CN111592595B - Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof - Google Patents

Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof Download PDF

Info

Publication number
CN111592595B
CN111592595B CN202010342471.6A CN202010342471A CN111592595B CN 111592595 B CN111592595 B CN 111592595B CN 202010342471 A CN202010342471 A CN 202010342471A CN 111592595 B CN111592595 B CN 111592595B
Authority
CN
China
Prior art keywords
cov
antibody
sars
novel coronavirus
heavy chain
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.)
Active
Application number
CN202010342471.6A
Other languages
Chinese (zh)
Other versions
CN111592595A (en
Inventor
高威
刘晓宇
高芳
苟黎明
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.)
Nanjing Medical University
Original Assignee
Nanjing Medical University
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 Nanjing Medical University filed Critical Nanjing Medical University
Priority to CN202010342471.6A priority Critical patent/CN111592595B/en
Publication of CN111592595A publication Critical patent/CN111592595A/en
Application granted granted Critical
Publication of CN111592595B publication Critical patent/CN111592595B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • 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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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 a neutralizing antibody for resisting novel coronavirus SARS-Cov-2 and application thereof. The antibody has at least one of a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR 3. The antibody can be used for preparing a diagnostic reagent or a diagnostic kit, a medicine or a pharmaceutical composition for detecting, preventing and treating COVID-19. The invention uses phage display technology to target SARS-Cov-2-RBD and SARS-Cov-1-RBD to carry out differential antibody screening, obtains a neutralizing antibody for resisting novel coronavirus SARS-Cov-2, can block the combination of SARS-Cov-2-RBD and ACE2 positive cells, has obvious virus neutralization effect on SARS-Cov-2 pseudovirus, and provides an effective alternative antibody medicament for the prevention and treatment of COVID-19.

