CN111718411B - Monoclonal antibody 1F2 for resisting SARS-CoV-2 - Google Patents
Monoclonal antibody 1F2 for resisting SARS-CoV-2 Download PDFInfo
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- CN111718411B CN111718411B CN202010566792.4A CN202010566792A CN111718411B CN 111718411 B CN111718411 B CN 111718411B CN 202010566792 A CN202010566792 A CN 202010566792A CN 111718411 B CN111718411 B CN 111718411B
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Abstract
The invention relates to a monoclonal antibody 1F2 for resisting SARS-CoV-2, six CDR areas of the antibody are: (1) heavy chain CDR1(VHCDR1) comprises the amino acid sequence shown in SEQ ID No. 1; (2) heavy chain CDR2(VHCDR2) comprises the amino acid sequence shown in SEQ ID No. 2; (3) heavy chain CDR3(VHCDR3) comprises the amino acid sequence shown in SEQ ID No. 3; (4) the light chain CDR1(VLCDR1) comprises the amino acid sequence shown in SEQ ID No. 4; (5) the light chain CDR2(VLCDR2) comprises the amino acid sequence shown in SEQ ID NO. 5; (6) the light chain CDR3(VLCDR3) comprises the amino acid sequence shown in SEQ ID NO. 6.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a monoclonal antibody 1F2 for resisting SARS-CoV-2.
Background
Convalescent plasma containing high concentrations of anti-SARS-CoV-2 antibody has shown positive effects in therapy, suggesting that anti-SARS-CoV-2 specific antibodies are effective in blocking virus binding to cells. In addition, a series of monoclonal antibodies having neutralizing activity have been developed during outbreaks of severe infectious diseases such as SARS and MERS, and have been proven to be safe and effective in the prevention and treatment of diseases. These suggest that in response to SARS-CoV-2 virus, it is possible to consider the preparation of monoclonal antibodies against SARS-CoV-2, particularly fully human monoclonal antibodies, which not only protect against virus invasion by blocking the binding of SARS-CoV-2 to the recipient cell, but also have the advantage of less side effects than humanized or murine chimeric antibodies, and will provide a novel means for the specific prevention and treatment of COVID-19.
Disclosure of Invention
The invention firstly relates to a monoclonal antibody 1F2 aiming at SARS-CoV-2 virus, which is characterized in that the six CDR regions of the antibody are:
(1) the heavy chain CDR1(VHCDR1) comprises the amino acid sequence shown in SEQ ID No. 1: GFTVSSNYMN, respectively;
(2) the heavy chain CDR2(VHCDR2) comprises the amino acid sequence shown in SEQ ID NO. 2: VIYSGGSTFYADSVKG, respectively;
(3) the heavy chain CDR3(VHCDR3) comprises the amino acid sequence shown in SEQ ID NO. 3: DLDILGGMDV, respectively;
(4) the light chain CDR1(VLCDR1) comprises the amino acid sequence shown in SEQ ID No. 4: RASQSVSSSYLA, respectively;
(5) the light chain CDR2(VLCDR2) comprises the amino acid sequence shown in SEQ ID NO. 5: a GASSRAT;
(6) the light chain CDR3(VLCDR3) comprises the amino acid sequence shown in SEQ ID NO. 6: QQYGSSPPT are provided.
Further, in the above-mentioned case,
the full length of the heavy chain variable region of the monoclonal antibody 1F2 comprises an amino acid sequence shown as SEQ ID NO. 7: EVQLLESGGGLVQPGGSLRLSCAASGFTVSSNYMNWVRQAPGKGLEWVSVIYSGGSTFYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARDLDILGGMDVWGQGTTVTVSS；
The full length of the variable region of the light chain of the monoclonal antibody 1F2 comprises an amino acid sequence shown as SEQ ID NO. 8: EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGSSPPTFGQGTKVEIK。
Further, the monoclonal antibody 1F2 is a human IgG type antibody.
Furthermore, the antigen bound by the monoclonal antibody 1F2 is spike protein S1 of SARS-CoV-2 virus, and specifically, the antigenic structural region bound by the monoclonal antibody 1F2 is RBD structural region in spike protein S1 of SARS-CoV-2 virus.
Most preferably, the light chain variable region and the heavy chain variable region of monoclonal antibody 1F2 are the amino acid sequences shown in SEQ ID NO.8 and SEQ ID NO.7, respectively.
The invention also relates to a nucleic acid fragment encoding the monoclonal antibody 1F 2.
The invention also relates to an antibody, wherein the light chain of the antibody is:
(1) the amino acid sequence shown in SEQ ID NO.8 is replaced, deleted or added with one or more amino acids to form a sequence with the same function;
or (2) an amino acid sequence with homology of more than 95 percent with the amino acid sequence shown in SEQ ID NO. 8;
the heavy chain of the antibody is:
(1) the amino acid sequence shown in SEQ ID NO.7 is replaced, deleted or added with one or more amino acids to form a sequence with the same function;
or (2) an amino acid sequence with homology of more than 95 percent with the amino acid sequence shown in SEQ ID NO. 7.
