CN113999301A

CN113999301A - anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody

Info

Publication number: CN113999301A
Application number: CN202111485922.2A
Authority: CN
Inventors: 廖高勇; 丁海剑; 陈海; 张怡
Original assignee: Xintrum Pharmaceuticals Ltd
Current assignee: Xintrum Pharmaceuticals Ltd
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-02-01
Anticipated expiration: 2041-12-07
Also published as: CN113999301B

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 SARS-CoV-2(COVID-19) S protein RBD and mutants thereof, can effectively inhibit the combination of the SARS-CoV-2(COVID-19) S protein RBD and mutants thereof (L452R, T478K) and ACE2, and has obvious neutralization activity on new crown pseudoviruses and mutant strains thereof (Delta) pseudoviruses.

Description

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

The technical field is as follows:

the invention relates to a genetic engineering antibody, in particular to a monoclonal antibody medicament developed aiming at a novel coronavirus S-RBD target spot.

Background art:

it is known that the novel coronavirus SARS-CoV-2(COVID-19) is infected by binding of its Receptor Binding Domain (RBD) of S protein to the host receptor angiotensin converting enzyme 2(ACE2) to mediate entry of the virus into host cells. The S protein comprises two functional subunits, S1 and S2, where S1 is responsible for binding to host cell receptors and the S2 subunit is responsible for viral membrane and cell membrane fusion. During infection, the S protein is cleaved by host proteases into an N-terminal S1 subunit and a C-terminal S2 subunit, and is converted from a pre-fusion state to a post-fusion state. S1 and S2 are composed 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) and is critical in determining tissue tropism and host range.

The Receptor Binding Domain (RBD) of the S protein of SARS-CoV-2(COVID-19) is immunodominant and is also the target for 90% of the neutralizing antibodies present in SARS-CoV-2 immune serum. Therefore, the development of antibody drugs aiming at the S-RBD target of the new coronavirus and the mutant thereof is an effective way for treating patients infected by the new coronavirus, and the developed monoclonal antibody has the potential to become a specific drug for treating the infection of the new coronavirus.

Disclosure of Invention

The invention aims to provide a monoclonal antibody which can effectively compete with ACE2 to bind to SARS-CoV-2(COVID-19) S protein RBD and mutants thereof (L452R, T478K) and is used for preparing a medicament for treating novel coronavirus related diseases.

The invention uses SARS-CoV-2(COVID-19) S protein RBD protein and mutant (L452R, T478K) protein to immunize BALB/c mice, and obtains a brand-new anti-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 the prior anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody.

The SARS-CoV-2(COVID-19) S protein RBD protein used was purchased from Biotech, Inc., Baipuseus, Beijing, and the mouse used was a BALB/c mouse.

Specifically, the present invention accomplishes the above-mentioned work by the following means:

a SARS-CoV-2(COVID-19) S protein RBD protein as antigen, immunized at 30 μ g/mouse BALB/c, three weeks later the same dose of boost;

b, measuring the antibody titer in the serum of the immunized mouse by an ELISA method, and performing impact immunization at a dose of 50 mu g after an ideal effect is achieved;

c, fusing the spleen cells of the mice successfully immunized with SP2/0 cells, carrying out supernatant titer detection after the cells grow into cell masses, and obtaining a positive monoclonal cell strain through three rounds of subcloning;

d, carrying out mouse intraperitoneal injection to prepare ascites after the monoclonal cell strain is subjected to amplification culture, and purifying the collected ascites to obtain a corresponding antibody;

e, utilizing an SPR technology to carry out affinity kinetic determination on the monoclonal antibody;

f, determining the competitive inhibition effect of the monoclonal antibody on the combination of SARS-CoV-2(COVID-19) S protein RBD and a mutant thereof (L452R, T478K) and ACE 2;

g determination of neutralizing Activity of monoclonal antibodies against New crown pseudoviruses and their mutant strains (Delta) pseudoviruses.

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 for the specificity of this antibody is primarily from its highly variable regions CDR1, CDR2, and CDR3, which are key sites for antigen binding.

The CDR1, CDR2 and CDR3 of the heavy chain and light chain of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody obtained by the invention are respectively the polypeptides with the amino acid sequences as shown in the following:

B15-5-B3: 1, 2 and 3; 4, 5 and 6;

the term "monoclonal antibody" as used herein should be construed to encompass any specific binding member having a binding domain of the desired specificity, either monovalent or single chain antibodies, diabodies, chimeric antibodies, as well as derivatives, functional equivalents and homologs of any of the foregoing, and also includes antibody fragments and any polypeptides comprising an antigen binding domain.

