CN112521494A - Monoclonal antibody 2B11 for resisting SARS-CoV-2 - Google Patents

Monoclonal antibody 2B11 for resisting SARS-CoV-2 Download PDF

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CN112521494A
CN112521494A CN202010567918.XA CN202010567918A CN112521494A CN 112521494 A CN112521494 A CN 112521494A CN 202010567918 A CN202010567918 A CN 202010567918A CN 112521494 A CN112521494 A CN 112521494A
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CN112521494B (en
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杨晓明
段凯
潘勇兵
桂芳
邓小杰
张囡
宋刚
敬兆飞
詹珊珊
王炯
吴小丽
杨溢民
陈莹
杜剑晖
刘建邦
张智
李新国
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China Biotechnology Co.,Ltd.
Wuhan Biological Products Research Institute Co., Ltd
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Abstract

The invention relates to a monoclonal antibody 2B11 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

Monoclonal antibody 2B11 for resisting SARS-CoV-2
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a monoclonal antibody 2B11 for resisting SARS-CoV-2.
Background
SARS-CoV-2 is a novel coronavirus which has caused a global pandemic since the outbreak in 2019, and seriously threatens global public health. At present, no vaccine or specific medicine which is proved to be effective for SARS-CoV-2 virus is available on the market, so that the development of new preventive and therapeutic measures is urgently needed.
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 prepare monoclonal antibodies against SARS-CoV-2, particularly fully human monoclonal antibodies. The antibody can prevent virus invasion by blocking the combination of SARS-CoV-2 and receptor cells to achieve the protection effect, has the advantage of smaller side effect compared with humanized or human-mouse chimeric antibody, and provides a new means for specific prevention and treatment of COVID-19.
Disclosure of Invention
The invention firstly relates to a monoclonal antibody 2B11 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: EITVSSNYMN, respectively;
(2) the heavy chain CDR2(VHCDR2) comprises the amino acid sequence shown in SEQ ID NO. 2: VIYSGGTTYYADSVKG, respectively;
(3) the heavy chain CDR3(VHCDR3) comprises the amino acid sequence shown in SEQ ID NO. 3: DLMEVGGMDV, respectively;
(4) the light chain CDR1(VLCDR1) comprises the amino acid sequence shown in SEQ ID No. 4: SGSSSNVENDNVN, respectively;
(5) the light chain CDR2(VLCDR2) comprises the amino acid sequence shown in SEQ ID NO. 5: NDRLRPS;
(6) the light chain CDR3(VLCDR3) comprises the amino acid sequence shown in SEQ ID NO. 6: VAWDASLQSYV are provided.
Further, in the above-mentioned case,
the full length of the heavy chain variable region of the monoclonal antibody 2B11 comprises an amino acid sequence shown as SEQ ID NO. 7: EVQLVESGGGLVQPGGSLRLSCAASEITVSSNYMNWVRQAPGKGLEWVSVIYSGGTTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDLMEVGGMDVWGQGTTVTVSS；
The full length of the variable region of the light chain of the monoclonal antibody 2B11 comprisesThe amino acid sequence shown as SEQ ID NO. 8: LPVLTQPPSASGTPGQRVTISCSGSSSNVENDNVNWFQQQVPGSTPKLVIYNDRLRPSGVPDRFSGSKSGTSAYLAISGLQSEDEADYYCVAWDASLQSYVFGTGTKVTVL。
Further, the monoclonal antibody 2B11 is a human IgG type antibody.
Furthermore, the antigen bound by the monoclonal antibody 2B11 is spike protein S1 of SARS-CoV-2 virus, and specifically, the antigenic structural region bound by the monoclonal antibody 2B11 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 2B11 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 2B 11.
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 2B11 in the preparation of a reagent for detecting SARS-CoV-2 virus.
The invention also includes the application of the monoclonal antibody 2B11 in the preparation of a reagent for inhibiting SARS-CoV-2 virus.
The invention also includes the application of the monoclonal antibody 2B11 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 2B11 antibody shows that the neutralizing activity and the blocking activity of the monoclonal antibody 2B11 are both high. Therefore, the human monoclonal antibody 2B11 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 2B 11: 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 2B 11.
FIG. 3, results of the detection of the binding activity of monoclonal antibody 2B 11. 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 shows the results of the detection of the blocking activity of monoclonal antibody 2B 11. A is the blocking activity against SARS-CoV-2 spike protein S1; b is blocking activity against the RBD domain of SARS-CoV-2 spike protein S1.