Description

Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof
Technical Field
The invention relates to a neutralizing antibody for resisting novel coronavirus SARS-Cov-2 and application thereof, belonging to the technical field of biological medicine.
Background
2019 the novel Coronavirus pneumonia (COVID-19) is caused by infection of novel Coronavirus (SARS-CoV-2, Severe Acute Respiratory Syndrome corona 2), and currently, more than 200 million people are infected globally, and the fatality rate is more than 6% (C: (S))https://covid19.who.int/) It is a serious infectious disease which endangers human health. SARS-CoV-2 is a positive strand single strand RNA virus belonging to the beta type coronavirus[1]The nucleic acid homology with SARS-Cov-1 is 79.5%[2]Its natural host is not known. Both SARS-CoV-2 and SARS-Cov-1 infect host cells by binding angiotensin converting enzyme 2(ACE2) on the host cell membrane[3]。
ACE2 belongs to the angiotensin converting enzyme, and the substrates are angiotensin I and II. Under physiological conditions, ACE2 counteracts the vasoconstrictive effects caused by ACE1 by hydrolyzing the substrate. SARS-Cov-2 binds to ACE2 through the Receptor Binding Domain (RBD) of the Spike glycoprotein S1 subunit on the viral coat, and then invades host cells[4]。
The protein structure of the SARS-Cov-2-RBD and ACE2 complex has been resolved, and compared to SARS-Cov-1, both RBDs bind to ACE2 at the same angle, and interact with 20 amino acid residues of ACE2 through 17 amino acid residues[3,5]. The subtle differences in the interface of contact of the two RBDs with ACE2 resulted in differences in the affinity of SARS-Cov-1 and SARS-Cov-2 for ACE2, which may be beneficial for subsequent studies.
Currently, no specific vaccine and neutralizing antibody for SARS-CoV-2 virus is available for clinical treatment. Therefore, the screening to obtain human monoclonal antibodies with neutralizing effect is an urgent need for preventing and treating COVID-19.
Disclosure of Invention
The main purposes of the invention are: overcomes the problems in the prior art, provides a neutralizing antibody for resisting novel coronavirus SARS-Cov-2, and has high-efficiency antiviral ability for the novel coronavirus SARS-Cov-2. Also, applications of the antibody are provided.
The technical scheme for solving the technical problems of the invention is as follows:
a neutralizing antibody against the novel coronavirus SARS-Cov-2, said antibody comprising a heavy chain and a light chain, characterized in that said antibody has at least one of the following technical characteristics:
i. the heavy chain includes a heavy chain CDR1 having the amino acid sequence: GFTFSSYA;
ii. The heavy chain includes a heavy chain CDR2 having the amino acid sequence: SIASSGYYTD, respectively;
iii, the heavy chain comprises heavy chain CDR3 with the amino acid sequence: KDADS;
iv, the light chain comprises a light chain CDR1 having the amino acid sequence: ISSYL;
v, the light chain includes a light chain CDR2 having the amino acid sequence: AASYL;
vi, the light chain comprises a light chain CDR3 having the amino acid sequence: AYSAPS.
Preferably, the antibody has at least one of the following technical features:
i. the heavy chain includes a heavy chain CDR1 having the amino acid sequence: GFTFSSYA; the heavy chain includes a heavy chain CDR2 having the amino acid sequence: SIASSGYYTD, respectively; the heavy chain further includes a heavy chain CDR3 having the amino acid sequence: KDADS;
ii. The light chain includes a light chain CDR1 having the amino acid sequence: ISSYL; the light chain includes a light chain CDR2 having the amino acid sequence: AASYL; the light chain also includes a light chain CDR3 having the amino acid sequence: AYSAPS.
Preferably, the amino acid sequence of the antibody is shown as SEQ ID NO. 2.
Preferably, the antibody is a VH single domain antibody, a Fab fragment, a Fab 'fragment, a F (ab)'2A fragment, a single chain variable fragment scFv, a disulfide stabilized variable region fragment dsFv, an IgG molecule, or a bispecific antibody.
Preferably, the antibody has a label including a fluorescent label, an enzymatic label, and a radioactive label.
The present invention also provides:
nucleic acid encoding a neutralizing antibody against the novel coronavirus SARS-Cov-2 as described hereinbefore.
Preferably, the sequence of the nucleic acid is shown as SEQ ID NO. 1.
The present invention also provides:
use of the neutralizing antibody against the novel coronavirus SARS-Cov-2 as described hereinbefore for the preparation of a diagnostic agent or diagnostic kit, a medicament or a pharmaceutical composition.
Use of the nucleic acid as hereinbefore described for the preparation of a neutralizing antibody, medicament or pharmaceutical composition against the novel coronavirus SARS-Cov-2.
Wherein the medicine or the medicine composition has a neutralizing antiviral effect against a novel coronavirus SARS-Cov-2.