Examples of the monoclonal antibody of the present invention are an immunoglobulin IgG subtype and a subtype subclass thereof;

although the molecular basis for antibody specificity arises primarily from its highly variable regions CDR1, CDR2, and CDR3, the sequences of the CDRs should be preserved as much as possible in order to maintain preferred binding properties. However, although it is possible to achieve the object of the invention, even more optimal binding properties, if there are individual amino acid changes. However, individual amino acid changes do not depart from the spirit and concept 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 by other sequences under conditions that do not affect the desired three-dimensional structure of the binding.

The following experiments prove that the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody B15-5-B3 obtained by the invention is used:

1. affinity kinetic analysis

The results show that the monoclonal antibody of the invention has high affinity with SARS-CoV-2(COVID-19) S protein RBD and its mutant (L452R, T478K) (see example 2 for details).

2. Competitive inhibition of binding to ACE2

Biochemical level experiment results show that the monoclonal antibody of the invention can obviously inhibit the combination of SARS-CoV-2(COVID-19) S protein RBD and mutants thereof (L452R, T478K) and ACE2 (see example 3 for details).

3. Detection of neutralizing Activity against New crown pseudovirus and mutant Strain (Delta) pseudovirus thereof

The results show that the monoclonal antibody of the invention has obvious neutralization inhibition activity on both the new crown pseudovirus and mutant strain (Delta) pseudovirus (see example 4 for details).

Drawings

FIG. 1 is a schematic diagram of the steps for obtaining an anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody according to the present invention;

FIGS. 2 and 3 are the results of measurement of the affinity of the antibody B15-5-B3 for SARS-CoV-2(COVID-19) S protein RBD and its mutant (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 are the results of experiments on the competitive inhibition of the antibody B15-5-B3 on the binding of SARS-CoV-2(COVID-19) S protein RBD and its mutant (L452R, T478K) to ACE2 in example 3;

FIGS. 6 and 7 show the results of experiments for detecting the neutralizing activity of the antibodies B15-5-B3 against the novel coronavirus and its mutant strain (Delta) pseudovirus in example 4.

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, CDR1, CDR2 and CDR3 of the heavy chain variable region of the RBD monoclonal antibody B15-5-B3 against SARS-CoV-2(COVID-19) S protein, respectively;

SEQ ID NO 4, 5 and 6, CDR1, CDR2 and CDR3 of the light chain variable region of the RBD monoclonal antibody B15-5-B3 against SARS-CoV-2(COVID-19) S protein;

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

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the following examples further illustrate the present invention in detail. It should be understood that the particular methods, reagents, etc. used in the examples are illustrative only and are not limiting upon the scope of the invention;

the invention provides heavy chain and light chain sequences of a specific anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody. The monoclonal antibody is expressed by a corresponding monoclonal cell strain obtained by screening hybridoma after a BALB/c mouse is immunized by SARS-CoV-2(COVID-19) S protein RBD protein; the monoclonal antibodies are IgG types;

the SARS-CoV-2(COVID-19) S protein RBD protein and each mutant protein, protein C-terminal containing 6 XHis tag, mentioned in the examples below, were purchased from Beijing Bethes Biotech Ltd.

The used immunologic adjuvant is a quick immunologic adjuvant for 5 weeks of Beijing Boolong, the immunization is strengthened once after 21 days of primary immunization, and the cell fusion can be carried out once by using antigen impact immunization before the cell fusion;

the fusion method is electrofusion, the electrofusion instrument is ECM2001 of BTX company, and the fusion buffer solution is original plant cell fusion solution;

after the fused cells grow out cell groups, ELISA is adopted to detect the expression of the supernatant antibody, an ELISA plate is coated with SARS-CoV-2(COVID-19) S protein RBD protein or His protein, and the His protein is used for eliminating false positive holes of Anti-His;

carrying out subcloning on the positive hole by adopting a limiting dilution method, carrying out three rounds of subcloning, and finally obtaining a positive monoclonal cell strain;

and (3) purifying the positive monoclonal antibody after ascites preparation to obtain a corresponding monoclonal antibody, and further using the monoclonal antibody for affinity determination, competitive binding experiments and pseudovirus neutralization experiments.

The B15-5-B3 mentioned in the following examples is the code number of the anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody provided by the present invention.

EXAMPLE 1 preparation of anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody

Antigen immunity, spleen cell fusion, screening of positive clone and preparation and purification of ascites antibody

Purpose of the experiment:

a monoclonal antibody was prepared using the RBD protein of SARS-CoV-2(COVID-19) S protein as an antigen.

