CN115710311A - Antibodies or antigen-binding fragments thereof to coronaviruses - Google Patents

Antibodies or antigen-binding fragments thereof to coronaviruses Download PDF

Info

Publication number
CN115710311A
CN115710311A CN202210890641.3A CN202210890641A CN115710311A CN 115710311 A CN115710311 A CN 115710311A CN 202210890641 A CN202210890641 A CN 202210890641A CN 115710311 A CN115710311 A CN 115710311A
Authority
CN
China
Prior art keywords
antibody
sequence
seq
antigen
binding fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210890641.3A
Other languages
Chinese (zh)
Inventor
黄竞荷
吴凡
刘梅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Super Extraordinary Shanghai Medical Technology Co ltd
Original Assignee
SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHANGHAI PUBLIC HEALTH CLINICAL CENTER filed Critical SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority to CN202210890641.3A priority Critical patent/CN115710311A/en
Publication of CN115710311A publication Critical patent/CN115710311A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • 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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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 an antibody or an antigen-binding fragment thereof of coronavirus, a nucleic acid molecule for coding the antibody or the antigen-binding fragment thereof, a carrier containing the nucleic acid molecule, a host cell containing the carrier, and application of the antibody or the antigen-binding fragment thereof in preparing a medicament for treating or preventing diseases caused by coronavirus, and application in detecting products; the invention obtains a series of coronavirus antibodies and antigen binding fragments thereof by using B cell in-vitro monoclonal culture and high-throughput antibody screening technology, the coronavirus antibodies and the antigen binding fragments thereof have strong binding capacity and neutralization capacity for SARS-CoV-2 virus, can recognize and bind S1 protein and RBD thereof of SARS-CoV-2 virus, and have very strong affinity, so that the coronavirus antibodies can be presumed to have binding capacity and neutralization capacity for other coronaviruses and coronaviruses which may appear in the future, and have good clinical application prospect in the future.