The invention utilizes the phage display technology, and differential antibody screening is carried out on targeting SARS-Cov-2-RBD and SARS-Cov-1-RBD, so as to obtain the neutralizing antibody for resisting novel coronavirus SARS-Cov-2, the antibody can block the combination of SARS-Cov-2-RBD and ACE2 positive cells, has obvious virus neutralization effect on SARS-Cov-2 pseudovirus, provides an effective alternative antibody medicament for preventing and treating COVID-19, and has potential clinical application prospect.
Drawings
FIG. 1 is a graph showing the binding of enriched phages to the antigen protein by ELISA in example 1 of the present invention.
FIG. 2 is a diagram of specific binding assay (ELISA) of the phage to SARS-Cov-2-RBD protein in example 2 of the present invention.
FIGS. 3 and 4 are schematic diagrams of expression vectors of example 3 of the present invention, respectively.
FIG. 5 is a diagram showing the result of SDS-PAGE in example 3 of the present invention.
FIG. 6 is a graph showing the results of affinity analysis in example 4 of the present invention.
FIG. 7 is a graph showing the analysis of the antibody blocking effect in example 5 of the present invention.
FIG. 8 is a graph showing the evaluation of neutralizing effect of the antibody against pseudovirus according to example 6 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the reagents and materials used are commercially available products unless otherwise specified.
Example 1 screening of fully human antibodies targeting SARS-Cov-2-RBD
By adopting phage display technology, SARS-Cov-2-RBD-his protein is used as positive antigen, SARS-Cov-1-RBD-hFc is used as negative antigen, and the antigen is displayed on Tomlinson I&J phage library (Genservice Ltd., Cambridge, UK, library size 1.47x108) Performing differential screening.
Coating the immune plate with 50 μ g/ml SARS-Cov-2-RBD his antigen and SARS-Cov-1-RBD hFc antigen at 4 deg.C overnight; blocking the immune plate for 1 hour at room temperature by using PBS (phosphate buffer solution) containing 5% of skimmed milk powder and 0.1% of Tween-20; phage library with 1012Mixing pfu with 10% skimmed milk powder PBS solution 1:1, incubating at room temperature for 2 hours, adding into a sealed SARS-Cov-1-RBD hFc antigen immune plate (100 μ l/hole), incubating at room temperature for 1 hour, and performing negative antigen pre-adsorption; pre-adsorbed supernatant transferred to addition sealClosed SARS-Cov-2-RBD his antigen immune plates (100. mu.l/well) were incubated for 1 hour at room temperature. The immune plate was washed 20 times with 0.1% Tween-20 in PBS; 100 μ l of 100mM Triethylamine was eluted at room temperature for 30 min; the eluted phage infected TG1 cells in log phase growth, which were expanded and recovered for the next round of panning. Positive phage enrichment was analyzed by ELISA after panning.
The specific process of ELISA detection is as follows: the immune plates were coated overnight at 4 ℃ with 5. mu.g/ml of SARS-Cov-2-RBD his antigen and negative control protein GPC5 his, respectively; blocking the immune plate for 1 hour at room temperature by using PBS (phosphate buffer solution) containing 3 percent of skimmed milk powder and 0.1 percent of Tween-20; the enriched phage were amplified and recovered in each round, and the ratio was 1:1 proportion and 6% skim milk powder PBS for 2 hours at room temperature, adding into a sealed immune plate (100 mul/hole), and incubating for 1 hour at room temperature; the immune plate was washed 5 times with 0.1% Tween-20 in PBS; HRP/Anti-M13 Monoclonal conjugate was treated at a rate of 1: mixing the mixture with PBS solution containing 5% skimmed milk powder and 0.05% Tween-20 at a ratio of 4000, adding the mixture into a washed immune plate (50 mu l/hole), and incubating the mixture at room temperature for 1 hour; the immune plate was washed 5 times with 0.05% Tween-20 in PBS; adding TMB color developing solution into an immune plate (100 μ l/well), developing at room temperature for 3 minutes, and adding 0.5M sulfuric acid to stop developing (100 μ l/well); detecting the light absorption value by an enzyme linked immunosorbent assay detector at the wavelength of 450nm, and analyzing the affinity of the phage after each round of amplification.
As a result, as shown in FIG. 1, the affinity of the enriched phage population for SARS-Cov-2-RBD-his antigen was significantly increased after four rounds of enrichment.
Single clones were randomly picked from the fourth round of enriched phage population and tested for their binding properties to SARS-Cov-2-RBD-his. As a result, 1 antibody sequence (4A3) was found to be significantly enriched.
Through sequencing identification, the DNA sequence of the antibody is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:2, respectively.
SEQ ID NO：1：
gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctattgcttcttctggttattatacagattacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaagatgctgattcttttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcggtggcgggtcggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcctatttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacaggcttattctgctccttctacgttcggccaagggaccaaggtggaaatcaaa。
SEQ ID NO：2：
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIASSGYYTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDADSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSAPSTFGQGTKVEIK。
In the amino acid sequence, the CDR regions are contained as follows: amino acid residues 26-33 (i.e., GFTFSSYA) are the heavy chain CDR1, amino acid residues 50-59 (i.e., SIASSGYYTD) are the heavy chain CDR2, and amino acid residues 98-102 (i.e., KDADS) are the heavy chain CDR 3; amino acid residues 160-164 (ISSYL) is the light chain CDR1, amino acid residues 181-185 (AASYL) is the light chain CDR2, and amino acid residues 222-227 (AYSAPS) is the light chain CDR 3.
Furthermore, the antibody format may be selected from VH single domain antibody, Fab fragment, Fab 'fragment, F (ab)'2A fragment, a single chain variable fragment (scFv), a disulfide stabilized variable region fragment (dsFv), an IgG molecule, or a bispecific antibody. The antibody may optionally have a label including fluorescent, enzymatic, and radioactive labels.
Example 2 antigen specificity analysis of antibodies
This example used ELISA to detect binding of the 4A3 phage of example 1 to SARS-Cov-2-RBD protein.
The specific process is as follows: the immune plates were coated overnight at 4 ℃ with 5. mu.g/ml of SARS-Cov-1-RBD-hFc and SARS-Cov-2-RBD-hFc, respectively; blocking the immune plate for 1 hour at room temperature by using PBS (phosphate buffer solution) containing 3 percent of skimmed milk powder and 0.05 percent of Tween-20; phage of 4A3 were added to blocked immunoplates (50. mu.l/well) and incubated for 1 hour at room temperature; wash the plate 3 times with 0.05% Tween-20 in PBS (340 ul/well); HRP/Anti-M13 Monoclonal conjugate was treated at a rate of 1: mixing the mixture with PBS solution containing 5% skimmed milk powder and 0.05% Tween-20 at a ratio of 4000, adding the mixture into a washed immune plate (50 mu l/hole), and incubating the mixture at room temperature for 1 hour; the immune plate was washed 5 times with 0.05% Tween-20 in PBS; adding TMB color developing solution into an immune plate (100 μ l/well), developing at room temperature for 3 minutes, and adding 0.5M sulfuric acid to stop developing (100 μ l/well); detecting the light absorption value by an enzyme linked immunosorbent assay detector at the wavelength of 450 nm.
As shown in FIG. 2, the results showed that the 4A3 antibody specifically recognized SARS-Cov-2-RBD-hFc protein, but not SARS-Cov-1-RBD-hFc protein.
Example 3 expression and purification of antibodies
The heavy chain variable region sequence and the light chain variable region sequence of 4A3 scFv sequence were inserted into pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk vectors (Invivogen, San Diego, Calif.), respectively, to construct an adult IgG1 expression plasmid, as shown in FIGS. 3 and 4. The above plasmid was co-transfected in 293T cells, and the supernatant was collected and purified using protein A-Agarose column separation, and the purity of the antibody was checked by SDS-PAGE, as shown in FIG. 5.
The specific process is as follows: the heavy chain variable region sequence and the light chain variable region sequence of 4A3 scFv sequence were inserted into pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk (Invivogen, San Diego, Calif.), respectively, to construct an adult IgG expression plasmid. 5 million HEK293T cells were seeded in cell culture dishes in DMEM medium supplemented with 10% fetal calf serum, 100U/ml penicillin, 0.1mg/ml streptomycin and cultured in a 5% CO2, 37 ℃ incubator. When the cell density reached 60-80%, 5. mu.g of pFUSE-4A3 VH and 5. mu.g of pFUSE-4A3 VL plasmid were co-transfected into HEK293T cells using PEI; the supernatant was collected.
Centrifuging the collected supernatant at 3500rpm and 4 deg.C for 20 min, and vacuum filtering with 0.45 μm microporous filter membrane to further remove debris; the supernatant was purified and separated from 4A3IgG recombinant Protein by passing through Protein A-Agarose (GE Healthcare, Piscataway, NJ) affinity column. Protein concentration was determined by BCA method and 3 μ g of 4A3IgG recombinant protein was subjected to polyacrylamide gel electrophoresis to give bands of 4A3IgG recombinant protein.
Example 4 antibody affinity assay
The affinity of 4A3 to SARS-Cov-2-RBD his protein was determined using SPR assay.
The 4A3 antibody affinity analysis was performed by Nanjing Kinsley and Surface Plasmon Resonance (SPR) analysis was performed using Biacore T200, GR18010468(GE Healthcare). The method comprises the following specific steps:
