CN113999301B - anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody - Google Patents

anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody Download PDF

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CN113999301B
CN113999301B CN202111485922.2A CN202111485922A CN113999301B CN 113999301 B CN113999301 B CN 113999301B CN 202111485922 A CN202111485922 A CN 202111485922A CN 113999301 B CN113999301 B CN 113999301B
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sars
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CN113999301A (en
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廖高勇
丁海剑
陈海
张怡
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Xintrum Pharmaceuticals Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides an anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody. Experiments prove that the antibody has high affinity with RBD and mutant thereof of SARS-CoV-2 (COVID-19) S protein, can effectively inhibit the combination of RBD and mutant thereof (L452R, T478K) of SARS-CoV-2 (COVID-19) S protein and ACE2, and has obvious neutralization activity on novel coronavirus and mutant strain thereof (Delta) pseudovirus.

Description

anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody
Technical field:
the invention relates to a genetic engineering antibody, in particular to a monoclonal antibody drug developed aiming at a novel coronavirus S-RBD target.
The background technology is as follows:
the novel coronavirus SARS-CoV-2 (COVID-19) is known to infect by binding of its Receptor Binding Domain (RBD) of S protein to the host receptor angiotensin converting enzyme 2 (ACE 2), mediating viral entry into host cells. The S protein comprises two functional subunits S1 and S2, wherein S1 is responsible for binding to host cell receptors and S2 subunit is responsible for viral membrane and cell membrane fusion. During infection, the S protein is cleaved by the host protease into the N-terminal S1 subunit and the C-terminal S2 subunit and is converted from the pre-fusion state to the post-fusion state. S1 and S2 consist of an extracellular domain (ECD) and a single transmembrane helix, mediating receptor binding and membrane fusion, respectively. S1 consists of an N-terminal domain (NTD) and a Receptor Binding Domain (RBD), which are critical for determining tissue tropism and host range.
The Receptor Binding Domain (RBD) of the S protein of SARS-CoV-2 (COVID-19) is immunodominant and also a target for 90% of the neutralizing antibodies present in SARS-CoV-2 immune serum. Therefore, developing antibody drugs against the S-RBD target of the novel coronavirus and mutants thereof is an effective way for treating patients with the novel coronavirus infection, and the monoclonal antibodies developed by the method have potential to become specific drugs for treating the novel coronavirus infection.
Disclosure of Invention
The invention aims to provide a monoclonal antibody which can effectively compete with ACE2 for combining with SARS-CoV-2 (COVID-19) S protein RBD and mutants (L452R, T478K) thereof, and is used for preparing medicaments for treating novel coronavirus related diseases.
The invention uses SARS-CoV-2 (COVID-19) S protein RBD protein and its mutant (L452R, T478K) protein to immunize BALB/c mouse, so as to obtain the invented SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody.
The heavy chain and light chain sequences of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody obtained by the invention are completely different from those of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody in the prior art.
The SARS-CoV-2 (COVID-19) S protein RBD protein was purchased from Beijing Baiposis Biotechnology Co., ltd. The mice used were BALB/c mice.
Specifically, the invention accomplishes the above-mentioned work by:
a SARS-CoV-2 (COVID-19) S protein RBD protein is used as antigen, and 30 mug/BALB/c mice are immunized, and the same dose is used for enhancing immunity after three weeks;
b ELISA method for determining antibody titer in serum of immunized mice, and after reaching ideal effect, the mice are immunized by impact at 50 mug dosage;
c, taking splenocytes of mice which are successfully immunized and fusing with SP2/0 cells, detecting the titer of the supernatant after the cells grow into cell clusters, and obtaining positive monoclonal cell strains through three rounds of subcloning;
d, performing intraperitoneal injection on the mice after the expanded culture of the monoclonal cell strain to prepare ascites, and purifying the collected ascites to obtain a corresponding antibody;
e, carrying out affinity kinetics measurement of the monoclonal antibody by utilizing an SPR technology;
f, determining the competitive inhibition effect of the monoclonal antibody on the combination of SARS-CoV-2 (COVID-19) S protein RBD and mutants (L452R, T478K) and ACE 2;
g the neutralizing activity of the monoclonal antibodies against the novel coronavirus and its mutant strain (Delta) pseudovirus was determined.
The anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody obtained by the invention is named as B15-5-B3. The molecular basis of this antibody specificity is mainly derived from its highly variable regions CDR1, CDR2 and CDR3, which are critical sites for antigen binding.