Description

Antibodies or antigen-binding fragments thereof to coronaviruses
Technical Field
The invention relates to an antibody of coronavirus or an antigen binding fragment thereof, a nucleic acid molecule for coding the antibody or the antigen binding fragment thereof, a vector containing the nucleic acid molecule, a host cell containing the vector, application of the antibody or the antigen binding fragment thereof in preparing a medicament for treating or preventing diseases caused by coronavirus, and application in detecting products, belonging to the field of biomedicine.
Background
The novel coronavirus pneumonia (2019-nCOV) is an acute respiratory infectious disease caused by SARS-COV-2 novel coronavirus. The virus has extremely strong transmission capability and can be transmitted to public health safety of the world through respiratory tract, contact and other ways to bring a serious challenge.
The SARS-CoV-2 virus belongs to the family of coronavirus, and has amino acid homology as high as 77.2% with SARS coronavirus of the same genus and beta genus, which has been developed in 2003. The main envelope protein of SARS-CoV-2 virus is its Spike protein (also called Spike protein, abbreviated as S protein), which is hydrolyzed into two parts, S1 and S2, by intracellular protease during virus infection. Wherein S2 is a transmembrane protein, and S1 has a Receptor Binding Domain (RBD) for recognizing and binding a cell receptor angiotensin converting enzyme-2 (ACE-2). The spike protein composed of S1 and S2 is a virus receptor which is specifically recognized by SARS-CoV-2 virus, binds to a target cell receptor and mediates virus infection, and is also a recognition target point of a neutralizing antibody to be developed.
So far, no effective medicine or vaccine for treating and preventing SARS-CoV-2 virus infection exists all over the world, and only supportive symptomatic treatment can be clinically adopted for patients with new coronary pneumonia. Research shows that clinically using the virus-specific recovered human plasma can effectively neutralize the virus, prevent the virus from diffusing in each organ in the body and play an important role in the outcome of the disease course of patients. However, not only is the source of polyclonal plasma limited, but clinical use is also limited by conditions such as poor quality control, differences in blood types of donors and recipients, and potential infectious agents. The method is one of the main directions for developing new coronavirus medicines at present, and can effectively overcome the problems by separating the fully human monoclonal antibody capable of neutralizing SARS-CoV-2 virus from the body of a new coronary pneumonia rehabilitator.
At present, a plurality of research teams at home and abroad report that a fully human monoclonal antibody which can be combined with SARS-CoV-2 virus S protein, such as BD-368-2, B38 and the like, is separated from peripheral blood of a new coronary pneumonia rehabilitator, and is still in an experimental development stage at present. The technical method adopted by these research teams is to use S protein or S protein receptor binding Region (RBD) of recombined expressed SARS-CoV-2 virus as bait, to screen and separate B cell (memory B cell) capable of binding these proteins from peripheral blood of convalescent person, to obtain heavy chain and light chain pairing gene of antibody expressed by single B cell by cell sequencing or single cell sequencing, to express antibody by means of in vitro recombination, and to verify the virus neutralizing capacity. Since this method uses a marker protein (the above-mentioned S protein or S protein receptor binding region of SARS-CoV-2 virus recombinantly expressed and called bait) to screen and enrich B cells in advance before antibody gene sequencing, only antibodies that specifically bind to the marker protein can be screened.
Huang Jinghe doctor (one of the inventors of the present application) initiated in 2013 a human B cell in vitro monoclonal culture and high throughput antibody screening technique (Huang J et al nature Protocols 2013), which is used for separating a fully human monoclonal antibody from peripheral blood of a convalescent coronary pneumonia patient, and the process is as follows: firstly, utilizing the SARS-CoV-2 and SARS-CoV pseudovirus neutralization system to detect the neutralizing antibody of serum of new coronary pneumonia convalescent person, and screening out the convalescent person having higher neutralization activity to SARS-CoV-2 and SARS-CoV at the same time; then collecting peripheral blood lymphocytes of the rehabilitee, and sorting out memory B lymphocytes by using flow cells; the single B cell is inoculated into a 384-well plate, and is added with cell factors and feeder cells for culture, and the cultured B cell secretes antibody into supernatant after being amplified and differentiated in vitro. Then, the neutralizing capacity of the antibody in the supernatant to SARS-CoV-2 and SARS-CoV viruses is detected by using an in vitro high-flux neutralization experiment, positive clones capable of simultaneously neutralizing the two viruses are screened out, heavy chain and light chain variable regions of the antibody are cloned by using an RT-PCR method, and the heavy chain and light chain variable regions are constructed to an antibody heavy chain and light chain expression vector, and then 293T cells are transfected to express and purify the monoclonal antibody.
Antibodies currently reported by other groups, although having a good neutralizing power against the tested SARS-CoV-2 virus strain, are prone to mutations in the viral genomic sequence during the spread of an epidemic, since SARS-CoV-2 virus is an RNA virus. When the non-conserved region sites recognized by these antibodies are mutated to generate new epidemic strains, the antibodies lose the protective effect on the mutant viruses.
Therefore, it is still desirable for those skilled in the art to be able to develop new antibodies having binding and neutralizing abilities against coronaviruses including SARS-CoV-2 virus.
Disclosure of Invention
In order to solve the above technical problems, an aspect of the present invention provides a coronavirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein:
the HCDR1 has a sequence general formula as follows: GX 1 TVSSNY wherein X 1 Is L, I or F;
the HCDR2 has a general sequence formula: x 2 YSGGSX 3 Wherein X is 2 Is any one amino acid of L or I, X 3 Is any one of A or T.
Preferably, the sequence formula of the HCDR3 is: ARDLIX 4 YGMDV of, wherein X 4 Any one amino acid that is D or T;
the sequence of the LCDR1 is QGISSY, and the sequence of the LCDR2 is AAS;
the sequence general formula of the LCDR3 is as follows: QQLNSYPPX 5 T, wherein, X 5 Is any one amino acid of L or Y.
In a preferred embodiment of the present invention, the sequence of HCDR1 is shown as SEQ ID NO.1, the sequence of HCDR2 is shown as SEQ ID NO.2, and the sequence of HCDR3 is shown as SEQ ID NO. 3; the sequence of the LCDR1 is shown as SEQ ID NO.5, the sequence of the LCDR2 is shown as SEQ ID NO.6, and the sequence of the LCDR3 is shown as SEQ ID NO. 7; alternatively, the first and second electrodes may be,
the sequence of the HCDR1 is shown as SEQ ID NO.11, the sequence of the HCDR2 is shown as SEQ ID NO.12, and the sequence of the HCDR3 is shown as SEQ ID NO. 13; and the sequence of the LCDR1 is shown as SEQ ID NO.15, the sequence of the LCDR2 is shown as SEQ ID NO.16, and the sequence of the LCDR3 is shown as SEQ ID NO. 17.
In another preferred embodiment of the present invention, the heavy chain variable region has the sequence shown in SEQ ID No.4 or a sequence having more than 80% sequence homology with the sequence shown in SEQ ID No.4, and the light chain variable region has the sequence shown in SEQ ID No.8 or a sequence having more than 80% sequence homology with the sequence shown in SEQ ID No. 8; alternatively, the first and second liquid crystal display panels may be,
the heavy chain variable region has a sequence shown as SEQ ID NO.14 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.14, and the light chain variable region has a sequence shown as SEQ ID NO.18 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO. 18.
In a preferred embodiment of the present invention, the sequence of HCDR1 is shown as SEQ ID NO.21, the sequence of HCDR2 is shown as SEQ ID NO.22, and the sequence of HCDR3 is shown as SEQ ID NO. 23; the sequence of the LCDR1 is shown as SEQ ID NO.25, the sequence of the LCDR2 is shown as SEQ ID NO.26, and the sequence of the LCDR3 is shown as SEQ ID NO. 27; alternatively, the first and second electrodes may be,
the sequence of the HCDR1 is shown as SEQ ID NO.31, the sequence of the HCDR2 is shown as SEQ ID NO.32, and the sequence of the HCDR3 is shown as SEQ ID NO. 33; the sequence of the LCDR1 is shown as SEQ ID NO.35, the sequence of the LCDR2 is shown as SEQ ID NO.36, and the sequence of the LCDR3 is shown as SEQ ID NO. 37; alternatively, the first and second electrodes may be,
the sequence of the HCDR1 is shown as SEQ ID NO.41, the sequence of the HCDR2 is shown as SEQ ID NO.42, and the sequence of the HCDR3 is shown as SEQ ID NO. 43; the sequence of the LCDR1 is shown as SEQ ID NO.45, the sequence of the LCDR2 is shown as SEQ ID NO.46, and the sequence of the LCDR3 is shown as SEQ ID NO. 47; alternatively, the first and second electrodes may be,
