WO2022110962A1 - Neutralizing antibody against severe acute respiratory syndrome type ii coronavirus (sars-cov-2) - Google Patents

Abstract

Disclosed is a neutralizing antibody against severe acute respiratory syndrome type II coronavirus (SARS-CoV-2), which belongs to the technical field of biomedicine. Peripheral immune cells are extracted from the blood of a patient who has recovered from COVID-19, and B cells that can bind to a novel coronavirus antigen protein-spike protein are screened therefrom. Analysis is then performed on single B cells producing antibodies at the single-cell level, thereby obtaining gene sequences which are in the B cells and encode the variable region heavy and light chains of the neutralizing antibody. These sequences can be used for the in vitro re-construction and expression of the neutralizing antibody that can neutralize novel coronavirus and are expected to be used for the treatment and prevention of diseases such as pneumonia caused by the novel coronavirus.

Description

Neutralizing antibodies against severe acute respiratory syndrome type II coronavirus SARS-COV-2 technical field

The invention relates to a neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2, belonging to the technical field of biomedicine.

Background technique

Pneumonia (COVID-19) caused by infection with severe acute respiratory syndrome type II coronavirus (SARS-CoV 2) is a serious infectious disease with severe impact worldwide. Finding effective treatments against the virus is an urgent need.

Neutralizing antibody refers to an antibody that can eliminate the ability of virus infection after binding to a virus. It is a corresponding antibody produced by B lymphocytes when pathogenic microorganisms invade the body. It is a soluble protein secreted by adaptive immune response cells. When pathogenic microorganisms invade cells, they need to rely on specific molecules expressed by the pathogens themselves to bind to receptors on the cells, in order to infect cells and further expand. Neutralizing antibodies can bind to antigens on the surface of pathogenic microorganisms, thereby preventing the pathogenic microorganisms from adhering to target cell receptors and preventing cell invasion. After the virus invades the human body, B cells secrete neutralizing antibodies into the blood, and the antibodies combine with the virus particles in the blood to prevent the virus from infecting cells and destroy the virus particles, thus "neutralizing" the virus. It can be seen that neutralizing antibodies play a major role in killing the free virus outside the cell.

Certain specific neutralizing antibodies from the blood of cured patients with viral infection have the effect of neutralizing the virus, so they can be used for the treatment of infectious diseases and make the virus lose its pathogenicity. With the advancement of antibody preparation technology, therapeutic antibodies have gradually played a good role in the treatment of various diseases. Existing vaccines, such as measles vaccine, polio vaccine, hepatitis B vaccine, and hepatitis A vaccine, all make vaccinated people produce neutralizing antibodies to prevent virus infection. Since neutralizing antibodies can destroy viruses before they enter cells and can remove free viruses outside cells in the body, neutralizing antibodies in the blood of recovered patients based on viral infections can be used for the treatment of viral infections.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention extracts peripheral immune cells from the blood of recovered patients with COVID-19, and selects B cells that can bind to the novel coronavirus antigen protein-spike protein from the peripheral immune cells. The analysis at the cellular level yielded the gene sequences encoding the heavy and light chains of the variable region of neutralizing antibodies in B cells. These sequences can be used to reconstruct and express neutralizing antibodies that can neutralize the new coronavirus in vitro, and are expected to be used for the treatment and prevention of diseases such as pneumonia caused by the new coronavirus.

The first object of the present invention is to provide a neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2, including: a light chain variable region DNA sequence, and a heavy chain variable region DNA sequence ;

The nucleotide sequence of the light chain variable region DNA sequence is one or more combinations in SEQ ID NO.1-5;

The nucleotide sequence of the heavy chain variable region DNA sequence is one or more of SEQ ID NO.6-8.

Further, the neutralizing antibody is a complete antibody comprising a constant region and a variable region, a partial antibody comprising only the variable region or a chimeric antibody comprising only the variable region.

The second object of the present invention is to provide a gene encoding the neutralizing antibody.

The third object of the present invention is to provide an expression vector carrying the gene.

The fourth object of the present invention is to provide a recombinant cell expressing the neutralizing antibody.

The fifth object of the present invention is to provide the application of the neutralizing antibody in the preparation of a medicine for treating pneumonia COVID-19.

The sixth object of the present invention is to provide a kit for treating pneumonia COVID-19, which contains the neutralizing antibody.

The beneficial effects of the present invention are:

The invention extracts peripheral immune cells from the blood of recovered patients with COVID-19, screens B cells that can bind to the novel coronavirus antigen protein-spike protein, and then analyzes the single B cells that produce antibodies at the single cell level, The gene sequences encoding the heavy and light chains of neutralizing antibody variable regions in B cells were obtained. These sequences can be used to reconstruct and express neutralizing antibodies that can neutralize the new coronavirus in vitro, and are expected to be used for the treatment and prevention of diseases such as pneumonia caused by the new coronavirus.