FIG. 5 shows the result of affinity detection of monoclonal antibody 2B 11.
FIG. 6, the results of the live virus neutralization assay of monoclonal antibody 2B 11.
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 2B11 expression vector
The coding gene segments containing the heavy chain and light chain variable regions of the 2B11 antibody are respectively integrated into pcDNA3.4 expression vectors containing the heavy chain and light chain constant region sequences of the human IgG1 antibody to obtain recombinant expression vectors capable of respectively expressing the heavy chain and the light chain of the target antibody.
Transfection of cells, antibody expression and purification:
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 2B11 with antigen
The binding ability of the antibody to the spike protein S1 and RBD domain of SARS-CoV-2 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 2B11 antibody to the antigen are shown in fig. 3, and it can be seen that the binding activity of monoclonal antibody 2B11 against S1 protein (fig. 3.a) is EC50 ═ 0.051 nM; the binding activity against the RBD domain (fig. 3.B) was EC50 ═ 0.047 nM.
2. Detection of blocking Activity of human anti-SARS-CoV-2 antibody 2B11
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, cat # 40591-V02H) or RBD recombinant protein (Sino company, cat # 40592-V05H) is used as coating antigen, 1.0 mug/mL antigen is coated on the ELISA 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 antibody blocking activity of 2B11 is shown in fig. 4, and it can be seen that the blocking activity of monoclonal antibody 2B11 against the binding of ACE2 and S1 (fig. 4.a) is IC50 ═ 2.792nM, and the maximum blocking rate is 95.43%; the blocking activity against ACE2 binding to RBD (fig. 4.B) was IC 50-1.956 nM with a maximum blocking rate of 99.21%.
3. Affinity detection of human anti-SARS-CoV-2 antibody 2B11
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 2B11 are shown in FIG. 5, which shows that the 2B11 has a binding constant ka of 1.70E + 51/Ms, a dissociation constant KD of 8.08E-41/s, and an affinity constant KD of 4.76E-9M.
4. Living virus neutralization assay for human anti-SARS-CoV-2 antibodies
The virus plaque reduction neutralization test was carried out using the SARS-CoV-2 virus BetacoV/Wuhan/IVDC-HB-envF13/2020 strain. The steps are briefly described as follows: a quantitative amount of SARS-CoV-2 virus and a dilution series of the monoclonal antibody were mixed and incubated, and then added to a previously prepared assay plate containing Vero cells, and after incubation, the number of viral plaques was observed and the virus-neutralizing activity (expressed as IC 50) was calculated.
As a result:
the result of the live virus neutralization test of the antibody 2B11 is shown in FIG. 6, and the monoclonal antibody can well neutralize SARS-CoV-2 virus (IC50<0.98 ng/mL).
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 2B11 for resisting SARS-CoV-2
<210> 1
<211> 10
<212> PRT
<213> Artificial sequence
<400> 1
Glu Ile 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 Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 3
<211> 10
<212> PRT
<213> Artificial sequence
<400> 3
Asp Leu Met Glu Val Gly Gly Met Asp Val
1 5 10
<210> 4
<211> 13
<212> PRT
<213> Artificial sequence
<400> 4
Ser Gly Ser Ser Ser Asn Val Glu Asn Asp Asn Val Asn
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence
<400> 5
Asn Asp Arg Leu Arg Pro Ser
1 5
<210> 6
<211> 11
<212> PRT
<213> Artificial sequence
<400> 6
Val Ala Trp Asp Ala Ser Leu Gln Ser Tyr Val
1 5 10
<210> 7
<211> 118
<212> PRT
<213> Artificial sequence
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Ile 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 Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu 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 Met Glu Val Gly Gly Met Asp Val Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 8
<211> 111
<212> PRT
<213> Artificial sequence
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Leu Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Glu Asn Asp
20 25 30
Asn Val Asn Trp Phe Gln Gln Gln Val Pro Gly Ser Thr Pro Lys Leu
35 40 45
Val Ile Tyr Asn Asp Arg Leu Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Tyr Leu Ala Ile Ser Gly Leu
65 70 75 80
Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Ala Ser
85 90 95
Leu Gln Ser Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu
100 105 110

