MONOCLONAL ANTIBODY AGAINST NOVEL CORONAVIRUS AND APPLICATION THEREOF
20230116587 · 2023-04-13
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
G01N2469/10
PHYSICS
G01N33/577
PHYSICS
C12N15/63
CHEMISTRY; METALLURGY
International classification
C12N15/63
CHEMISTRY; METALLURGY
Abstract
Provided are a monoclonal antibody against a novel coronavirus and a composition that comprises said antibody. The antibody can be used to diagnose, prevent and/or treat novel coronavirus infections and/or diseases caused by an infection.
Claims
1. A monoclonal antibody or an antigen-binding fragment thereof, comprising complementarity determining regions 1-3 (CDRs 1-3) of a heavy chain variable region (VH) having amino acid sequences as shown in SEQ ID NOs: 1-3, respectively; and/or, complementarity determining regions 1-3 (CDRs 1-3) of a light chain variable region (VL) having amino acid sequences as shown in SEQ ID NOs: 4-6, respectively; preferably, the monoclonal antibody comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 7, and/or a light chain variable region (VL) as shown in SEQ ID NO: 8; preferably, the monoclonal antibody comprises: VH CDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 1-3, respectively, and VL CDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 4-6, respectively; preferably, the monoclonal antibody comprises: VH as shown in SEQ ID NO: 7 and VL as shown in SEQ ID NO: 8; preferably, the monoclonal antibody or the antigen-binding fragment thereof is selected from a Fab, Fab′, F(ab′).sub.2, Fd, Fv, dAb, a complementarity determining region fragment, a single chain antibody (e.g., scFv), a human antibody, a chimeric antibody or a bispecific or multispecific antibody; preferably, the monoclonal antibody further comprises a heavy chain constant region; preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9; preferably, the monoclonal antibody further comprises a light chain constant region; and preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 10.
2. An isolated nucleic acid molecule, comprising a nucleic acid sequence that can encode a heavy chain variable region of an antibody, wherein the heavy chain variable region of the antibody comprises: VH CDRs 1-3 having amino acid sequences of SEQ ID NOs:1-3, respectively; for example, the isolated nucleic acid molecule comprises nucleotide sequences as shown in SEQ ID NOs:11-13; for example, the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO: 7; and for example, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 17.
3. An isolated nucleic acid molecule, comprising a nucleic acid sequence that can encode a light chain variable region of an antibody, wherein the light chain variable region of the antibody comprises: VL CDRs 1-3 having amino acid sequences of SEQ ID NOs: 4-6, respectively; for example, the isolated nucleic acid molecule comprises nucleotide sequences as shown in SEQ ID NOs: 14-16; for example, the light chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO: 8; and for example, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 18.
4. An isolated nucleic acid molecule, which encodes the monoclonal antibody or the antigen-binding fragment thereof of claim 1.
5. A vector, comprising the isolated nucleic acid molecule of any one of claims 2-4.
6. A host cell, comprising the isolated nucleic acid molecule of any one of claims 2-4 or the vector of claim 5.
7. A method for preparing the monoclonal antibody or the antigen-binding fragment thereof of claim 1, comprising culturing the host cell of claim 6 under a suitable condition, and recovering the monoclonal antibody or the antigen-binding fragment thereof from a cell culture.
8. A composition, comprising the monoclonal antibody or the antigen-binding fragment thereof of claim 1, the isolated nucleic acid molecule of any one of claims 2-4, the vector of claim 5, or the host cell of claim 6.
9. A kit, comprising the monoclonal antibody or the antigen-binding fragment thereof of claim 1; for example, the monoclonal antibody or the antigen-binding fragment thereof further comprises a detectable label, such as a radioisotope, a fluorescent material, a luminescent material, a colored material and an enzyme; for example, the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or the antigen-binding fragment thereof; and optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent material, a luminescent material, a colored material and an enzyme.