the sequence of the HCDR1 is shown as SEQ ID NO.51, the sequence of the HCDR2 is shown as SEQ ID NO.52, and the sequence of the HCDR3 is shown as SEQ ID NO. 53; the sequence of the LCDR1 is shown as SEQ ID NO.55, the sequence of the LCDR2 is shown as SEQ ID NO.56, and the sequence of the LCDR3 is shown as SEQ ID NO. 57; alternatively, the first and second electrodes may be,
the sequence of the HCDR1 is shown as SEQ ID NO.61, the sequence of the HCDR2 is shown as SEQ ID NO.62, and the sequence of the HCDR3 is shown as SEQ ID NO. 63; the sequence of the LCDR1 is shown as SEQ ID NO.65, the sequence of the LCDR2 is shown as SEQ ID NO.66, and the sequence of the LCDR3 is shown as SEQ ID NO. 67.
In another preferred embodiment of the present invention, said heavy chain variable region has the sequence shown as SEQ ID NO.24 or a sequence having more than 80% sequence homology with the sequence shown as SEQ ID NO.24, and said light chain variable region has the sequence shown as SEQ ID NO.28 or a sequence having more than 80% sequence homology with the sequence shown as SEQ ID NO. 28; alternatively, the first and second electrodes may be,
the heavy chain variable region has a sequence shown as SEQ ID NO.34 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.34, and the light chain variable region has a sequence shown as SEQ ID NO.38 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO. 38; alternatively, the first and second electrodes may be,
the heavy chain variable region has a sequence shown as SEQ ID NO.44 or a sequence which has more than 80 percent of sequence homology with the sequence shown as SEQ ID NO.44, and the light chain variable region has a sequence shown as SEQ ID NO.48 or a sequence which has more than 80 percent of sequence homology with the sequence shown as SEQ ID NO. 48; alternatively, the first and second electrodes may be,
the heavy chain variable region has a sequence shown as SEQ ID NO.54 or a sequence having more than 80% of sequence homology with the sequence shown as SEQ ID NO.54, and the light chain variable region has a sequence shown as SEQ ID NO.58 or a sequence having more than 80% of sequence homology with the sequence shown as SEQ ID NO. 58; alternatively, the first and second electrodes may be,
the heavy chain variable region has a sequence shown as SEQ ID NO.64 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.64, and the light chain variable region has a sequence shown as SEQ ID NO.68 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO. 68.
With respect to the percentage of "sequence homology," the number of matched positions is generated by determining the number of amino acid residues present in both sequences, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the structure by 100 to generate the percentage of sequence identity.
In a specific embodiment of the present invention, the heavy chain variable region may be added, decreased or substituted with amino acids based on the first amino acid sequence, and the light chain variable region may be added, decreased or substituted with amino acids based on the second amino acid sequence, such as the substitution of similar amino acids or the addition, decrease or substitution of a small amount of amino acids, especially the addition, decrease or substitution of amino acids in the conserved sequence portion, to obtain antibody variants having high homology (80% or more homology) and retaining the original antibody function, i.e., the function and property of binding specifically to coronavirus, which variants also fall within the scope of the present invention.
In a preferred embodiment of the invention, the heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is set forth in SEQ ID No.9, and the light chain amino acid sequence is set forth in SEQ ID No. 10; alternatively, the first and second electrodes may be,
the heavy chain amino acid sequence of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.19, and the light chain amino acid sequence is shown as SEQ ID NO. 20; alternatively, the first and second electrodes may be,
the heavy chain amino acid sequence of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.29, and the light chain amino acid sequence is shown as SEQ ID NO. 30; alternatively, the first and second electrodes may be,
the heavy chain amino acid sequence of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.39, and the light chain amino acid sequence is shown as SEQ ID NO. 40; alternatively, the first and second electrodes may be,
the heavy chain amino acid sequence of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.49, and the light chain amino acid sequence is shown as SEQ ID NO. 50; alternatively, the first and second electrodes may be,
the heavy chain amino acid sequence of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.59, and the light chain amino acid sequence is shown as SEQ ID NO. 60; alternatively, the first and second electrodes may be,
the heavy chain amino acid sequence of the antibody or the antigen binding fragment thereof is shown as SEQ ID NO.69, and the light chain amino acid sequence is shown as SEQ ID NO. 70.
In a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof of a coronavirus.
The term "neutralizing antibody" is an antibody or antigen-binding fragment that specifically binds to a viral receptor protein, which specifically binds to inhibit a biological function of the viral receptor protein, e.g., prevents the receptor protein from binding to its target cell receptor, which specifically reduces the ability of the virus to infect the target cell; in the present application, a neutralizing antibody or antigen-binding fragment thereof of a coronavirus refers to an antibody or antigen-binding fragment thereof that binds to the S protein of a coronavirus.
The term "antibody" is used herein in the broadest sense and encompasses natural and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, and antibody fragments, antigen binding proteins, fusion proteins, and the like, which exhibit the desired antigen binding activity.
In a preferred embodiment of the invention, the antibody is a monoclonal antibody.
In a more preferred embodiment of the invention, the antibody is a fully human monoclonal antibody.
In a preferred embodiment of the invention, the Fc domain of the antibody is derived from an Fc domain of an immunoglobulin, including a native sequence Fc domain or a variant Fc domain.
In a preferred embodiment of the invention, the antibody is any one or combination of IgG1, igG2, igG3 or IgG 4.
Preferably, the antibody may be a whole antibody selected from IgG1, igG2, igG3 or IgG 4.
In a preferred embodiment of the invention, the antigen binding fragment is an Fv, fab, F (ab ') 2, fab', dsFv, scFv, sc (Fv) 2 or single chain antibody.
In a preferred embodiment of the invention, the antibody, or antigen-binding fragment thereof, may be combined with other antibodies, or antigen-binding fragments thereof, to form an antibody, or antigen-binding fragment thereof, having at least two antigen-binding sites, i.e., a multispecific antibody, or antigen-binding fragment thereof; the two antigen binding sites may be different epitopes of the same antigen or different epitopes of different antigens.
In a preferred embodiment of the invention, the antibody, or antigen-binding fragment thereof, described above may be further chemically modified, e.g., one or more chemical groups may be attached to the antibody to increase one or more functional properties of the antibody. For example, glycosylation modification, pegylation modification, and the like are common chemical modifications. For example, the heavy chain or light chain variable region may be modified by glycosylation, and one or more glycosylation sites may be added to improve a part of the function of the antibody, for example, enhance the immunogenicity of the antibody or improve the pharmacokinetics of the antibody. For example, the antibody or antigen-binding fragment thereof is subjected to acylation or alkylation with an active polyethylene glycol (e.g., an active ester or aldehyde derivative of polyethylene glycol) under suitable conditions to effect pegylation modification to improve a portion of the antibody's function, e.g., increase the biological (e.g., serum) half-life of the antibody, etc. The above chemical modifications do not significantly alter the basic function and properties of the antibody or antigen-binding fragment thereof of the invention, i.e., the function and properties of specific binding to coronaviruses; such chemically modified variants also fall within the scope of the present invention.
In a preferred embodiment of the present invention, the above-described antibody or antigen-binding fragment thereof may be conjugated with other factors by a chemical method or a genetic engineering method; for example, these factors may provide the effect or other property of targeting the antibody to the desired functional site; for example, the agent may be one or more heterologous molecules, preferably, the heterologous molecule is a cytotoxic agent. The antibody or the antigen binding fragment thereof is conjugated with other factors to form a complex, and the complex falls into the protection scope of the invention.
In another aspect, the invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody, or an antigen-binding fragment thereof, as described above.
In a preferred embodiment of the invention, the nucleic acid molecule comprises the nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO.71 and the nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO. 72; alternatively, the first and second electrodes may be,