first, antibody 4A3 was immobilized on a Series S Sensor Chip Protein A Chip (GE Healthcare), and then SARS-Cov-2-RBD Protein with a concentration gradient of 1.25nM to 40nM was injected separately into the Chip, and the analysis was performed at a constant temperature of 25 ℃ using HBS-EP +:10mM HEPES,150mM NaCl,3mM EDTA, 0.05% P20, pH 7.4(Lot. No.30393) (GE Healthcare); the flow rate was 10. mu.l/min. The binding curve is shown in fig. 6. The 5 curves in the figure show antibody concentrations of 40, 20, 10, 5, 2.5, 1.25nM from top to bottom, respectively, with curves at different concentrations constituting the kinetic curves shown in the figure. The calculation of binding kinetic constants was performed using Biacore T200 Evaluation software version 3.1 software. The result shows that the affinity detection result of the 4A3 antibody and SARS-Cov-2-RBD his protein is that ka is 1.91E +06, KD is 1.90E-03, and KD is 3.62 nM.
Example 5 blocking of binding of antibodies to SARS-Cov-2-RBD and ACE2-CHO cells
After pre-incubation of 4A3IgG, control IgG, M396 antibody (this is a SARS-Cov-1 neutralizing antibody) with SARS-Cov-2-RBD-hFc protein for 1 hour at room temperature in advance, 10 was added6ACE2-CHO cells were added to the mixed system and incubated on ice for 1 hour. PBS washed cells, 1: goat anti-human PE was added to 200 cells and incubated on ice for 1 hour. PBS washed cells, BD FACS Calibur machine detects the binding of SARS-Cov-2-RBD-hFc and ACE2-CHO cells.
As shown in FIG. 7, the 4A3 antibody significantly inhibited the binding of SARS-Cov-2-RBD to ACE2-CHO cells after incubation with SARS-Cov-2-RBD protein, while the SARS-Cov-1-RBD neutralizing antibody M396 failed to block the binding of SARS-Cov-2-RBD to ACE2-CHO cells
Example 6 virus neutralization assay
At a slow speedThe VSV-G protein gene is replaced by SARS-Cov-2 spike gene in a virus packaging system, 293T cells are cotransfected with pLVX-EGFP-Luciferase reporter gene (namely, transfection is carried out by pseudovirus), and virus supernatant is collected for 48 hours, wherein the weight ratio of 1:1 diluting for later use. ACE2-CHO cells were treated as 104Perwell was inoculated in 96-well plates overnight. The antibody diluted in a gradient was incubated with the virus supernatant for 1 hour at 37 ℃ in advance, and then added to an ACE2-CHO cell culture plate. After 48 hours, the Luciferase activity was assayed.
As shown in FIG. 8, the results indicate that the 4A3 antibody can significantly inhibit infection of ACE2-CHO cells by pseudoviruses and IC50It was 0.28. mu.g/ml.
Each antibody of the invention can specifically combine/identify SARS-Cov-2-RBD his antigen, has better affinity to SARS-Cov-2-RBD his protein, can effectively inhibit SARS-Cov-2 invading cells, and has important application value as a medicament for preventing and treating COVID-19.
In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.
Reference to the literature
[1].Zhu,N.,et al.,A Novel Coronavirus from Patients with Pneumonia in China,2019.N Engl J Med,2020.382(8):727-733.
[2].Zhou,P.,et al.,A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature,2020.579(7798):270-273.
[3].Lan,J.,et al.,Structure of the SARS-CoV-2spike receptor-binding domain bound to the ACE2 receptor.Nature,2020.
[4].Hoffmann,M.,et al.,SARS-CoV-2Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.Cell,2020.181(2):271-280e278.
[5].Li,F.,et al.,Structure of SARS coronavirus spike receptor-binding domain complexed with receptor.Science,2005.309(5742):1864-1868.
Sequence listing
<110> Nanjing university of medical science
<120> neutralizing antibody against novel coronavirus SARS-Cov-2 and use thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 714
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatct attgcttctt ctggttatta tacagattac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatgct 300
gattcttttg actactgggg ccagggaacc ctggtcaccg tctcgagcgg tggaggcggt 360
tcaggcggag gtggcagcgg cggtggcggg tcggacatcc agatgaccca gtctccatcc 420
tccctgtctg catctgtagg agacagagtc accatcactt gccgggcaag tcagagcatt 480
agcagctatt taaattggta tcagcagaaa ccagggaaag cccctaagct cctgatctat 540
gctgcatcct atttgcaaag tggggtccca tcaaggttca gtggcagtgg atctgggaca 600
gatttcactc tcaccatcag cagtctgcaa cctgaagatt ttgcaactta ctactgtcaa 660
caggcttatt ctgctccttc tacgttcggc caagggacca aggtggaaat caaa 714
<210> 2
<211> 238
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Glu Val Gln Leu Leu 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 Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ala Ser Ser Gly Tyr Tyr Thr Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Ala Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
130 135 140
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
145 150 155 160
Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
165 170 175
Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg
180 185 190
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
195 200 205
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser
210 215 220
Ala Pro Ser Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
225 230 235