10. A method for detecting presence of a novel coronavirus, an S protein thereof or a RBD of the S protein, or a level thereof in a sample, comprising using the monoclonal antibody or the antigen-binding fragment thereof of claim 1; for example, the monoclonal antibody or the antigen-binding fragment thereof further comprises a detectable label, such as a radioisotope, a fluorescent material, a chemiluminescence material, a colored material and an enzyme; and for example, the method further comprises detecting the monoclonal antibody or the antigen-binding fragment thereof by using a second antibody carrying a detectable label (such as a radioisotope, a fluorescent material, a luminescent material, a colored material and an enzyme).
11. Use of the monoclonal antibody or the antigen-binding fragment thereof of claim 1 in the preparation of a kit, wherein the kit is used for detecting presence of a novel coronavirus, an S protein thereof or a RBD of the S protein, or a level thereof in a sample, or for diagnosing whether a subject is infected with the novel coronavirus; preferably, the sample is an excrement, an oral or nasal secretion, or an alveolar lavage fluid from the subject (e.g., a mammal, preferably a human).
12. A pharmaceutical composition, comprising the monoclonal antibody or the antigen-binding fragment thereof of claim 1, and a pharmaceutically acceptable carrier and/or excipient; and preferably, the pharmaceutical composition further comprises a second pharmacologically active agent, such as favipiravir, remdesivir and interferon.
13. A method for neutralizing virulence of a novel coronavirus in a sample, comprising contacting the sample comprising the novel coronavirus with the monoclonal antibody or the antigen-binding fragment thereof of claim 1.
14. Use of the monoclonal antibody or the antigen-binding fragment thereof of claim 1 in the preparation of a drug, wherein the drug is used for neutralizing virulence of a novel coronavirus in a sample or for preventing or treating a novel coronavirus infection or a disease related to the novel coronavirus infection (e.g., Corona Virus Disease 2019) in a subject; preferably, the subject is a mammal, for example, a human; and preferably, the drug is used alone or in combination with a second pharmacologically active agent (such as favipiravir, remdesivir and interferon).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0073]
[0074]
[0075]
DETAILED DESCRIPTION OF EMBODIMENTS
[0076] The present invention is described with reference to the following examples, which are meant to illustrate the present invention (but not limit the present invention).
[0077] Unless specifically stated, the molecular biology experimental methods and immunodetection methods used in the present invention were basically carried out with reference to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989 and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; and restriction enzymes were used according to the conditions recommended by the product manufacturer. If no specific conditions are indicated in the examples, conventional conditions or the conditions suggested by the manufacturer shall be followed. The reagents or instruments used without indicating the manufacturers are commercially available conventional products. It is known to a person skilled in the art that the examples illustrate the present invention by way of example and are not intended to limit the claimed scope of the present invention.
EXAMPLE 1
Isolation of Memory B Cell
[0078] Blood was collected from people once infected with SARS-CoV-2 virus but recovered and discharged (provided by Beijing Youan Hospital), and PBMCs were extracted using STEMCELL SepMate™-15 (Stemcell Technologies, Cat #86415) in a Biosafety
[0079] Physical Containment Level-2+ Laboratory. Then, memory B cells were enriched from the extracted PBMCs using STEMCELL EasySep Human Memory B Cell Isolation Kit (Stemcell Technologies, Cat #17864) according to the manufacturer's instructions.
EXAMPLE 2
Acquisition and Identification of Antibody Sequence
[0080] Single-cell transcriptome VDJ sequencing of the above-mentioned enriched memory B cells was performed using Chromium Single Cell V(D)J Reagent Kits (purchased from 10× genomics, Cat #100006) according to the manufacturer's instructions. The sequencing results were analyzed, and an antibody was obtained and named as BD23. The sequence information for BD23 antibody is as follows: [0081] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7 (the encoding gene thereof is as shown in SEQ ID NO: 17), wherein the amino acid sequences of the CDRs 1-3 of the heavy chain variable region are as shown in SEQ ID NOs: 1-3 (the encoding genes thereof are as shown in SEQ ID NOs: 11-13, respectively); [0082] the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8 (the encoding gene thereof is as shown in SEQ ID NO: 18), wherein the amino acid sequences of the CDRs 1-3 of the light chain variable region are as shown in SEQ ID NOs: 4-6 (the encoding genes thereof are as shown in SEQ ID NOs: 14-16, respectively); [0083] the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9 (the encoding gene thereof is as shown in SEQ ID NO: 19); [0084] and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 10 (the encoding gene thereof is as shown in SEQ ID NO: 20).