The experimental method comprises the following steps:

hybridoma technology is used, including antigen immunization, spleen cell fusion, screening of positive clones and preparation and purification of ascites antibodies. The specific method comprises the following steps:

female BALB/c mice at 4-6 weeks were immunized with 30 μ g of protein;

the same method is adopted to strengthen the immunity once on the 21 st day after the first immunization;

ELISA determination of serum titer by canthus blood collection serum was performed on day 35 after the first immunization;

after the antibody titer meets the requirement, 50 mu g of SARS-CoV-2(COVID-19) S protein RBD protein is adopted for antigen impact immunization;

splenocytes taken 3 days after the impact immunization were fused with SP2/0 cells, and the supernatant of the hybridomas were tested for antibodies against the SARS-CoV-2(COVID-19) S protein RBD by ELISA after cell clumping.

The experimental results are as follows:

after three rounds of subcloning, through double screening of affinity and biochemical level competition binding experiments, a SARS-CoV-2(COVID-19) S protein RBD antibody high expression monoclonal cell strain is finally obtained, which is named as B15-5-B3. Amplifying the monoclonal cells, and then preparing the purified antibody from ascites for subsequent affinity determination, competitive binding experiments and pseudovirus neutralization experiments.

Through verification (see the following examples 2, 3 and 4 for details), 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 thereof (L452R, T478K) and ACE2, and meanwhile, a pseudovirus neutralization experiment shows that the antibody has good neutralizing activity on new crown pseudoviruses and mutant strains thereof (Delta) pseudoviruses.

Example 2 analysis of the affinity kinetics of monoclonal antibody B15-5-B3 with the SARS-CoV-2(COVID-19) S protein RBD and its mutants (L452R, T478K)

Purpose of the experiment:

the affinity of the monoclonal antibody of the invention and the new coronavirus S protein RBD and the mutant thereof is detected.

Reagents and methods:

the affinity kinetic constants of monoclonal Antibody B15-5-B3 were determined using the Biacore T200 system using the Mouse Antibody Capture Kit, a commercial Kit from GE.

The anti-mouse Fc IgG is fixed on a CM5 sensor chip by adopting an amino coupling method, the 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) protein thereof are injected. After each cycle, a Glycine-HCl regeneration chip with the pH of 1.7 is adopted.

The running buffer was HBS-EP + (10mM HEPES, pH7.4, 150mM NaCl, 3mM EDTA and 0.05% P20), measured at 25 ℃;

using Biacore T200 evaluation software, according to 1: 1 binding model the data were fitted and the binding (Ka) and dissociation (Kd) rate constants and equilibrium constants (Kd) were calculated.

The experimental results are as follows:

the specific results of the affinity data for each antibody, based on the assay results, are shown in fig. 2, fig. 3, and the following table:

and (4) experimental conclusion:

the monoclonal antibody B15-5-B3 for resisting the SARS-CoV-2(COVID-19) S protein RBD, which is obtained by the invention, has high affinity with the SARS-CoV-2(COVID-19) S protein RBD and mutants thereof (L452R, T478K).

EXAMPLE 3 competitive inhibition of the binding of monoclonal antibody B15-5-B3 to SARS-CoV-2(COVID-19) S protein RBD and its mutants (L452R, T478K) and ACE2

Purpose of the experiment:

the competitive inhibition of the monoclonal antibody B15-5-B3 on the binding of SARS-CoV-2(COVID-19) S protein RBD and its mutant (L452R, T478K) to ACE2 was examined at the biochemical level.

The experimental method comprises the following steps:

the ACE2 protein is coated into an enzyme label plate at the amount of 2 mu g/mL and 100 mu L/hole, and is coated overnight at 4 ℃; the next day, the coating solution in the ELISA plate was discarded, PBST was washed 3 times and incubated with 2% BSA blocking solution in a microplate constant temperature shaker at 37 ℃ for 1.5 h; 1.5h later, the blocking solution is discarded, PBST is washed for 3 times, prepared 0.25 mu g/mL SARS-CoV-2(COVID-19) S protein RBD or a mutant thereof (L452R, T478K) and 2 times concentration of monoclonal antibody B15-5-B3 gradient solution (4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0 mu g/mL) are added into an enzyme label plate, 50 mu L of each hole is added, the plate is sealed by a sealing film, and the plate is incubated for 1h in a 37 ℃ incubator; after 1h the liquid was discarded, PBST washed 4 times, and Anti His-HRP antibody was added as 1: diluting according to the proportion of 4000, adding the diluted solution into an enzyme label plate, sealing each hole by using a sealing film with the volume of 100 mu L, and incubating for 1h in a constant-temperature incubator at 37 ℃; discarding the liquid after 1h, washing with PBST for 4 times, adding 100 μ L of color development liquid into each well, and developing for 15-20 min; adding stop solution 50 mu L per well after color development is finished; and measuring the OD value of each hole of the ELISA plate at the wavelength of 450 nm. And the experimental results were processed and analyzed using GraphPad Prism 6 software.