The invention also includes the application of the monoclonal antibody 1F2 in the preparation of a reagent for detecting SARS-CoV-2 virus.
The invention also includes the application of the monoclonal antibody 1F2 in the preparation of a reagent for inhibiting SARS-CoV-2 virus.
The invention also includes the application of the monoclonal antibody 1F2 in preparing medicaments for preventing and/or treating diseases caused by SARS-CoV-2 virus infection.
The invention has the beneficial effects that: because there are no vaccines and specific drugs on the market for the diseases caused by SARS-CoV-2 virus, although the convalescent plasma treatment has been proved to be a promising treatment method, the large-scale preparation is limited, and the method mainly aims at patients with severe and critical illness, while the monoclonal antibody has the advantages of high purity, strong targeting property, small side effect, large-scale preparation and the like. The experimental result of the 1F2 antibody shows that the neutralizing activity and the blocking activity of the monoclonal antibody 1F2 are both high. Therefore, the human monoclonal antibody 1F2 obtained by the invention provides a new candidate drug for specific prevention and treatment of COVID-19.
Drawings
FIG. 1, SDS-PAGE electrophoresis detection of monoclonal antibody 1F 2: lane 1 is a non-reducing SDS-PAGE electrophoresis, and lane 2 is a reducing SDS-PAGE electrophoresis.
FIG. 2 SEC-HPLC detection of monoclonal antibody 1F 2.
FIG. 3, results of the detection of the binding activity of monoclonal antibody 1F 2. A is the binding activity against SARS-CoV-2 spike protein S1; b is the binding activity against the RBD domain of the SARS-CoV-2 spike protein S1.
FIG. 4, results of the live virus neutralization assay of monoclonal antibody 1F 2.
FIG. 5 shows the results of affinity detection of monoclonal antibody 1F 2.
Detailed Description
Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagent consumables are commercially available.
Example 1 construction, expression and purification of monoclonal antibody 1F2 expression vector
The coding gene segments containing the variable regions of the heavy chain and the light chain of the 1F2 antibody are respectively integrated into pcDNA3.4 expression vectors containing the constant region sequences of the heavy chain and the light chain of the human IgG1 antibody to obtain recombinant expression vectors for respectively expressing the heavy chain and the light chain of the target antibody.
Transfection, antibody expression and purification of cells
1. Transfection: transfection was performed using the transfection kit from Gibco, Inc. according to the instructions, and the procedure is briefly described as follows:
mixing the two recombinant expression vector total DNAs with a transfection reagent to form a DNA Expifeactine (TM) 293 complex;
then 40mL of 2.94X10 was added6one/mL in Expi293 cell culture;
finally, the cells were incubated at 37 ℃ and 125rpm with 8% CO 2.
2. And (3) purification:
after 5 days of incubation, the supernatant was collected by centrifugation at 4000rpm for 10 minutes at 25 ℃ and then purified by a MabSelect Sure affinity column. The purification steps are briefly described as follows:
equilibrating with 0.1M Tris buffer, pH 7.0;
after loading, eluting with 0.1M Tris buffer solution with pH7.0;
elution was then carried out with 1.0M Tris buffer, pH 8.0.
The eluate was collected and further dialyzed against PBS buffer. The purified antibody was subjected to SDS-PAGE and HPLC-SEC detection.
3. And (4) analyzing results: the results of SDS-PAGE are shown in FIG. 1, which shows that the human SARS-CoV-2 antibody exhibits a band with a molecular weight of about 150kDa under non-reducing conditions; two bands with molecular weights of approximately 50kDa and 25kDa are present under reducing conditions, corresponding to the heavy and light chains of the antibody, respectively. The SEC-HPLC results are shown in FIG. 2, which shows that the purity of the purified monoclonal antibody reaches more than 99%. The amino acid sequence of the purified monoclonal antibody obtained by peptide map analysis is consistent with the expected amino acid sequence. Specifically, the sequences of the light chain variable region and the heavy chain variable region are respectively the same as SEQ ID NO.8 and SEQ ID NO. 7.