EXAMPLE 3
Preparation and Purification of Antibody BD23
[0085] According to the sequence information of BD23 antibody identified in example 2, Sino Biological Inc. was entrusted to express and purify BD23 antibody, and the antigenic reactivity of BD23 antibody was detected.
[0086] In short, nucleic acid molecules encoding the heavy and light chains of the antibody were synthesized in vitro and then cloned into expression vectors, respectively, thereby obtaining recombinant expression vectors encoding the heavy and light chains of the antibody, respectively. HEK293 cells were co-transfected with the above-mentioned recombinant expression vectors encoding the heavy and light chains of the antibody, respectively. 4-6 hours after the transfection, the cell culture solution was changed to a serum-free medium, which was cultured at 37° C. for another 6 days. After cultivation, the antibody protein expressed by the cells was purified from the culture by an affinity purification column. Then, the purified protein of interest was detected by reducing and non-reducing SDS-PAGE. The results are as shown in
[0087] Then, the antigenic reactivity of the purified BD23 antibody was detected by ELISA experiments using the RBD of the recombinantly expressed S protein as a coating antigen and using Goat anti-human IgG Fc labeled with horseradish peroxidase (HRP) as a secondary antibody. In short, a 96-well plate was coated with the RBD of the recombinantly expressed S protein (with an amino acid sequence as shown in SEQ ID NO: 21 and a concentration of 0.01 μg/ml or 1 μg/ml), and then the 96-well plate was blocked with a blocking solution. Then, the monoclonal antibodies to be detected (irrelevant control antibody or BD23 antibody; at a concentration of 0.1 μg/ml) were added respectively and incubated. After the plate was washed with an ELISA washing liquid, Goat anti-human IgG Fc labeled with horseradish peroxidase (HRP) was added as a secondary antibody (diluted at 1:500); and the plate was again incubated. Then, the ELISA plate was washed with PBST, and a color developing agent was added to develop the color. Then, the absorbance at OD450 nm was read on a microplate reader. The results are as shown in Table 2. The results in Table 2 show that BD23 antibody can specifically recognize and bind to the RBD of the S protein.
TABLE-US-00002 TABLE 2 Reactivity of BD23 antibody with RBD of S protein detected by ELISA (OD450 reading) Sample to Concentration of RBD protein be detected 0.01 μg/ml 1 μg/ml Irrelevant antibody 0.006 0.025 BD23 antibody 0.017 3.026
EXAMPLE 4
Evaluation of Binding Ability of Antibody BD23 to S Protein
[0088] In this example, a high-sensitivity microscale thermophoresis-based molecular interaction analysis system was used to detect the binding ability of antibody BD23 to S protein. The analysis system can be directly used for simple, rapid and precise quantitative analysis of the affinity of biomolecular interactions in a solution.
(1) S Protein with Histidine Tag (His-Tag) Being Labeled with Cy5 Fluorescent Dye
[0089] According to the manufacturer's instructions, the recombinantly expressed S protein with His-tag (the amino acid sequence thereof is as shown in SEQ ID NO: 22) was labeled with Cy5 fluorescent dye using a Monolith His-tag labeling kit (Cat#MO-L018). In short, the Cy5 fluorescent dye was diluted to 100 nM using lx PBS-T buffer. Then, 90 μL of S protein with His-tag (at a concentration of 200 nM) was mixed well with 90 μL of the diluted dye (100 nM) and incubated at room temperature for 30 minutes. Then, the incubated sample was centrifuged at 4° C., at 15000 g for 10 minutes. The supernatant was collected into a new tube for use.