the nucleic acid sequence for coding the heavy chain variable region is shown as SEQ ID NO.75, and the nucleic acid sequence for coding the light chain variable region is shown as SEQ ID NO. 76; alternatively, the first and second electrodes may be,
the nucleic acid sequence for coding the heavy chain variable region is shown as SEQ ID NO.79, and the nucleic acid sequence for coding the light chain variable region is shown as SEQ ID NO. 80; alternatively, the first and second electrodes may be,
the nucleic acid sequence for coding the heavy chain variable region is shown as SEQ ID NO.83, and the nucleic acid sequence for coding the light chain variable region is shown as SEQ ID NO. 84; alternatively, the first and second electrodes may be,
the nucleic acid sequence for coding the heavy chain variable region is shown as SEQ ID NO.87, and the nucleic acid sequence for coding the light chain variable region is shown as SEQ ID NO. 88; alternatively, the first and second electrodes may be,
the nucleic acid sequence for coding the heavy chain variable region is shown as SEQ ID NO.91, and the nucleic acid sequence for coding the light chain variable region is shown as SEQ ID NO. 92; alternatively, the first and second electrodes may be,
the nucleic acid sequence for coding the heavy chain variable region is shown as SEQ ID NO.95, and the nucleic acid sequence for coding the light chain variable region is shown as SEQ ID NO. 96.
In a more preferred embodiment of the present invention, in the nucleic acid molecule,
the nucleic acid sequence of the coding heavy chain is shown as SEQ ID NO.73, and the nucleic acid sequence of the coding light chain is shown as SEQ ID NO. 74; alternatively, the first and second electrodes may be,
the nucleic acid sequence of the coding heavy chain is shown as SEQ ID NO.77, and the nucleic acid sequence of the coding light chain is shown as SEQ ID NO. 78; alternatively, the first and second electrodes may be,
the nucleic acid sequence of the coding heavy chain is shown as SEQ ID NO.81, and the nucleic acid sequence of the coding light chain is shown as SEQ ID NO. 82; alternatively, the first and second liquid crystal display panels may be,
the nucleic acid sequence of the coding heavy chain is shown as SEQ ID NO.85, and the nucleic acid sequence of the coding light chain is shown as SEQ ID NO. 86; alternatively, the first and second electrodes may be,
the nucleic acid sequence of the coding heavy chain is shown as SEQ ID NO.89, and the nucleic acid sequence of the coding light chain is shown as SEQ ID NO. 90; alternatively, the first and second electrodes may be,
the nucleic acid sequence of the coding heavy chain is shown as SEQ ID NO.93, and the nucleic acid sequence of the coding light chain is shown as SEQ ID NO. 94; alternatively, the first and second electrodes may be,
the nucleic acid sequence of the coding heavy chain is shown as SEQ ID NO.97, and the nucleic acid sequence of the coding light chain is shown as SEQ ID NO. 98.
In a further aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In a preferred embodiment of the present invention, the vector further comprises an expression control sequence linked to the nucleic acid molecule described above.
The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed within the host cell. The vector may contain various elements for controlling expression, such as a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, a reporter gene, and the like. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively. In an embodiment of the present invention, the carrier may be selected from, but is not limited to: plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosome YAC, bacterial artificial chromosome BAC or P1-derived artificial chromosome PAC), bacteriophages (e.g., lambda phage or M13 phage), and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papilloma polyomaviruses (e.g., SV 40).
In yet another aspect, the present invention provides a host cell comprising the vector described above.
With respect to "host cells," one can select, but is not limited to: prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast cells and Aspergillus, insect cells such as S2 Drosophila cells and Sf9, and animal cell models such as fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, and HEK293 cell.
Preferably, the host cell is a HEK293 cell.
In a further aspect, the present invention provides a method for producing an antibody, or an antigen-binding fragment thereof, as described above, wherein the host cell described above is cultured to produce the antibody, or an antigen-binding fragment thereof.
In a further aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the antibody, or an antigen-binding fragment thereof, as described above.
In a preferred embodiment of the invention, the pharmaceutical composition comprises a therapeutically effective amount of a neutralizing antibody, or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent. One skilled in the art can administer to a patient a therapeutically effective amount of the neutralizing antibody, or antigen-binding fragment thereof, in combination with a suitable pharmaceutical carrier or diluent for the treatment or prevention of a disease caused by a coronavirus.
In a further aspect, the present invention provides the use of the antibody, or antigen-binding fragment thereof, or the pharmaceutical composition as described above, in the preparation of a medicament for the treatment or prevention of a disease caused by a coronavirus.
In a preferred embodiment of the present invention, the use refers to the use in the preparation of a medicament for the treatment or prevention of a disease caused by SARS-CoV-2, SARS-CoV or SARS-like coronavirus.
In one aspect, the invention also provides a method of treating or preventing a disease caused by a coronavirus by administering to a patient a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, described above; or administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of the above-described antibody, or antigen-binding fragment thereof. Preferably, the disease caused by coronavirus is a SARS-CoV-2, SARS-CoV or SARS-like coronavirus caused disease.
In a further aspect, the invention provides a test product comprising an antibody, or antigen-binding fragment thereof, as described above.
The test product is useful for detecting the presence or level of coronavirus in a sample.
In one embodiment of the present invention, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or test paper, and the like.
The antibody or the antigen-binding fragment thereof of the present invention may be labeled by a chemical method or a genetic engineering method, and the labeled antibody or the antigen-binding fragment thereof may be used for detection; the labeled antibody or antigen binding fragment thereof falls within the scope of the present invention.
The specific detection method can adopt the following steps of 1) providing a sample; 2) Contacting said sample with an antibody or antigen-binding fragment thereof of the coronavirus of the invention described above; 3) Detecting an immune reaction between the sample and the antibody or antigen-binding fragment thereof.
The invention obtains a series of coronavirus antibodies and antigen binding fragments thereof by using B cell in-vitro monoclonal culture and high-throughput antibody screening technology, the antibodies and the antigen binding fragments thereof have strong binding capacity and neutralization capacity for SARS-CoV-2 virus, can recognize and bind S1 protein and RBD thereof of SARS-CoV-2 virus, and have very strong affinity, so that the invention can be speculated that the antibodies and the antigen binding fragments thereof of the series coronavirus can also have binding capacity and neutralization capacity for other coronavirus and coronavirus which may appear in the future, and have good clinical application prospect in the future.
Drawings
FIG. 1 shows the result of detecting the S1 protein and its RBD of SARS-CoV-2 virus and the S2 protein by monoclonal antibody 4L 12;
FIG. 2 shows the result of detecting the S1 protein and its RBD of SARS-CoV-2 virus and the S2 protein by monoclonal antibody 12F 5;
FIG. 3 shows the result of detecting the S1 protein and its RBD, and S2 protein of SARS-CoV-2 virus recognized by monoclonal antibody 3D 13;
FIG. 4 shows the result of detecting the S1 protein and its RBD, and the S2 protein of SARS-CoV-2 virus recognized by monoclonal antibody 10C 2;
FIG. 5 shows the result of detecting S1 protein and its RBD, and S2 protein of SARS-CoV-2 virus recognized by monoclonal antibody 16L 9;
FIG. 6 shows the result of detecting the S1 protein and its RBD, and S2 protein of SARS-CoV-2 virus recognized by monoclonal antibody 20E 21;
FIG. 7 shows the result of detecting the S1 protein and its RBD, and S2 protein of SARS-CoV-2 virus recognized by monoclonal antibody 22H 22;
FIG. 8 shows the result of affinity detection of RBD of monoclonal antibody 4L12 binding to S1 protein of SARS-CoV-2 virus;
FIG. 9 shows the result of affinity detection of RBD of monoclonal antibody 12F5 binding to S1 protein of SARS-CoV-2 virus;
FIG. 10 shows the result of affinity detection of RBD of monoclonal antibody 3D13 binding to S1 protein of SARS-CoV-2 virus;
FIG. 11 shows the result of affinity detection of RBD of monoclonal antibody 10C2 binding to S1 protein of SARS-CoV-2 virus;
FIG. 12 shows the result of affinity detection of RBD of monoclonal antibody 16L9 binding to S1 protein of SARS-CoV-2 virus;