Claims (9)

1. A neutralizing antibody against the novel coronavirus SARS-Cov-2, said antibody comprising a heavy chain and a light chain, characterized in that said antibody has all the following technical characteristics:
i. the heavy chain includes a heavy chain CDR1 having the amino acid sequence: GFTFSSYA; the heavy chain includes a heavy chain CDR2 having the amino acid sequence: SIASSGYYTD, respectively; the heavy chain further includes a heavy chain CDR3 having the amino acid sequence: KDADS;
ii. The light chain includes a light chain CDR1 having the amino acid sequence: ISSYL; the light chain includes a light chain CDR2 having the amino acid sequence: AASYL; the light chain also includes a light chain CDR3 having the amino acid sequence: AYSAPS.
2. The neutralizing antibody of claim 1, wherein the amino acid sequence of said antibody is set forth in SEQ ID NO 2.
3. The neutralizing antibody according to claim 1, wherein said antibody is a Fab fragment, a Fab 'fragment, F (ab)'2A fragment, a single chain variable fragment scFv, a disulfide stabilized variable region fragment dsFv, an IgG molecule, or a bispecific antibody.
4. The neutralizing antibody of claim 1, wherein said antibody has a label comprising a fluorescent label, an enzymatic label, and a radioactive label.
5. Nucleic acid encoding a neutralizing antibody against the novel coronavirus SARS-Cov-2 according to any one of claims 1 to 4.
6. The nucleic acid of claim 5, wherein the sequence of the nucleic acid is as shown in SEQ ID NO. 1.
7. Use of the neutralizing antibody against the novel coronavirus SARS-Cov-2 according to any one of claims 1 to 4 for the preparation of a diagnostic reagent or diagnostic kit against the novel coronavirus SARS-Cov-2, a medicament or pharmaceutical composition against the novel coronavirus SARS-Cov-2.
8. Use of the nucleic acid according to claim 5 or 6 for the preparation of neutralizing antibodies against the novel coronavirus SARS-Cov-2, medicaments or pharmaceutical compositions against the novel coronavirus SARS-Cov-2.
9. Use according to claim 7 or 8, characterized in that the medicament or pharmaceutical composition has a neutralizing antiviral effect against the novel coronavirus SARS-Cov-2.
CN202010342471.6A 2020-04-27 2020-04-27 Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof Active CN111592595B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010342471.6A CN111592595B (en) 2020-04-27 2020-04-27 Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010342471.6A CN111592595B (en) 2020-04-27 2020-04-27 Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof

Publications (2)

Publication Number Publication Date
CN111592595A CN111592595A (en) 2020-08-28
CN111592595B true CN111592595B (en) 2021-02-19

Family

ID=72190736

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010342471.6A Active CN111592595B (en) 2020-04-27 2020-04-27 Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof

Country Status (1)

Country Link
CN (1) CN111592595B (en)

Families Citing this family (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2023517234A (en) * 2020-03-09 2023-04-24 アブセレラ バイオロジクス インコーポレイテッド Anti-coronavirus antibodies and methods of use
CR20220552A (en) 2020-04-02 2023-01-17 Regeneron Pharma Anti-sars-cov-2-spike glycoprotein antibodies and antigen-binding fragments
JP7038367B2 (en) * 2020-05-01 2022-03-18 花王株式会社 Anti-SARS-CoV-2 antibody
US20220056111A1 (en) * 2020-08-11 2022-02-24 Yurogen Biosystems Llc MONOCLONAL ANTIBODY AGAINST SPIKE S1 PROTEIN OF SARS-CoV-2 AND USE THEREOFOF
CN112010964A (en) * 2020-09-02 2020-12-01 安第斯抗体生物技术衡水有限公司 Novel coronavirus alpaca antibody and preparation method and application thereof
WO2022048624A1 (en) * 2020-09-03 2022-03-10 Nanjing GenScript Biotech Co., Ltd. Antibodies against receptor binding domain of sars-cov-2 spike protein and uses thereof
CN111995674B (en) * 2020-09-03 2022-02-11 中国人民解放军军事科学院军事医学研究院 anti-COVID-19 virus neutralizing antibody mhC3, humanized antibody and application thereof
AU2021209282B2 (en) 2020-09-04 2022-06-02 Newsoara Biopharma Co., Ltd. Anti-Sars-Cov-2 Neutralizing Antibodies
CN114181301B (en) * 2020-09-14 2023-04-28 复旦大学 ADE-effect-free neutralizing antibodies against SARS-CoV-2
EP4211158A1 (en) * 2020-09-14 2023-07-19 Nanjing Vazyme Biotech Co., Ltd. Neutralizing antibodies against sars-cov-2
WO2022056712A1 (en) * 2020-09-16 2022-03-24 The Third People's Hospital Of Shenzhen Anti-sars-cov-2 antibodies and uses thereof
CN113769121B (en) * 2020-09-18 2022-10-28 中国原子能科学研究院 Radiotherapeutic medicine for diseases caused by coronavirus or influenza virus and preparation method thereof
GB202014851D0 (en) * 2020-09-21 2020-11-04 Iontas Ltd SARS-COV-2 antibodies
CN112175071A (en) * 2020-09-22 2021-01-05 通用生物系统（安徽）有限公司 Preparation method of novel coronavirus spike protein monoclonal antibody
CN112062839A (en) 2020-09-22 2020-12-11 石河子大学 Nano antibody based on novel coronavirus S protein S1 subunit and application thereof
WO2022062803A1 (en) * 2020-09-24 2022-03-31 The University Of Hong Kong Neutralizing antibodies against covid-19 and methods of use thereof
CN116249711A (en) * 2020-09-25 2023-06-09 庆应义塾 SARS-CoV-2 neutralizing antibodies or fragments thereof
CN112175073B (en) * 2020-09-30 2022-08-02 上海市公共卫生临床中心 Neutralizing antibodies or antigen-binding fragments thereof to coronaviruses
CN115710311A (en) * 2020-09-30 2023-02-24 上海市公共卫生临床中心 Antibodies or antigen-binding fragments thereof to coronaviruses
CN112409488B (en) * 2020-10-23 2022-07-01 中国科学院上海药物研究所 Monoclonal antibody aiming at various coronaviruses and application
CN114426574A (en) * 2020-10-29 2022-05-03 中国科学院武汉病毒研究所 Novel coronavirus neutralizing human monoclonal antibody and application thereof
WO2022095968A1 (en) * 2020-11-06 2022-05-12 Shanghaitech University ANTIBODIES AGAINST SARS-CoV-2 SPIKE PROTEIN
WO2022095045A1 (en) * 2020-11-09 2022-05-12 上海济煜医药科技有限公司 Sars-cov-2 antibody and application thereof
WO2022105772A1 (en) * 2020-11-18 2022-05-27 三优生物医药(上海)有限公司 Bispecific antibody having neutralizing activity against coronavirus, and use thereof
CN112409479B (en) * 2020-11-23 2022-04-26 中国疾病预防控制中心病毒病预防控制所 Humanized anti-neocoronavirus neutralizing antibody nCoV-121 and application thereof
CN112430265B (en) * 2020-11-23 2022-04-12 中国疾病预防控制中心病毒病预防控制所 Humanized anti-neocoronavirus neutralizing antibody nCoV-61 and application thereof
WO2022112392A1 (en) * 2020-11-26 2022-06-02 Memo Therapeutics Ag Anti-sars-cov-2 antibody molecules
CN115087667B (en) * 2020-12-14 2023-06-23 杰科（天津）生物医药有限公司 Antigen binding proteins that specifically bind SARS-CoV-2
WO2022154267A2 (en) * 2021-01-13 2022-07-21 재단법인대구경북과학기술원 Antiviral antibody against sars-cov2 binding to ace2, and use thereof
AU2021209287B1 (en) * 2021-01-19 2022-03-24 Newsoara Biopharma Co., Ltd. Expression Vector for Anti-Sars-Cov-2 Neutralizing Antibodies
WO2022156670A1 (en) * 2021-01-19 2022-07-28 Hifibio (Hk) Limited Multispecific antibodies against sars-cov-2 and methods of use thereof
CN112390879B (en) * 2021-01-21 2021-04-02 上海科技大学 Antibody targeting SARS-CoV-2 and its preparation method and use
CN114805556A (en) * 2021-01-29 2022-07-29 中国科学院上海巴斯德研究所 Neutralizing antibody for SARS-CoV-2 virus
CN115141271A (en) * 2021-01-31 2022-10-04 中南大学湘雅医院 Novel coronavirus monoclonal antibody XY7 and application thereof
CN112834746B (en) * 2021-02-19 2022-12-16 南京大学深圳研究院 Method for evaluating antiviral adhesion and application
CN112552399B (en) * 2021-02-24 2021-07-20 恒翼生物医药科技(上海)有限公司 anti-SARS-COV-2 neutralizing antibody
CN113009153B (en) * 2021-02-25 2023-12-15 山东莱博生物科技有限公司 New coronavirus neutralizing antibody detection kit based on magnetic particle chemiluminescence and application thereof
CN115073593B (en) * 2021-03-10 2024-02-23 上海君实生物医药科技股份有限公司 Novel coronavirus antibodies and uses thereof
CN112980885B (en) * 2021-03-18 2022-04-15 恒翼生物医药科技(上海)有限公司 Expression vector of anti-SARS-COV-2 neutralizing antibody
CN113045647B (en) * 2021-03-22 2022-04-26 南京传奇生物科技有限公司 Neutralizing antibody of novel coronavirus SARS-CoV-2 and application thereof
WO2022226539A1 (en) * 2021-04-23 2022-10-27 Immunome, Inc. Methods of administering antibodies against sars-cov-2 spike protein
CN113512114B (en) * 2021-08-09 2022-08-02 北京大学 Antibodies against SARS-CoV-2 mutant strains and uses thereof
CN113433328B (en) * 2021-08-30 2021-11-02 南京立顶医疗科技有限公司 Virus neutralizing antibody and non-neutralizing antibody combined detection method, detection card and application
CN113980135B (en) * 2021-12-27 2022-04-19 三优生物医药(上海)有限公司 Anti-drug antibody combined with coronavirus bispecific antibody, preparation method and application thereof
CN114031685B (en) * 2022-01-10 2022-03-25 中国人民解放军军事科学院军事医学研究院 Fully human anti-new coronavirus neutralizing antibody ZW2G10 and application
CN115043933B (en) * 2022-03-31 2023-08-08 深圳市人民医院 Nanometer antibody targeting novel coronavirus and preparation method and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1300172C (en) * 2003-11-25 2007-02-14 中国人民解放军军事医学科学院微生物流行病研究所 Immunoglobulin antibody against SARS-CoV and its preparing method
US20110159001A1 (en) * 2008-01-17 2011-06-30 Institute For Research In Biomedicine CROSS-NEUTRALIZING HUMAN MONOCLONAL ANTIBODIES TO SARS-CoV AND METHODS OF USE THEREOF
CN109666070B (en) * 2017-10-13 2021-02-19 清华大学 Monoclonal antibody MERS-4V2 and coding gene and application thereof