(2) Detection of Binding Affinity of Antibody BD23 to S Protein
[0090] According to the manufacturer's instructions, a microscale thermophoresis (MO NT.115PICO) was used to detect the affinity of BD23 antibody to S protein. The specific steps are as follows: [0091] a. antibody BD23 was serially double-diluted (16 concentrations in total), and the initial concentration of dilution was 1 μM. The dilution method is as follows: 16 PCR tubes were prepared, and 10 μl of PBST buffer (PBS+0.005% Tween 20) was added to PCR tubes Nos 2-16; 20 μl of BD23 antibody (at a concentration of 1 μM) was added to tube No 1; 10 μl of the liquid was pipetted from tube No 1, added to tube No 2 and mixed well; and then, 10 μl of the well-mixed liquid was pipetted from tube No 2, added to tube No 3 and mixed well; the operation was performed in sequence, and finally 10 μl of the well-mixed liquid was taken from tube No. 16 and discarded. [0092] b. 10 μl of fluorescent molecules (the S protein labeled with Cy5 fluorescent dye, prepared in step (1)) were added to each PCR tube (tube Nos. 1-16) and mixed well. [0093] c. The mixture was placed at room temperature for 5 minutes, and then the sample was loaded into a microscale thermophoresis using a capillary (cat#MO-K025). [0094] d. The Kd value of the interaction between antibody BD23 and S protein was measured in a microscale thermophoresis using the Binding affinity mode.
[0095] The measurement results are as shown in
EXAMPLE 5
Assessment of Ability of BD23 Antibody to Neutralize SARS-CoV-2 Pseudovirus
[0096] In this example, the cell microneutralization assay was used to detect the neutralizing activity of monoclonal antibody BD23 against SARS-CoV-2 pseudovirus with reference to the description of Temperton N J et al., Emerg Infect Dis, 2005, 11(3), 411-416. The SARS-CoV-2 pseudovirus used in this example was provided by China National Institutes for Food and Drug Control, has similar cell infection characteristics to the euvirus, can be used to simulate the early process of euvirus infection of a cell, and carries reporter gene luciferase, which can be quickly and easily detected and analyzed. The safety for operating the pseudovirus is high, and the neutralization experiment can be completed in Biosafety Physical Containment Level-2 Laboratory to detect the neutralization activity (Neutralization titer) of the antibody. The specific steps of the experiment method are as follows: [0097] 1. Reagent for Equilibration
[0098] The reagent (0.25% trypsin-EDTA, DMEM complete medium) stored at 2° C.-8° C. was taken out and equilibrated at room temperature for more than 30 minutes. [0099] 2. Experimental Operation [0100] (1) A 96-well plate was taken, and the arrangement of the samples was set up as shown in Table 3; A2-H2 wells were set as cell control wells (CC), which only contains experimental cells; A3-H3 wells were set as virus control wells (VV), which contains experimental cells and pseudovirus; A4-A11, B4-B11, C4-C11, D4-D11, E4-E11, F4-F11, G4-G11, H4-H11 wells were set as experimental wells, which contain experimental cells, pseudovirus and different concentrations of antibody to be detected; and other wells were set as blank. The experimental cells and pseudovirus used in this example were Huh-7 cells and SARS-CoV-2 virus (both provided by China National Institutes for Food and Drug Control), respectively.
TABLE-US-00003 TABLE 3 Arrangement of samples in 96-well plate 1 2 3 4 5-10 11 12 A — CC VV Dilution 1 Dilution 1 Dilution 1 — B — CC VV Dilution 2 Dilution 2 Dilution 2 — C — CC VV Dilution 3 Dilution 3 Dilution 3 — D — CC VV Dilution 4 Dilution 4 Dilution 4 — E — CC VV Dilution 5 Dilution 5 Dilution 5 — F — CC VV Dilution 6 Dilution 6 Dilution 6 — G — CC VV Dilution 7 Dilution 7 Dilution 7 — H — CC VV Dilution 8 Dilution 8 Dilution 8 — [0101] (2) DMEM complete mediums (containing 1% antibiotic, 25 mM HEPES, 10% FBS) were added at 100 μl/well to the cell control wells; DMEM complete mediums were added at 100 μl/well to the virus control wells; and the indicated concentration of the antibody to be detected diluted in DMEM complete mediums was added to the experimental wells at 50 μ/well. The antibody concentrations of dilutions 1-8 used in Table 3 were 1/30 μg/μl, 1/90 