FIG. 13 shows the result of affinity detection of RBD of monoclonal antibody 20E21 binding to S1 protein of SARS-CoV-2 virus;
FIG. 14 shows the result of affinity assay of monoclonal antibody 22H22 binding to RBD of S1 protein of SARS-CoV-2 virus.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments shown in the drawings. These embodiments are not intended to limit the present invention, and structural, methodological, or functional changes in accordance with the embodiments are within the scope of the present invention.
Example embodiments will now be described more fully with reference to the accompanying drawings. Example embodiments may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of example embodiments to those skilled in the art.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The examples do not show the specific techniques or conditions, according to the literature in the field of technology or conditions (for example refer to J. SammBruk et al, huang Peitang, translation of molecular cloning laboratory Manual, third edition, scientific Press) or according to the product instructions.
Example 1: screening and detection of antibodies to coronaviruses
The inventor carries out pseudovirus neutralization experimental screening on the plasma of a novel coronavirus pneumonia patient (follow-up visit after two weeks of recovery and discharge) who is treated in a unit (public health clinical center in Shanghai city) from 20 days at 1 month and 26 days at 2020 to 26 months at 2020, finds that the serum of three mild patients has strong neutralizing activity on SARS-CoV-2 pseudovirus, and extracts the peripheral blood of the patients after written consent of an ethical committee of the unit of the inventor and the patient.
1. Sorting of peripheral blood memory B cells
1) Isolation of peripheral blood lymphocytes: peripheral blood of the patient in the convalescent period is collected and mixed with an equal amount of physiological saline, and then lymphocyte is separated from peripheral blood by using lymphocyte separation solution Lymphoprep (Stemcell Technologies, cat. No. 07851), and the operation process is described in the specification of the lymphocyte separation solution.
2) Sorting peripheral blood memory B cells: staining peripheral blood lymphocytes separated in the above step 1) with an antibody mixture for 30min at 4 ℃ in the dark, wherein the antibody mixture is a mixture of anti-CD 19-PE-Cy7 (BD Bioscience), igA-APC (Jackson Immunoresearch), igD-FITC (BD Bioscience) and IgM-PE (Jackson Immunoresearch); after staining, washed with 10ml PBS-BSA buffer and resuspended in 500. Mu.l PBS-BSA; finally, CD19+ IgA-IgD-IgM-memory B cells were sorted out using a FACSAria III cell sorter (Becton Dickinson).
2. Incubation of peripheral blood memory B cells
Resuspending said sorted CD19+ IgA-IgD-IgM-memory B cells in a medium containing 10% FBS and 100U/ml IL-2, 50ng/ml IL-21 and irradiated 3T3-msCD40L feeder cells; memory B cells were seeded at a density of 4 cells/well in 384-well microtiter plates (final volume 50 μ Ι) and incubated for 13 days; growth factors IL-2 and IL-21 stimulate the growth of memory B cells by dividing, and secreting antibodies into the incubated culture. Specific culture methods are described in Huang J et al, nature Protocols 2013,8 (10): 1907-15.
3. Production of SARS-CoV-2 and SARS-CoV pseudovirus
SARS-CoV-2 and SARS-CoV pseudoviruses are non-replication defective retrovirus particles having SARS-CoV-2 and SARS-CoV Spike membrane protein (Spike, S) on their surfaces, respectively, and carrying a luciferase reporter gene, which can mimic the infection process of SARS-CoV-2 and SARS-CoV viruses on host cells (e.g., human hepatoma cell line Huh-7, 293T cell line 293T-ACE2 stably expressing human ACE2 receptor), respectively, and express the luciferase reporter gene in infected cells. Since pseudoviral infection does not produce mature viral particles, the relevant procedures can be safely performed in biosafety secondary laboratories.
SARS-CoV-2 and SARS-CoV pseudoviruses were obtained by co-transfection of 293T cells with respective S protein expression plasmids and HIV Env-deficient backbone plasmids with luciferase reporter (pNL 4-3.Luc. R-E-). SARS-CoV-2 and SARS-CoV S gene sequence are designed based on NCBI GenBank sequence NC-045512 and ABD72979.1, and the gene sequences are synthesized by Nanjing Jinslei company after codon optimization and connected to pcDNA3.1 eukaryotic expression vector to construct SARS-CoV-2 and SARS-CoV S protein expression plasmid. pNL4-3.Luc. R-E-backbone plasmid was derived from NIH AIDS ReagenProgram. All plasmids were amplified by transformation of DH 5. Alpha. Competent cells and purified using a plasmid purification kit from the production of the organism, the purification procedure being referred to the kit instructions.
293T cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco) and plated onto 10cm cell plates prior to transfection. After 24 hours of culture, the backbone plasmid (pNL 4-3.Luc. R-E-) was co-transfected with the plasmid expressing SARS-CoV or SARS-CoV-2 at a ratio of 3:1 into 293T cells using EZ Trans cell transfection reagent (Liji organism), see instructions for use of EZ Trans cell transfection reagent for details of transfection. After 48 hours of transfection, the supernatant containing pseudovirus was collected, centrifuged at 1500 rpm for 10 minutes to remove cell debris, and then frozen in a freezer at-80 ℃ for detection of neutralizing antibodies.
4. Neutralization screening
After 13 days of in vitro culture of peripheral blood memory B cells, 40. Mu.l of culture supernatant per well was collected for detection of SARS-CoV-2 and neutralizing antibodies to SARS-CoV. The detection method comprises the following steps: mu.l of the culture supernatant was mixed with 20. Mu.l of the supernatant of the pseudovirus produced as described above in a 384-well cell culture plate, and after incubation at room temperature for 30 minutes, 50. Mu.l of 5000T-ACE 2 cells per well was added and the culture was continued in a cell culture chamber. After 48 hours, the cells were lysed using a Luciferase Assay kit (Luciferase Assay System, promega Cat. # E1500) and Luciferase activity was assayed per well, using the kit instructions for the specific Assay protocol. The chemiluminescent RLU values per well were measured using a multifunctional microplate reader (Perkin Elmer). And calculating the neutralization inhibition percentage of the culture supernatant to the pseudovirus according to the ratio of the culture supernatant to the virus control RLU value, and screening out the wells with the inhibition percentage of more than 90 percent as virus neutralization positive wells.
5. RT-PCR amplification of heavy and light chain genes
Virus neutralizes positive well B cells and RT-PCR is used to amplify the variable regions of the heavy and light chains of the immunoglobulin genes. Primer design and specific procedures for RT-PCR are described in reference Tiller, t.et al.j.immunol Methods 2018, 329:112-124. the heavy chain and light chain variable region genes of the antibody obtained by amplification are purified and recovered by agarose gel electrophoresis, and are cloned into a PMD19-T vector by utilizing a PMD19-T vector cloning kit (Takara 6013), the specific operation process refers to the kit specification, and a single clone is selected for gene sequencing.
6. Expression and purification of monoclonal antibodies
The antibody heavy chain variable region gene with correct sequencing and the pCMV/R-10E8 heavy chain gene (NIHAIDS RegentProgram Cat 12290) are subjected to enzyme digestion by Age I and Sal I respectively, and then the target fragment obtained after the purification and recovery of the connecting gel is transformed into DH5 alpha competent cells to construct an antibody expression heavy chain plasmid; sequencing a correct antibody Lambda or Kappa light chain variable region gene and a pCMV/R-10E8 Lambda light chain gene expression plasmid (NIHAIDS Reagent Program Cat 12291) or a pCMV/R-N6 Kapp light chain gene expression plasmid (NIH AIDS Reagent Program Cat 12966) respectively, carrying out enzyme digestion on the obtained product by Age I and Xho I or the obtained product I and BsiwI, purifying the recovered target fragment by using a connecting gel, and transforming DH5 alpha competent cells to construct an antibody expression light chain plasmid; the heavy chain and light chain plasmids of the antibody are purified by a plasmid purification kit (Meiji organisms) (see the SDS-PAGE detection result of the expression and purification antibody in figure 1), and are co-transfected into 293T cells for expression by utilizing an EZ Trans cell transfection reagent (Liji organisms) according to the proportion of 1:1. After 72 hours, cell transfection supernatants were collected and antibody IgG in the supernatants was purified using protein-G column (Tiandi and Biotech, inc., changzhou) according to the instructions for the protein-G column. The purified antibody IgG was measured for absorbance at 280nm using Nanodrop 2000 (Thermo Fisher) and the antibody concentration was calculated.
From the above sections 1-6, the inventors obtained several IgG antibodies, 7 of which (named in order: 4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H 22) are disclosed.
The amino acid sequence numbering information for the 7 antibodies is shown in table 1 below:
TABLE 1