Claims (8)

1. Monoclonal antibody 2B11 directed against SARS-CoV-2 virus, comprising the six CDR regions:
(1) the heavy chain CDR1(VHCDR1) comprises the amino acid sequence shown in SEQ ID No. 1: EITVSSNYMN, respectively;
(2) the heavy chain CDR2(VHCDR2) comprises the amino acid sequence shown in SEQ ID NO. 2: VIYSGGTTYYADSVKG, respectively;
(3) the heavy chain CDR3(VHCDR3) comprises the amino acid sequence shown in SEQ ID NO. 3: DLMEVGGMDV, respectively;
(4) the light chain CDR1(VLCDR1) comprises the amino acid sequence shown in SEQ ID No. 4: SGSSSNVENDNVN, respectively;
(5) the light chain CDR2(VLCDR2) comprises the amino acid sequence shown in SEQ ID NO. 5: NDRLRPS;
(6) the light chain CDR3(VLCDR3) comprises the amino acid sequence shown in SEQ ID NO. 6: VAWDASLQSYV are provided.
2. The antibody of claim 1,
the full length of the heavy chain variable region of the monoclonal antibody 2B11 comprises an amino acid sequence shown as SEQ ID NO. 7:
the full length of the variable region of the light chain of the monoclonal antibody 2B11 comprises an amino acid sequence shown in SEQ ID NO. 8.
3. The antibody of claim 1 or 2, wherein monoclonal antibody 2B11 is a human IgG-type antibody.
4. The antibody of claim 1 or 2, wherein the antigen bound by monoclonal antibody 2B11 is spike protein S1 of SARS-CoV-2 virus, and specifically, the antigenic domain bound by monoclonal antibody 2B11 is RBD domain of spike protein S1 of SARS-CoV-2 virus.
5. A nucleic acid fragment encoding the antibody of any one of claims 1-4.
6. Use of the monoclonal antibody 2B11 of any one of claims 1-4 in the preparation of a reagent for detecting SARS-CoV-2 virus.
7. Use of the monoclonal antibody 2B11 of any one of claims 1-4 in the preparation of a reagent for inhibiting SARS-CoV-2 virus.
8. Use of the monoclonal antibody 2B11 of any one of claims 1-4 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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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113274494A (en) * 2021-06-07 2021-08-20 武汉生物制品研究所有限责任公司 Liquid preparation of recombinant fully human monoclonal antibody for resisting SARS-CoV-2
CN113354733A (en) * 2021-06-04 2021-09-07 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
WO2022044573A1 (en) * 2020-08-26 2022-03-03 国立大学法人熊本大学 Human antibody or antigen-binding fragment thereof against coronavirus spike protein
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WO2022044573A1 (en) * 2020-08-26 2022-03-03 国立大学法人熊本大学 Human antibody or antigen-binding fragment thereof against coronavirus spike protein
US11999777B2 (en) 2021-06-02 2024-06-04 Regeneron Pharmaceuticals, Inc. Methods for treating or preventing SARS-CoV-2 infections and COVID-19 with anti-SARS-CoV-2 spike glycoprotein antibodies
CN113354733A (en) * 2021-06-04 2021-09-07 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
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