Also Published As

Publication number Publication date
CN111592595A (en) 2020-08-28

Similar Documents

Publication Publication Date Title
CN111592595B (en) Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof
CN111647076B (en) Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof
CN113444170B (en) Monoclonal antibody F10 against novel coronavirus SARS-CoV-2
CN113264998B (en) Single-chain antibody of S1 protein on surface of anti-new coronavirus SARS-CoV-2 and application thereof
CA2790949C (en) Human monoclonal antibodies derived from human b cells and having neutralizing activity against influenza a viruses
KR101732056B1 (en) Neutralizing anti-influenza a virus antibodies and uses thereof
CN111690058A (en) Antibodies with neutralizing activity against coronaviruses and uses thereof
KR102184151B1 (en) Fully humanized antibody of anti-respiratory syncytial virus
US9856312B2 (en) Binding molecule having influenza A virus-neutralizing activity produced from human B cell
CN110016079B (en) Neutralizing antibody for resisting respiratory syncytial virus and application thereof
CN113045647B (en) Neutralizing antibody of novel coronavirus SARS-CoV-2 and application thereof
CN113150129A (en) Single-chain antibody of S2 protein on surface of anti-new coronavirus SARS-CoV-2 and application thereof
CN112062838B (en) Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof
CN114763380B (en) Construction body of nano antibody S43 and application thereof
CN114805560B (en) Construction body of nano antibody R14 and application thereof
CN105985433B (en) Fully human monoclonal antibody aiming at H7N9 subtype avian influenza virus and application
Duty et al. Discovery and intranasal administration of a SARS-CoV-2 broadly acting neutralizing antibody with activity against multiple Omicron subvariants
Duty et al. Discovery of a SARS-CoV-2 broadly-acting neutralizing antibody with activity against Omicron and Omicron+ R346K variants
Chen et al. Potent Monoclonal antibodies neutralize Omicron Sublineages and other SARS-Cov-2 variants
CN114805579B (en) Anti-human ACE2 protein monoclonal antibody, nucleic acid molecule and application
Cao et al. Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies
CN115087667B (en) Antigen binding proteins that specifically bind SARS-CoV-2
Guo et al. A SARS-CoV-2 neutralizing antibody with exceptional spike binding coverage and optimized therapeutic potentials
US9834594B2 (en) Human monoclonal antibodies derived from human B cells and having neutralizing activity against influenza A viruses
WO2023173577A1 (en) Coronavirus antibody and use 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
GR01 Patent grant
GR01 Patent grant