Figure SMS_1
Figure SMS_2
The nucleotide sequence numbering information for the 7 antibodies is shown in table 2 below:
TABLE 2
Figure SMS_3
7. Detection of neutralizing Activity of 7 mAbs (4L 12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H 22) of the present application against SARS-CoV-2 coronavirus
Different concentrations of monoclonal antibody were tested on 96-well cell plates to inhibit pseudovirus infection of Huh-7 cells to test the neutralizing ability of the monoclonal antibody against SARS-CoV-2 coronavirus.
The detection method comprises the following steps: 1) Huh-7 cells were seeded in 96-well cell plates at 1X10 per well 4 37 ℃ C., 5% CO 2 Culturing in a cell culture box for 24 hours; 2) Diluting the monoclonal antibody with a cell culture medium to different concentrations, mixing with a pseudovirus diluent containing 100TCID50 in equal volume, and incubating at 37 ℃ for 1 hour; 3) Discarding the cell culture solution, adding 50 μ l of virus-antibody complex into each well, and setting multiple wells, and setting antibody-free group, virus-free group and positive serum control group; 4) After culturing for 12 hours, adding 150 mul of maintenance liquid into each hole, and continuously culturing for 48 hours at 37 ℃; 5) Using a Luciferase Assay kit (Luciferase Assay System, promega Cat. # E1500) to lyse cells and detect Luciferase activity of each well, wherein the specific detection method refers to the kit instructions; detecting the chemiluminescence RLU value of each hole by using a multifunctional microplate reader (Perkin Elmer); 6) The percent of neutralization inhibition of pseudovirus by different concentrations of mabs was calculated from the ratio of mab to virus control RLU values, and the median inhibitory dose IC50 of mab-inhibited virus was calculated using PRISM7 software (GraphPad).
See table 3 below for results.
TABLE 3
IC50(ng/mL)
4L12 4.5
12F5 11.1
3D13 16.1
10C2 30.1
16L9 4.1
20E21 2.3
22H22 60.6
As can be seen from Table 3, 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 can neutralize SARS-CoV-2 virus well at a concentration of the order of ng/ml, and the neutralizing activity is very strong. The stronger the neutralizing activity, the less the antibody dosage and the lower the cost. Therefore, the 7 antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 have better clinical application prospect.
8. Detection of the S1 protein and its RBD protein of SARS-CoV-2 Virus recognized by 7 monoclonal antibodies (4L 12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H 22) of the present application
The 7 monoclonal antibodies obtained by the purification recognize S1 and RBD proteins of SARS-CoV-2 virus, and are detected by an enzyme-linked immunosorbent assay (ELISA) method in turn.
The detection method comprises the following steps: 1. Mu.g/ml of antigenic protein (Cassia, yinqiao) was coated in 96-well ELISA plates overnight at 4 ℃. The plate was washed 5 times with PBS-T solution (0.2% Tween-20) and 300. Mu.l of blocking solution (PBS, 1% FBS,5% mil) was added to each well and blocked for 1 hour at room temperature. The plate was washed 3 times with PBS, and after 5-fold serial dilutions of the mAbs with PBS diluent (PBS, 5% FBS,2% BSA,1% Tween-20), 100. Mu.l of the sample was added to the ELISA plate and incubated at 37 ℃ for 1 hour. The plate was washed 5 times with PBS-T, and 100. Mu.l of horseradish peroxidase-labeled goat anti-human IgG antibody (Jackson Immunoresearch) diluted with PBS dilution 1. The plate was washed 5 times with PBS-T, 150. Mu.l of ABTS chromogenic substrate (Thermo Fisher) was added, and after 30 minutes of development in the dark at room temperature, the absorbance value at 405nm was read by a microplate reader.
Referring to FIG. 1, monoclonal antibody 4L12 recognizes S1 protein of SARS-CoV-2 virus and its RBD, and the detection result of S2 protein;
referring to FIG. 2, monoclonal antibody 12F5 recognizes S1 protein of SARS-CoV-2 virus and its RBD, and the result of detection of S2 protein;
referring to FIG. 3, monoclonal antibody 3D13 recognizes S1 protein of SARS-CoV-2 virus and its RBD, and the detection result of S2 protein;
referring to FIG. 4, monoclonal antibody 10C2 recognizes S1 protein of SARS-CoV-2 virus and its RBD, and the result of detection of S2 protein;
referring to FIG. 5, monoclonal antibody 16L9 recognizes S1 protein of SARS-CoV-2 virus and its RBD, and the result of detection of S2 protein;
referring to FIG. 6, monoclonal antibody 20E21 recognizes S1 protein of SARS-CoV-2 virus and its RBD, and the result of detection of S2 protein;
referring to FIG. 7, monoclonal antibody 22H22 recognizes S1 protein of SARS-CoV-2 virus and its RBD, and the results of the detection of S2 protein.
As can be seen from FIGS. 1-7, mAbs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 were all able to recognize and bind the S1 protein of SARS-CoV-2 virus and its RBD (conserved region); considering that the RBD of S1 protein of coronavirus is a region to which ACE2 receptor binds, it is highly conserved, it can be assumed that mabs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application may have binding and neutralizing abilities to other coronaviruses as well as coronaviruses that may appear in the future, in addition to having strong binding and neutralizing abilities to SARS-CoV-2 virus.
8. Bio-membrane layer interference technology for detecting the binding ability of 7 monoclonal antibodies (4L 12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H 22) of the application to RBD of S1 protein of SARS-CoV-2 virus
In order to detect the interaction between 7 monoclonal antibodies of the present application and the RBD of S1 protein of SARS-CoV-2 virus, the binding kinetics between them was detected by biofilm interference technique, and the detection process was performed on OctetRED96 (Fortebio) instrument.
The detection method comprises the following steps: the AHC probe is soaked in sterile water for 10 minutes in advance for balancing, the detection process is carried out under the reaction condition of 30 ℃, and the detection process can be divided into the following five steps, namely 1) zero setting: immersing the probe in sterile water for 60 seconds to obtain a detection baseline; 2) Capture antibody: immersing the probe into 10 mu g/ml monoclonal antibody solution to act for 200 seconds to capture the antibody; 3) And (4) zeroing again: immersing the probe in a buffer (0.02% in Tween20 in PBS) for 120 seconds to remove unbound antibody; 4) Combining RBD: immersing the probe into RBD protein solution with the initial concentration of 100nM and 3 times of gradient dilution, and acting for 300 seconds to obtain a dynamic curve of the combination of the monoclonal antibody and the RBD; 5) And (3) association and dissociation: the probe was placed in buffer for 300 seconds. The combination of protein causes the change of the thickness of the biological membrane, so that the interference light waves generate relative displacement, and are detected by the spectrometer to form an interference spectrum which is displayed by the real-time displacement (nm) of the interference spectrum. This is used to determine the dynamic curve of binding and dissociation of RBD and the monoclonal antibody of the present application. Data from sample wells were subtracted from data from buffer control wells at the time of data analysis, and nonspecific interference from buffer solutions was subtracted using a 1:1, performing overall curve fitting on the combination of the RBD and the monoclonal antibody under different RBD dilution concentrations to obtain an average combination constant K on Dissociation constant K off And affinity constant K D The value is obtained.
The detection results are shown in FIGS. 8-14, which are the results of affinity detection of RBD of monoclonal antibody 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 binding to S1 protein of SARS-CoV-2 virus; five curves are presented in each figure, representing the kinetic binding dissociation curves for the mab with five different concentrations of RBD.
As can be seen from FIGS. 8-14, 7 mAbs of this application, 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22, all were concentration gradient dependent on binding to RBD of S1 protein of SARS-CoV-2 virus; dissociation is performed after binding, and the dissociated RBD is very little; k for 7 mAbs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 D The values are (1.49 + -0.06) nM, (2.22 + -0.07) nM, (4.17 + -0.15) nM, (3.36 + -0.18) nM, (1.21 + -0.06) nM, (2.3 + -0.07) nM, (5.07 + -0.2) nM, in this order; it is shown that 7 mabs of the present application have very strong affinity to the RBD conserved region of the S1 protein of SARS-CoV-2. It is concluded that the 7 mabs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application demonstrated in section 7 above have strong neutralizing activity against the RBD of the S1 protein of SARS-CoV-2 virus, because the 7 mabs of the present application have very strong affinity for the RBD conserved region of the S1 protein of SARS-CoV-2 virus. The results of Table 1 and FIGS. 1-14 taken together further demonstrate that 7 mAbs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application, in addition to having strong binding and neutralizing capacity for SARS-CoV-2, may also have binding and neutralizing capacity for other coronaviruses, as well as for coronaviruses that may appear in the future.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims (10)