The heavy chain and light chain CDR1, CDR2 and CDR3 of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody obtained by the invention are polypeptides with the amino acid sequences shown as follows:
B15-5-B3: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
the term "monoclonal antibody" as used herein is to be construed as encompassing any specific binding factor having a binding domain of the desired specificity, and may be monovalent or single chain antibodies, diabodies, chimeric antibodies, and derivatives, functional equivalents and homologs of the above antibodies, as well as antibody fragments and any polypeptide comprising an antigen binding domain.
Examples of monoclonal antibodies of the invention are immunoglobulin IgG subtypes and subclasses thereof;
although the molecular basis for antibody specificity is primarily derived from its highly variable regions CDR1, CDR2 and CDR3, the sequences of the CDRs should be as preserved as possible in order to maintain the preferred binding properties. However, although individual amino acid changes, it is possible to achieve the object of the invention and even optimize the binding properties. However, individual amino acid changes do not depart from the spirit and scope of the present invention.
In addition to the highly variable regions CDR1, CDR2 and CDR3 in the heavy and light chains as described above, the others are framework regions. The framework regions may be replaced with other sequences without affecting the three-dimensional structure required for binding.
The anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody B15-5-B3 obtained by the invention is subjected to the following experimental verification:
1. affinity kinetic analysis
The results show that the monoclonal antibodies of the invention have high affinity with SARS-CoV-2 (COVID-19) S protein RBD and mutants thereof (L452R, T478K) (see example 2 for details).
2. Competitive inhibition of ACE2 binding
Biochemical level experiment results show that the monoclonal antibody can obviously inhibit the combination of SARS-CoV-2 (COVID-19) S protein RBD and its mutant (L452R, T478K) and ACE2 (see example 3).
3. Detection of neutralizing Activity against novel coronavirus and mutant (Delta) pseudoviruses thereof
The results show that the monoclonal antibodies of the invention have obvious neutralizing and inhibitory activity on both the novel coronavirus and the mutant strain (Delta) pseudovirus thereof (see example 4 for details).
Drawings
FIG. 1 is a schematic diagram showing the steps for obtaining an anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody of the present invention;
FIGS. 2 and 3 show the results of the detection of the affinity of the antibody B15-5-B3 for SARS-CoV-2 (COVID-19) S protein RBD and its mutants (L452R, T478K) in example 2, respectively, wherein Ka, kd and KD are the binding constant, dissociation constant and affinity constant, respectively;
FIGS. 4 and 5 show the results of experiments on the competitive inhibition of SARS-CoV-2 (COVID-19) S protein RBD and its mutant (L452R, T478K) binding to ACE2 by the B15-5-B3 antibody of example 3, respectively;
FIGS. 6 and 7 show the results of experiments for detecting neutralizing activity of the B15-5-B3 antibody of example 4 on a novel coronavirus and its mutant strain (Delta) pseudovirus, respectively.
The above-mentioned B15-5-B3 is the name of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody obtained by the present invention.
Sequence information:
SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 are CDR1, CDR2 and CDR3 of the heavy chain variable region of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody B15-5-B3, respectively;
SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 are CDR1, CDR2 and CDR3 of the light chain variable region of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody B15-5-B3, respectively;
SEQ ID NO. 7 and SEQ ID NO. 8 are the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody B15-5-B3, respectively.
Detailed Description
The following examples are provided to illustrate the present invention in further detail in order to make the objects, technical solutions and effects of the present invention more clear and clarified. It should be understood that the particular methods, reagents, etc. used in the examples are illustrative of the present invention and are not intended to limit the scope of the invention;
the invention provides heavy and light chain sequences of specific anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibodies. The monoclonal antibody is expressed by a corresponding monoclonal cell strain obtained by hybridoma screening after a BALB/c mouse is immunized by SARS-CoV-2 (COVID-19) S protein RBD protein; the monoclonal antibody type is of an IgG type;
the SARS-CoV-2 (COVID-19) S protein RBD protein and each mutant protein, mentioned in the examples below, contained a 6 XHis tag at the C-terminus, all available from Beijing Baiposis Biotech Co.
The used immune adjuvant is Beijing bololone 5-week rapid immune adjuvant, the primary immunization is carried out for 21 days, the primary immunization is carried out, and the antigen is used for carrying out the impact immunization once before the cell fusion, so that the cell fusion can be carried out;
the fusion method is an electrofusion method, the electrofusion instrument is ECM2001 type of BTX company, and the fusion buffer solution is cell fusion solution of a primary plant;
ELISA is adopted to detect the expression of the supernatant antibody after the fused cells grow out of the cell mass, and an ELISA plate is coated with SARS-CoV-2 (COVID-19) S protein RBD protein or His protein which is used for eliminating false positive holes of Anti-His;
subcloning the positive hole by adopting a limiting dilution method, and subcloning for three times to finally obtain a positive monoclonal cell strain;
and (3) carrying out ascites preparation on the positive monoclonal antibody, and purifying to obtain a corresponding monoclonal antibody, wherein the monoclonal antibody is further used for affinity determination, competition binding experiments and pseudo virus neutralization experiments.