Example 2 functional analysis of antibodies
1. Detection of binding Activity of human anti-SARS-CoV-2 antibody 1F2 with antigen
The binding ability of the antibody to the spike protein S1 and RBD domain of SARS-CoV-2(2019-nCoV) virus was determined by ELISA. The steps are briefly described as follows:
(1) SARS-CoV-2 spike S1-His recombinant protein (Sino company, goods number: 40591-V08H) or RBD structure domain recombinant protein (Sino company, goods number 40592-V05H) is used as coating antigen, 1.0 mu g/mL antigen is coated on an enzyme label plate by bicarbonate buffer solution, and the temperature is kept at 4 ℃ overnight;
(2) blocking with casein buffer at 37 ℃ for 1 h; adding the serially diluted antibodies to be detected, and keeping the temperature at 37 ℃ for 1 h;
(3) adding 1: 10000 diluted goat anti-human IgG-HRP (Bethyl company, cat # A80-304P), 37 ℃ for 1 h;
(4) after developing with developing solution, terminating the reaction by 2M HCl; and detecting absorbance A450 and A540 values by a microplate reader.
As a result:
the results of the detection of the binding activity of the antibody 1F2 to the antigen are shown in fig. 3, and it can be seen that the binding activity of the monoclonal antibody 1F2 against S1 protein (fig. 3A) is EC50 ═ 0.068 nM; the binding activity against the RBD domain (fig. 3B) was EC50 ═ 0.048 nM.
2. Detection of blocking Activity of human anti-SARS-CoV-2 antibody 1F2
ELISA assay was used to test the ability of the antibody to block the binding of ACE2 recombinant protein (His-tagged) to the spike protein S1 and RBD domain of SARS-CoV-2(2019-nCoV) virus. The steps are briefly described as follows:
(1) SARS-CoV-2 spike S1 subunit protein (Sino company, goods number: 40591-V02H) or recombinant protein of RBD structure domain (Sino company, goods number 40592-V05H) is used as coating antigen, 1.0 mu g/mL antigen is coated on an enzyme label plate by bicarbonate buffer solution, and the temperature is kept at 4 ℃ overnight;
(2) blocking with casein buffer at 37 ℃ for 1 h; adding the antibody to be detected after serial dilution and ACE recombinant protein with the concentration of 0.5 mu g/mL or 0.02 mu g/mL for co-incubation at 37 ℃ for 1 h;
(3) adding 1: 3000 diluted biotin-labeled mouse anti-His tag antibody (GenScript, Cat: A00613), 1h at 37 ℃;
(4) adding 1: 20000 diluted streptavidin-HRP (Thermofeisher Co., Ltd., cat # SNN1004), 37 ℃ for 1 h; after developing with developing solution, terminating the reaction by 2M HCl; and detecting absorbance A450 and A540 values by a microplate reader.
As a result:
the detection result of the blocking activity of the 1F2 antibody is shown in fig. 4, and it can be seen that the blocking activity of the monoclonal antibody 1F2 on the binding of ACE2 and S1 (fig. 4.a) is 4.34nM at IC50, and the maximum blocking rate is 92%; the blocking activity against ACE2 binding to RBD was (fig. 4.B) 2.65nM with an IC50, with a maximum blocking rate of 98%.
3. Affinity detection of human anti-SARS-CoV-2 antibody 1F2
A Biacore 8K instrument of GE company is used for carrying out a surface plasma resonance experiment to detect the affinity of the antibody.
The results of the affinity assay for monoclonal antibody 1F2 are shown in FIG. 5, and it can be seen that 1F2 has a binding constant ka value of 7.54E + 041/Ms, a dissociation constant KD value of 2.97E-031/s, and an affinity constant KD value of 3.94E-08M.
4. Living virus neutralization assay for human anti-SARS-CoV-2 antibodies
The virus plaque reduction neutralization assay was performed using the SARS-CoV-2 virus BetacoV/IVDC-HB-envF13/2020 strain. The steps are briefly described as follows: mixing and incubating a quantitative SARS-CoV-2 virus and a series of monoclonal antibodies diluted in multiple proportion, adding the mixture into a detection plate containing Vero cells prepared in advance, observing and recording the number of virus plaques after culturing, and calculating the virus neutralization activity.
As a result: the detection result of the live virus neutralization experiment of the antibody 1F2 shows that the monoclonal antibody can well neutralize SARS-CoV-2 virus.