1. An antibody of a coronavirus, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; the method is characterized in that:
the sequence of the HCDR1 is shown as SEQ ID NO.31, the sequence of the HCDR2 is shown as SEQ ID NO.32, and the sequence of the HCDR3 is shown as SEQ ID NO. 33; and the sequence of the LCDR1 is shown as SEQ ID NO.35, the sequence of the LCDR2 is shown as SEQ ID NO.36, and the sequence of the LCDR3 is shown as SEQ ID NO. 37.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein:
the heavy chain variable region has a sequence shown as SEQ ID NO.34 or a sequence which has more than 80% of sequence homology with the sequence shown as SEQ ID NO.34, and the light chain variable region has a sequence shown as SEQ ID NO.38 or a sequence which has more than 80% of sequence homology with the sequence shown as SEQ ID NO. 38.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein:
the antibody is a monoclonal antibody; preferably, the antibody is a fully human monoclonal antibody; preferably, the antibody is any one or combination of more of IgG1, igG2, igG3 or IgG 4; the antigen binding fragment is Fv, fab, F (ab ') 2, fab', dsFv, scFv or sc (Fv) 2.
4. A nucleic acid molecule, wherein: the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.
5. A vector comprising the nucleic acid molecule of claim 4.
6. A host cell comprising the vector of claim 5.
7. A pharmaceutical composition characterized by: the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
8. An assay product, comprising: the assay product comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
9. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: culturing the host cell of claim 6 to produce the antibody or antigen-binding fragment thereof.
10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment or prevention of a disease caused by a coronavirus.
CN202210890641.3A 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses Pending CN115710311A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210890641.3A CN115710311A (en) 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011065506.2A CN112159469B (en) 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses
CN202210890641.3A CN115710311A (en) 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN202011065506.2A Division CN112159469B (en) 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses

Publications (1)

Publication Number Publication Date
CN115710311A true CN115710311A (en) 2023-02-24

Family

ID=73862418

Family Applications (3)

Application Number Title Priority Date Filing Date
CN202011065506.2A Active CN112159469B (en) 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses
CN202210890641.3A Pending CN115710311A (en) 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses
CN202210841040.3A Pending CN116063464A (en) 2020-09-30 2020-09-30 Antibodies or antigen binding fragments thereof to coronaviruses

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202011065506.2A Active CN112159469B (en) 2020-09-30 2020-09-30 Antibodies or antigen-binding fragments thereof to coronaviruses

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN202210841040.3A Pending CN116063464A (en) 2020-09-30 2020-09-30 Antibodies or antigen binding fragments thereof to coronaviruses

Country Status (1)

Country Link
CN (3) CN112159469B (en)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2020340881A1 (en) 2020-04-02 2021-10-21 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
WO2021249547A1 (en) * 2020-06-12 2021-12-16 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Anti-coronavirus antibodies and uses thereof
CN115141272A (en) * 2021-01-31 2022-10-04 中南大学湘雅医院 Novel coronavirus monoclonal antibody XY1 and application thereof
WO2022188829A1 (en) * 2021-03-10 2022-09-15 上海君实生物医药科技股份有限公司 Sars-cov-2 antibody and application thereof
CN113880947B (en) * 2021-07-26 2023-07-04 中国人民解放军军事科学院军事医学研究院 Small molecule antibody, coding gene thereof, preparation method and application thereof, and pharmaceutical composition
CN113563464B (en) * 2021-08-01 2023-02-03 中国疾病预防控制中心性病艾滋病预防控制中心 Humanized high-neutralization-activity anti-novel coronavirus monoclonal antibody and application thereof
CN114106191A (en) * 2021-12-20 2022-03-01 复旦大学 Bispecific antibody for neutralizing coronavirus
WO2023131262A1 (en) * 2022-01-10 2023-07-13 杰库(上海)生物医药研究有限公司 Antigen-binding protein specifically bound to sars-cov-2
CN114751986A (en) * 2022-01-27 2022-07-15 复旦大学 Multispecific antibodies for neutralizing novel coronaviruses
CN114409774B (en) * 2022-02-07 2024-04-02 浙江大学医学院附属第一医院 Broad-spectrum humanized anti-novel coronavirus monoclonal antibody and application thereof
CN114349855B (en) * 2022-03-18 2022-06-28 百斯医学诊断科技(北京)有限公司 Novel coronavirus Delta mutant strain specific antibody and application thereof
CN114957455B (en) * 2022-05-06 2023-05-16 深圳国家感染性疾病临床医学研究中心 Novel coronavirus monoclonal antibody and application thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111592594B (en) * 2020-03-13 2022-05-10 北京大学 Monoclonal antibody for resisting novel coronavirus and application thereof
CN111690058B (en) * 2020-03-30 2021-02-05 三优生物医药(上海)有限公司 Antibodies with neutralizing activity against coronaviruses and uses thereof
AU2020340881A1 (en) * 2020-04-02 2021-10-21 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
CN111647076B (en) * 2020-04-27 2021-02-26 南京医科大学 Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof
CN111592595B (en) * 2020-04-27 2021-02-19 南京医科大学 Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof

Also Published As

Publication number Publication date
CN116063464A (en) 2023-05-05
CN112159469B (en) 2022-08-02
CN112159469A (en) 2021-01-01

Similar Documents

Publication Publication Date Title
WO2022022445A1 (en) Antibody that specifically binds to coronavirus or antigen-binding fragment thereof
CN115710311A (en) Antibodies or antigen-binding fragments thereof to coronaviruses
CN116023478A (en) Neutralizing antibodies or antigen binding fragments thereof for coronaviruses
US7736648B2 (en) Anti-RSV G protein antibodies
KR100337069B1 (en) Anti-HIV Monoclonal Antibodies
CN113354729B (en) Monoclonal antibody for resisting novel coronavirus and application thereof
CA3175110A1 (en) Human monoclonal antibodies to severe acute respiratory syndrome coronavirus 2 (sars-cov-2)
US20170158753A1 (en) Monoclonal antibodies directed against envelope glycoproteins from multiple filovirus species
US20120020980A1 (en) High affinity human antibodies to human cytomegalovirus (cmv) gb protein
CN114075289A (en) anti-CD 73 antibodies and uses thereof
WO2021174594A1 (en) Anti-novel coronavirus monoclonal antibody and application thereof
RU2603262C2 (en) Composition for preventing and/or treating infection, caused by hiv-1
CN114478755B (en) Fully human antibody against novel coronavirus, composition and application thereof
TWI785583B (en) Monoclonal antibody against new coronavirus and use thereof
WO2022150740A1 (en) Cross-reactive antibodies recognizing the coronavirus spike s2 domain
WO2022132887A1 (en) Human monoclonal antibodies targeting the sars-cov-2 spike protein
WO2021235514A1 (en) Human anti-tetanus-toxin antibody
US20240067704A1 (en) Antibody cocktail against sars-cov-2 spike protein
WO2021228135A1 (en) Method for preparing antigen-binding unit
US20230235029A1 (en) Broadly neutralizing binding molecules against marburgviruses
Kim et al. Enhancing HIV-1 neutralization by increasing the local concentration of MPER-directed bnAbs
Kim et al. Enhancing HIV-1 Neutralization by Increasing the Local Concentration of Membrane-Proximal External Region-Directed Broadly Neutralizing Antibodies
CN117430716A (en) Recombinant interferon drug targeting conserved HIV gp41 subunit near-membrane-end outer region and application thereof
WO2022165434A2 (en) Fc-enhanced antibodies for prevention and treatment of ebola virus infection
CN117645673A (en) Fusion protein of IL-21, sCD4 and Fc and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20230726

Address after: 201108 Room 411, Building 14, No. 3333, Huaning Road, Minhang District, Shanghai

Applicant after: Super Extraordinary (Shanghai) Medical Technology Co.,Ltd.

Address before: 201508 no.2901 Caolang Road, Jinshan District, Shanghai

Applicant before: SHANGHAI PUBLIC HEALTH CLINICAL CENTER