence)
<400> 3
Cys Ala Arg Asp Leu Val Tyr Tyr Gly Met Asp Val Trp
1 5 10
<210> 4
<211> 6
<212> PRT
<213> (Artificial sequence)
<400> 4
Gln Gly Ile Ser Ser Tyr
1 5
<210> 5
<211> 3
<212> PRT
<213> (Artificial sequence)
<400> 5
Ala Ala Ser
1
<210> 6
<211> 9
<212> PRT
<213> (Artificial sequence)
<400> 6
Gln His Leu Asn Ser Asp Arg Tyr Thr
1 5
<210> 7
<211> 25
<212> PRT
<213> (Artificial sequence)
<400> 7
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 8
<211> 17
<212> PRT
<213> (Artificial sequence)
<400> 8
Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
1 5 10 15
Leu
<210> 9
<211> 37
<212> PRT
<213> (Artificial sequence)
<400> 9
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Gly Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr
35
<210> 10
<211> 10
<212> PRT
<213> (Artificial sequence)
<400> 10
Gly Gln Gly Thr Thr Val Thr Val Ala Ser
1 5 10
<210> 11
<211> 26
<212> PRT
<213> (Artificial sequence)
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
20 25
<210> 12
<211> 17
<212> PRT
<213> (Artificial sequence)
<400> 12
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
1 5 10 15
Tyr
<210> 13
<211> 36
<212> PRT
<213> (Artificial sequence)
<400> 13
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
1 5 10 15
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
20 25 30
Thr Tyr Tyr Cys
35
<210> 14
<211> 9
<212> PRT
<213> (Artificial sequence)
<400> 14
Phe Gly Gln Gly Thr Lys Leu Glu Ile
1 5
<210> 15
<211> 117
<212> PRT
<213> (Artificial sequence)
<400> 15
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Val Ser Arg Asn
20 25 30
Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Leu Ile Tyr Ala Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Gly Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Leu Val Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ala Ser
115
<210> 16
<211> 106
<212> PRT
<213> (Artificial sequence)
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Asn Ser Asp Arg Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105
<210> 17
<211> 351
<212> DNA
<213> (Artificial sequence)
<400> 17
gaagtgcagc tgcaggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60
agctgcgcgg cgagcggcct gaccgtgagc cgcaactata tgacctgggt gcgccaggcg 120
ccgggcaaag gcctggaatg ggtgagcctg atttatgcgg gcggcagcac ctattatgcg 180
gatagcgtga aaggccgctt taccattagc cgcgataaca gcaaaaacac cctgtatctg 240
cagatgaaca gcctgggcgc ggaagatacc gcggtgtatt attgcgcgcg cgatctggtg 300
tattatggca tggatgtgtg gggccagggc accaccgtga ccgtggcgag c 351
<210> 18
<211> 318
<212> DNA
<213> (Artificial sequence)
<400> 18
gatattcaga tgacccagag cccgtttagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca gggcattagc agctatctgg cgtggtatca gcagaaaccg 120
ggcaaagcgc cgaaactgct gatttatgcg gcgagcaccc tgcagagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcat ctgaacagcg atcgctatac ctttggccag 300
ggcaccaaac tggaaatt 318
<210> 19
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 19
acaggtgccc actcccaggt gcag 24
<210> 20
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 20
aaggtgtcca gtgtgargtg cag 23
<210> 21
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 21
cccagatggg tcctgtccca ggtgcag 27
<210> 22
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 22
caaggagtct gttccgaggt gcag 24
<210> 23
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 23
ggaaggaagt cctgtgcgag gc 22
<210> 24
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 24
ggaaggtgtg cacgccgctg gtc 23
<210> 25
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 25
tgggaagttt ctggcggtca cg 22
<210> 26
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 26
ctgcaaccgg tgtacattcc caggtgcagc tggtgcag 38
<210> 27
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 27
ctgcaaccgg tgtacattcc gaggtgcagc tggtgcag 38
<210> 28
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 28
ctgcaaccgg tgtacattcc caggttcagc tggtgcag 38
<210> 29
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 29
ctgcaaccgg tgtacattcc caggtccagc tggtacag 38
<210> 30
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 30
ctgcaaccgg tgtacattct gaggtgcagc tggtggag 38
<210> 31
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 31
ctgcaaccgg tgtacattct gaggtgcagc tgttggag 38
<210> 32
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 32
ctgcaaccgg tgtacattct caggtgcagc tggtggag 38
<210> 33
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 33
ctgcaaccgg tgtacattct gaagtgcagc tggtggag 38
<210> 34
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 34
ctgcaaccgg tgtacattcc caggtgcagc tgcaggag 38
<210> 35
<211> 40
<212> DNA
<213> (Artificial sequence)
<400> 35
ctgcaaccgg tgtacattcc caggtgcagc tacagcagtg 40
<210> 36
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 36
ctgcaaccgg tgtacattcc cagctgcagc tgcaggag 38
<210> 37
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 37
ctgcaaccgg tgtacattcc caggtacagc tgcagcag 38
<210> 38