The B15-5-B3 mentioned in each example below is the code of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody provided by the invention.
EXAMPLE 1 preparation of anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody
Antigen immunization, spleen cell fusion, screening of positive clones and preparation and purification of ascites antibodies
The purpose of the experiment is as follows:
monoclonal antibodies were prepared using the SARS-CoV-2 (COVID-19) S protein RBD protein as antigen.
The experimental method comprises the following steps:
hybridoma techniques are used, including antigen immunization, spleen cell fusion, screening of positive clones, and purification of ascites antibodies. The specific method comprises the following steps:
female BALB/c mice were immunized with 30 μg protein for 4-6 weeks;
boosting once by adopting the same method on the 21 st day after the first immunization;
ELISA determination of serum titer was performed by collecting blood from the canthus at day 35 after the first immunization;
after the antibody titer meets the requirement, 50 mug SARS-CoV-2 (COVID-19) S protein RBD protein is adopted for antigen impact immunization;
spleen cells and SP2/0 cells are fused after 3 days of impact immunization, and after the cells are clustered, anti-SARS-CoV-2 (COVID-19) S protein RBD antibodies in hybridoma supernatant are detected by ELISA.
Experimental results:
through three rounds of subcloning and double screening of affinity and biochemical level competition binding experiments, a monoclonal cell strain with high expression of SARS-CoV-2 (COVID-19) S protein RBD antibody is finally obtained and named as B15-5-B3. The monoclonal cells are amplified and then ascites is carried out to prepare purified antibodies for subsequent affinity determination, competitive binding experiments and pseudovirus neutralization experiments.
Through verification (see the examples 2, 3 and 4 described below in detail), the obtained monoclonal antibody B15-5-B3 has high affinity, can effectively compete and inhibit the combination of SARS-CoV-2 (COVID-19) S protein RBD and mutants (L452R, T478K) and ACE2, and meanwhile, a pseudovirus neutralization experiment shows that the antibody has good neutralizing activity on a novel coronavirus and a mutant strain (Delta) pseudovirus thereof.
EXAMPLE 2 analysis of affinity kinetics of monoclonal antibody B15-5-B3 with SARS-CoV-2 (COVID-19) S protein RBD and its mutant (L452R, T478K)
The purpose of the experiment is as follows:
the affinity of the monoclonal antibody of the invention with the novel coronavirus S protein RBD and mutants thereof was examined.
Reagents and methods:
the affinity kinetic constants of monoclonal antibody B15-5-B3 were measured using the Biacore T200 system using Mouse Antibody Capture Kit, a commercial kit purchased from GE company.
The anti-mouse Fc IgG is immobilized on a CM5 sensor chip by adopting an amino coupling method, monoclonal antibody B15-5-B3 is captured by the coupled anti-mouse Fc IgG, and then a series of concentration gradient SARS-CoV-2 (COVID-19) S protein RBD and mutant (L452R, T478K) proteins thereof are injected. After each cycle, a Glycine-HCl regeneration chip with a pH of 1.7 of the kit is adopted.
The running buffer was HBS-EP+ (10mM HEPES,pH7.4, 150mM Nacl,3mM EDTA and 0.05% P20) and the assay temperature was 25 ℃;
using Biacore T200 evaluation software, according to 1:1 binding model fitting data, binding (Ka) and dissociation (Kd) rate constants and equilibrium constants (Kd) were calculated.
Experimental results:
specific results for each antibody affinity data based on the detection results are shown in fig. 2, 3 and the following table:
conclusion of experiment:
the monoclonal antibody B15-5-B3 of SARS-CoV-2 (COVID-19) S protein RBD and its mutant (L452R, T478K) have high affinity.
EXAMPLE 3 competitive inhibition of SARS-CoV-2 (COVID-19) S protein RBD and its mutant (L452R, T478K) binding to ACE2 by monoclonal antibody B15-5-B3
The purpose of the experiment is as follows:
the competitive inhibition of SARS-CoV-2 (COVID-19) S protein RBD and its mutant (L452R, T478K) binding to ACE2 by monoclonal antibody B15-5-B3 was examined from biochemical levels.