μg/μl, 1/270 μg/μl, 1/810 μg/μl, 1/2430 μg/μl, 1/7290 μg/μl, 1/21870 μg/μl, and 1/65610 μg/μl, respectively. [0102] (3) The SARS-CoV-2 pseudovirus was diluted to about 1.3×10.sup.4/ml (TCID50) with DMEM complete mediums; and then, the SARS-CoV-2 pseudovirus was added at 50 μl/well to the virus control wells and the experimental wells. [0103] (4) The 96-well plate was placed in a cell incubator (37° C., 5% CO.sub.2) and incubated for 1 hour. [0104] (5) The pre-cultured Huh-7 cells were diluted to 2×10.sup.5 cells/ml with DMEM complete mediums. After the incubation in the previous step, cells were added at 100 μl/well to the cell control wells, virus control wells and experimental wells. [0105] (6) The 96-well plate was placed in a cell incubator (37° C., 5% CO.sub.2) and cultured for 20-28 hours. [0106] (7) The 96-well plate was taken out from the cell incubator; 150 μl of the supernatant was aspirated from each well and discarded; and then 100 μl of luciferase detection reagents were added, and reacted at room temperature for 2 minutes in the dark. [0107] (8) After the reaction was completed, the liquid in each well was pipetted 6 to 8 times repeatedly using a pipette until the cells were fully lysed. Then, 150 μl of liquid was aspirated from each well and transferred to the corresponding 96-well chemiluminescence detection plate, and the luminescence value was read with a chemiluminescence detector (Perkinelmer EnSight multimode microplate reader). [0108] (9) Calculation of neutralization inhibition rate:
Inhibition rate=[1−(mean luminescence intensity of experimental wells−mean luminescence intensity of CC wells)/(mean luminescence intensity of VV wells−mean luminescence intensity of CC wells)]×100%. [0109] (10) IC50 of the antibody to be detected was calculated by Reed-Muench method according to the result of the neutralization inhibition rate.
[0110] The experimental results are as shown in
EXAMPLE 6
Assessment of Ability of BD23 Antibody to Neutralize SARS-CoV-2 Euvirus
[0111] The SARS-CoV-2 virus used in this example was provided by Academy of Military Medical Sciences, the titer thereof (TCID50) was 10.sup.5/ml, and all experimental operations were completed in a BSL-3 laboratory. The specific steps of the neutralizing experiment method are as follows: [0112] (1) 100 μl of Vero E6 cells were added to each well of a 96-well culture plate at a concentration of 5×10.sup.4/ml, and cultured at 37° C., 5% CO2 for 24 hours. [0113] (2) The antibody to be detected was diluted to 3 concentrations: 50 μg/ml, 10 μg/ml and 2 μg/ml. 100 μl of the antibody to be detected at a specified concentration was taken out; an equal volume of SARS-CoV-2 euvirus (100 TCID50) was added; and the mixture was incubated at 37° C., 5% CO.sub.2 for 1 h. [0114] (3) After cultivation in step (1), the cell culture solution in the 96-well culture plate was discarded, and the mixture solution (200 μl) containing the antibody to be detected and the euvirus prepared in step (2) was added as an experimental group. After the mixture was incubated for 1 h, the supernatant was aspirated from the wells, and 200 μl of DMEM mediums (containing 2% antibiotic and 16 μg/ml of trypsin) were added to each well.
[0115] During the experiment, the cell control group and the virus control group were set in parallel. In the cell control group (4 replicate wells), after the cell culture solution in the wells was discarded; 200 μl of DMEM mediums (containing 2% antibiotic and 16 μg/ml of trypsin) were added to each well. In the virus control group (4 duplicate wells), after the cell culture solution in the wells was discarded; 100 TCID50 of euvirus (100 μl) was added to each well, and the mixture was incubated at 37° C. for 1 h; After the incubation, the supernatant was aspirated from the wells, and 200 μl of DMEM mediums (containing 2% antibiotic and 16 μg/ml of trypsin) were added to each well. [0116] (4) The cells were cultured for 4-5 days at 37° C., 5% CO.sub.2. [0117] (5) The cytopathic effect (CPE) was observed under the optical microscope, and the inhibitory activities of different concentrations of monoclonal antibody BD23 on CPE were evaluated according to conditions of the cytopathic effect.
[0118] The experimental results are as shown in