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Biometrics institute of Biotechnology, Inc
<120> monoclonal antibody 1F2 for anti-SARS-CoV-2
<210> 1
<211> 10
<212> PRT
<213> Artificial sequence
<400> 1
Gly Phe Thr Val Ser Ser Asn Tyr Met Asn
1 5 10
<210> 2
<211> 16
<212> PRT
<213> Artificial sequence
<400> 2
Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 3
<211> 10
<212> PRT
<213> Artificial sequence
<400> 3
Asp Leu Asp Ile Leu Gly Gly Met Asp Val
1 5 10
<210> 4
<211> 12
<212> PRT
<213> Artificial sequence
<400> 4
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence
<400> 5
Gly Ala Ser Ser Arg Ala Thr
1 5
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence
<400> 6
Gln Gln Tyr Gly Ser Ser Pro Pro Thr
1 5
<210> 7
<211> 118
<212> PRT
<213> Artificial sequence
<400> 7
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 Val Ser Ser Asn
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Leu Asp Ile Leu Gly Gly Met Asp Val Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 8
<211> 108
<212> PRT
<213> Artificial sequence
<400> 8
Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
Claims (9)
1. A monoclonal antibody against SARS-CoV-2 virus, wherein the antibody has six CDR regions:
(1) heavy chain CDR1(VHCDR1) is the amino acid sequence shown in seq id No. 1: GFTVSSNYMN, respectively;
(2) heavy chain CDR2(VHCDR2) is the amino acid sequence shown in seq id No. 2: VIYSGGSTFYADSVKG, respectively;
(3) heavy chain CDR3(VHCDR3) is the amino acid sequence shown in seq id No. 3: DLDILGGMDV, respectively;
(4) the light chain CDR1(VLCDR1) has an amino acid sequence shown in SEQ ID NO. 4: RASQSVSSSYLA, respectively;
(5) the light chain CDR2(VLCDR2) has an amino acid sequence shown as SEQ ID NO. 5: a GASSRAT;
(6) the light chain CDR3(VLCDR3) has an amino acid sequence shown as SEQ ID NO. 6: QQYGSSPPT are provided.
2. The monoclonal antibody according to claim 1,
the full length of the heavy chain variable region of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 7:
the full length of the light chain variable region of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 8.
3. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody is a human IgG-type antibody.
4. The monoclonal antibody of claim 1 or 2, wherein the antigen bound by the monoclonal antibody is spike protein S1 of SARS-CoV-2 virus.
5. The monoclonal antibody of claim 4, wherein the antigenic domain to which said monoclonal antibody binds is the RBD domain of the spike protein S1 of SARS-CoV-2 virus.
6. A nucleic acid fragment encoding the monoclonal antibody of any one of claims 1-5.
7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of a reagent for detecting SARS-CoV-2 virus.
8. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of an agent for inhibiting SARS-CoV-2 virus.
9. Use of the monoclonal antibody of any one of claims 1-5 in the manufacture of a medicament for the prevention and/or treatment of a disease caused by SARS-CoV-2 virus infection.
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| AU2020340881A1 (en) | 2020-04-02 | 2021-10-21 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
| CN116057069A (en) | 2020-06-03 | 2023-05-02 | 瑞泽恩制药公司 | Methods of treating or preventing SARS-CoV-2 infection and COVID-19 using anti-SARS-CoV-2 spike glycoprotein antibodies |
| EP4206224A1 (en) * | 2020-08-26 | 2023-07-05 | National University Corporation Kumamoto University | Human antibody or antigen-binding fragment thereof against coronavirus spike protein |
| CN116419971A (en) * | 2020-09-30 | 2023-07-11 | 南京金斯瑞生物科技有限公司 | Monoclonal antibody against SARS-CoV-2 spike protein outer region and application thereof |
| CN112326962B (en) * | 2020-11-03 | 2021-10-12 | 山西康健恩生物科技有限公司 | Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof |
| WO2022101839A1 (en) * | 2020-11-12 | 2022-05-19 | Cadila Healthcare Limited | Anti-sars-cov-2 monoclonal antibodies and cocktail thereof |
| WO2022112392A1 (en) * | 2020-11-26 | 2022-06-02 | Memo Therapeutics Ag | Anti-sars-cov-2 antibody molecules |
| CN117924467A (en) * | 2020-12-02 | 2024-04-26 | 深圳先进技术研究院 | Broad-spectrum anti-coronavirus fully human monoclonal antibody and preparation method and application thereof |
| WO2022179561A1 (en) * | 2021-02-24 | 2022-09-01 | The University Of Hong Kong | Neutralizing antibodies against covid-19 and methods of use 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 |
| CN113354733B (en) * | 2021-06-04 | 2022-02-18 | 武汉生物制品研究所有限责任公司 | Monoclonal antibody 20D8 for resisting SARS-CoV-2 epidemic mutant strain |
| CN114478755B (en) * | 2021-06-30 | 2023-05-12 | 军事科学院军事医学研究院军事兽医研究所 | Fully human antibody against novel coronavirus, composition and application thereof |
| WO2023070310A1 (en) * | 2021-10-26 | 2023-05-04 | 中国科学院深圳先进技术研究院 | Anti-sar-cov-2 fully humanized monoclonal antibody and preparation method therefor 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 |
| CN114231497B (en) * | 2022-02-24 | 2022-05-20 | 广州伯尼兹生物科技有限公司 | Monoclonal antibody hybridoma cell line expressing SARS-CoV-2S 1 protein and neutralizing active antibody |
| CN117025548B (en) * | 2023-10-08 | 2024-02-02 | 北京博奥森生物技术有限公司 | Mouse anti SARS-CoV-2 Spike protein hybridoma cell strain, antibody, kit and application |
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