The experimental method comprises the following steps:
coating ACE2 protein into an ELISA plate at the amount of 2 mug/mL and 100 mug/hole, and coating at 4 ℃ overnight; the next day, the coating liquid in the ELISA plate is discarded, PBST is washed 3 times and then incubated for 1.5 hours in a microplate constant temperature oscillator at 37 ℃ by using 2% BSA blocking liquid; 1.5h later, the blocking solution is discarded, PBST is washed 3 times, 0.25 mug/mL SARS-CoV-2 (COVID-19) S protein RBD or mutant thereof (L452R, T478K) and 2 times concentration monoclonal antibody B15-5-B3 gradient solution (4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0 mug/mL) are added into the ELISA plate, 50 mug of each hole is added, each hole is sealed by a sealing film, and incubation is carried out in a constant temperature incubator at 37 ℃ for 1h; after 1h, the liquid was discarded, PBST was washed 4 times, and Anti His-HRP antibody was purified according to 1: diluting 4000, adding 100 mu L of the diluted 4000 into an ELISA plate, sealing with a sealing film, and incubating in a constant-temperature incubator at 37 ℃ for 1h; after 1h, the liquid is discarded, PBST is washed for 4 times, 100 mu L of color development liquid is added into each hole, and the color development is carried out for 15-20min; adding a stop solution after the color development is completed, wherein 50 mu L of stop solution is added into each hole; OD values of wells of the ELISA plate were measured at a wavelength of 450 nm. And the experimental results were analyzed by treatment with GraphPad Prism 6 software.
Experimental results: see fig. 4, 5;
the IC50 of each antibody competition inhibitory effect is summarized in the following table:
antibody name S-RBD S-RBD(L452R-T478K)
EC50(μg/mL) B15-5-B3 0.1636 0.05837
Conclusion of experiment:
the monoclonal antibody B15-5-B3 obtained by the invention has obvious competitive inhibition effect on the combination of SARS-CoV-2 (COVID-19) S protein RBD and mutants (L452R, T478K) thereof and ACE 2.
EXAMPLE 4 detection of neutralizing Activity of monoclonal antibody B15-5-B3 against New coronavirus and mutant (Delta) pseudovirus thereof
The purpose of the experiment is as follows: the neutralizing activity of monoclonal antibody B15-5-B3 against the novel coronavirus and its mutant strain (Delta) was tested.
Reagents and methods:
new crown original pseudoviruses were purchased from Beijing Boolone immunotechnology Co., ltd (accession number BDAA 0026);
new coronavirus Delta mutant was purchased from Fubaiao (Suzhou) biotechnology Co., ltd (cat# FNV 3718)
HEK293-ACE2 cells in logarithmic growth phase were taken at 6X 10 5 The wells were plated into 96 well white plates at 37℃with 5% CO 2 Culturing overnight in an incubator; the next day, taking a sample, diluting the sample according to an experimental design, taking the pseudovirus out of a liquid nitrogen tank after the sample is diluted, quickly and gently thawing in a water bath at 37 ℃, and placing the sample on ice; mixing the diluted monoclonal antibody B15-5-B3 sample with pseudoviruses in a 96-well plate, and then incubating for 1h at room temperature; after incubation, the mixture was added to 96 Kong Baiban pre-plated HEK293-ACE2 cells the day before, and placed at 37℃with 5% CO 2 Culturing in an incubator for 48 hours; after 48h, the culture solution in the pore plate is discarded, 50 mu L of luciferase chromogenic solution is added into each pore, the mixture is incubated for 5min at room temperature, and the value is read by a multifunctional enzyme-labeling chemiluminescence method.
Experimental results: see fig. 6, 7;
the IC50 of neutralizing effect of the antibody novel coronavirus and Delta mutant pseudovirus is summarized in the following table:
antibody name FNV-SARS-CoV-2-S B.1.617.2(Delta)
EC50(ng/mL) B15-5-B3 36.92 269.6
Conclusion of experiment:
the monoclonal antibody B15-5-B3 obtained by the invention has obvious neutralization inhibition activity on the novel coronavirus and the mutant strain (Delta) pseudovirus thereof.
Sequence listing
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Claims (2)

1. An anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody, wherein CDR1, CDR2 and CDR3 in the heavy chain variable region are polypeptides of the amino acid sequences shown below, respectively: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; CDR1, CDR2 and CDR3 in the light chain variable region are polypeptides of the amino acid sequences shown below, respectively: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
2. Use of the monoclonal antibody of claim 1 in the preparation of novel coronavirus SARS-CoV-2 (covd-19) and mutant Delta neutralizing antibody drugs thereof.
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