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 38
gggaattctc acaggagacg a 21
<210> 39
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 39
gttcggggaa gtagtccttg ac 22
<210> 40
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 40
gtccgctttc gctccaggtc acact 25
<210> 41
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 41
ggtcctgggc ccagtctgtg ctg 23
<210> 42
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 42
ggtcctgggc ccagtctgcc ctg 23
<210> 43
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 43
gctctgtgac ctcctatgag ctg 23
<210> 44
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 44
ggtctctctc scagcytgtg ctg 23
<210> 45
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 45
gttcttgggc caattttatg ctg 23
<210> 46
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 46
ggtccaattc ycaggctgtg gtg 23
<210> 47
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 47
gagtggattc tcagactgtg gtg 23
<210> 48
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 48
caccagtgtg gccttgttgg cttg 24
<210> 49
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 49
ctgctaccgg ttcctgggcc cagtctgtgc tgackcag 38
<210> 50
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 50
ctgctaccgg ttcctgggcc cagtctgccc tgactcag 38
<210> 51
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 51
ctgctaccgg ttctgtgacc tcctatgagc tgacwcag 38
<210> 52
<211> 37
<212> DNA
<213> (Artificial sequence)
<400> 52
ctgctaccgg ttctctctcs cagcytgtgc tgactca 37
<210> 53
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 53
ctgctaccgg ttcttgggcc aattttatgc tgactcag 38
<210> 54
<211> 38
<212> DNA
<213> (Artificial sequence)
<400> 54
ctgctaccgg ttccaattcy cagrctgtgg tgacycag 38
<210> 55
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 55
ctcctcactc gagggyggga acagagtg 28
<210> 56
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 56
atgaggstcc cygctcagct gctgg 25
<210> 57
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 57
ctcttcctcc tgctactctg gctcccag 28
<210> 58
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 58
atttctctgt tgctctggat ctctg 25
<210> 59
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 59
gtttctcgta gtctgctttg ctca 24
<210> 60
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 60
atgacccagw ctccabycwc cctg 24
<210> 61
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 61
gtgctgtcct tgctgtcctg c 21

Claims (10)

1. A fully human monoclonal antibody against a novel coronavirus,
(I) three CDR regions of the heavy chain variable region of the monoclonal antibody: the amino acid sequences of CDR1, CDR2 and CDR3 are respectively shown as SEQ ID No.1, 2 and 3; and is
(II) three CDR regions of the light chain variable region of the monoclonal antibody: the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID Nos. 4, 5 and 6, respectively.
2. The monoclonal antibody according to claim 1,
(III), 4 FR regions of the heavy chain of the monoclonal antibody: the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID Nos. 7, 8, 9 and 10, respectively; and is
(IV), 4 FR regions of the light chain of the monoclonal antibody: the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID Nos. 11, 12, 13 and 14, respectively.
3. The monoclonal antibody according to claim 1,
(V) the heavy chain has an amino acid sequence shown as SEQ ID No.15, and
(VI), the light chain has an amino acid sequence shown as SEQ ID No. 16.
4. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1 to 3.
5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule encoding the heavy chain has:
(VII) a nucleotide sequence shown as SEQ ID No. 17; or
(VIII) a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID No. 17; or
A nucleotide sequence of (IX) which encodes the same protein as the nucleotide sequence of (VII) or (VIII) but which differs from the nucleotide sequence of (VII) or (VIII) due to the degeneracy of the genetic code.
6. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule encoding the light chain has:
(X) a nucleotide sequence shown as SEQ ID No. 18; or
(XI) the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID No. 18; or
(XII), a nucleotide sequence which encodes the same protein as the nucleotide sequence of (X) or (XI) but which differs from the nucleotide sequence of (X) or (XI) due to the degeneracy of the genetic code.
7. An expression vector comprising a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3.
8. A host cell comprising the nucleic acid molecule of any one of claims 4 to 6, or comprising the expression vector of claim 7.
9. A drug against a novel coronavirus, which comprises a safe and effective amount of the monoclonal antibody of any one of claims 1 to 3, and a pharmaceutically acceptable excipient.
10. A diagnostic kit for diagnosing COVID-19, comprising the monoclonal antibody according to any one of claims 1 to 3.
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