The experimental results are as follows: see fig. 4, 5;

the competitive inhibitory effect IC50 for each antibody is summarized in the following table:

	name of antibody	S-RBD	S-RBD(L452R-T478K)
				EC50(μg/mL)	B15-5-B3	0.1636	0.05837

And (4) experimental conclusion:

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 its mutant (L452R, T478K) and ACE 2.

EXAMPLE 4 detection of neutralizing Activity of monoclonal antibody B15-5-B3 against New crown pseudovirus and its mutant (Delta) pseudovirus

Purpose of the experiment: monoclonal antibody B15-5-B3 was tested for neutralizing activity against the novel coronaviruses and mutants thereof (Delta).

Reagents and methods:

the new crown original pseudovirus is purchased from Beijing Boolong immune technology Co., Ltd (cargo number BDAA 0026);

new crown pseudovirus Delta mutant purchased from Bai' ao (Suzhou) Biotechnology Co., Ltd. (Cat: FNV3718)

Taking HEK293-ACE2 cells in logarithmic growth phase at 6 × 10⁵The amount of each well was spread on a 96-well white plate at 37 ℃ with 5% CO₂Culturing in an incubator overnight; the next day, samples were taken, samples were diluted according to the experimental design, pseudoviruses were removed from the liquid nitrogen tank after sample dilution was complete, and water bath at 37 ℃ was used to rapidly soften the virusThawing and placing on ice; mixing the diluted monoclonal antibody B15-5-B3 sample with pseudovirus in a 96-well plate, and then incubating for 1h at room temperature; after incubation, the mixture was added to a 96-well white plate on which HEK293-ACE2 cells had been previously plated the day before, and placed at 37 ℃ in 5% CO₂Continuously culturing for 48h in the incubator; and after 48 hours, discarding the culture solution in the pore plate, adding 50 mu L of luciferase developing solution into each pore, incubating at room temperature for 5min, and reading the value by a chemiluminescence method of a multifunctional microplate reader.

The experimental results are as follows: see fig. 6, 7;

the neutralizing effect IC50 of the antibody new crown pseudovirus and Delta mutant pseudovirus is summarized in the following table:

	name of antibody	FNV-SARS-CoV-2-S	B.1.617.2(Delta)
				EC50(ng/mL)	B15-5-B3	36.92	269.6

And (4) experimental conclusion:

the monoclonal antibody B15-5-B3 obtained by the invention has obvious neutralization inhibition activity on new crown pseudoviruses and mutant strains (Delta) pseudoviruses thereof.

Sequence listing

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Claims

1. An anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody, which contains one or more of the polypeptides with the amino acid sequences as shown in the following:

1, 2, 3, 4, 5 or 6.

2. A polypeptide having the amino acid sequence shown below:

1, 2, 3, 4, 5 or 6.

3. Use of one or more of the polypeptides of claim 2 for the preparation of an anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody.

4. Use of one or more of the polypeptides of claim 2 for the preparation of a medicament of neutralizing antibodies against novel coronaviruses and mutants thereof.

5. Two groups are providedMultiple purposePeptides, each group comprising three polypeptides, the amino acid sequence of which is shown below:

group 1: 1, 2 and 3;

and 2, group: 4, 5 and 6.

6. Monoclonal antibodies comprising either or both of the two sets of polypeptides of claim 5.

7. Use of either or both of the two sets of polypeptides of claim 5 in the preparation of an anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody.

8. Use of either or both of the two sets of polypeptides of claim 5 in the preparation of a medicament for neutralizing antibodies against novel coronaviruses and mutants thereof.

9. An anti-SARS-CoV-2 (COVID-19) S protein RBD monoclonal antibody, wherein CDR1, CDR2 and CDR3 in the heavy chain and light chain variable region are respectively the polypeptide with the amino acid sequences as shown in the following:

heavy chain variable region SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; light chain variable regions SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6.

10. Use of the monoclonal antibody of claim 9 for the preparation of a medicament for neutralizing antibodies against novel coronaviruses and mutants thereof.