Description of drawings

Figure 1 is a schematic diagram of the process of screening neutralizing antibodies using single B cells in the blood of patients who have recovered from COVID-19;

Figure 2 is the experimental result of using SDS-PAGE to analyze the antibody after expression and purification;

Figure 3 shows the pseudovirus neutralization test results of negative control antibody Pmab (no neutralizing effect) and neutralizing antibody HC5K VH/B5K VL (IC ₅₀ ～0.1273 μg/mL);

Figure 4 shows the results of the neutralization experiment of the neutralizing antibody D5K VH/B5K VL pseudovirus (IC ₅₀ ～0.00033 μg/mL);

Figure 5 shows the pseudovirus neutralization test results of neutralizing antibody HC5K VH/D4K VL (IC ₅₀ ～0.00084 μg/mL) and neutralizing antibody HF4L VH/D5K VL (IC ₅₀ ～0.0046 μg/mL);

Figure 6 shows the pseudovirus neutralization test results of neutralizing antibody HC5K VH/D5K VL (IC ₅₀ ～0.0066 μg/mL) and neutralizing antibody HD5K VH/A5K VL (IC ₅₀ ～0.0017 μg/mL);

Figure 7 shows the pseudovirus neutralization test results of neutralizing antibody HF4L VH/A5K VL (IC ₅₀ ～0.6049 μg/mL) and neutralizing antibody HF4L VH/B5K VL (IC ₅₀ ～0.6077 μg/mL);

Fig. 8 is the result of pseudovirus neutralization experiment of neutralizing antibody HD5K VH/D4K VL (IC ₅₀ ～0.1996 μg/mL).

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Example 1: Isolation and extraction of single B cells that can be combined with SARS-COV-2

About 15 mL of blood was drawn from multiple recovered patients with new coronavirus pneumonia, and the peripheral blood mononuclear cells (PBMCs) in the blood of each recovered patient were separated and extracted by the Ficoll gradient method, and the obtained PBMCs were washed twice before use.

Fc block was added to each PBMC sample first, and after 15 minutes of action, APC-H7-labeled anti-human CD3 antibody, BV421-labeled anti-human CD19 antibody, BB700-labeled anti-human CD27 antibody, and Biotin-labeled SARS-COV- 2 Spike protein, Biotin-tagged SARS-COV-2 Spike RBD. Streptavidin-APC was then added and allowed to act for 30 minutes. Afterwards, FACS AriaTMIII flow cytometer was used to load and analyze the antibody-labeled PBMC samples. CD3 is a specific surface marker for T cells, CD19 is a specific surface marker for B cells, and CD27 is a specific surface marker for memory B cells. Biotin-labeled two antigens can be specific to APC-labeled Streptavidin. Therefore, APC-positive cells are cells that can specifically bind to SARS-COV-2. The cells selected from CD3 ^- CD19 ⁺ CD27 ⁺ APC ⁺ are the memory B cells we are looking for that can specifically bind to SARS-COV-2. The B cells of this type were isolated from single B cells by flow cytometry and directly injected into a 96-well plate pre-loaded with cell lysate, sealed immediately, and quickly frozen on dry ice, and then stored at -80 °C until later. use. In this way, single B cells that can specifically bind to SARS-COV-2 can be isolated.

Example 2: Amplification and sequencing of variable region light and heavy chains in single B cells

Because B cells are cells that secrete antibodies, the sequence information of antibodies is stored in B cells that can bind to SARS-COV-2. Divide the lysed single B cell lysate in the 96-well plate into 3 parts. One for Kappa light chain sequence analysis, one for lamda light chain sequence analysis, and one for heavy chain sequence analysis.

In this part of the experiment, RT-PCR was used to reverse transcription to obtain a cDNA library, and then nested PCR technology was used to amplify the DNA sequence in a single cell. Since the primers used in the experiment are specific primers for the variable region of the light chain or the variable region of the heavy chain, the obtained sequences are the sequences of the light chain and heavy chain portions of the antibody variable region in the single B cell. The amplified sequences are subjected to DNA sequencing analysis to obtain the sequence information of the variable region light chain and heavy chain of the antibody contained in a single B cell.

RT-PCR amplification of single B cell antibody light and heavy chain variable region genes

Design RT-PCR primers (SEQ ID NO.9-25), as shown in Table 1:

Table 1

Single B cell RT-PCR was performed using the sorted single B cells as templates. The PCR system configuration and sample addition were completed in a biological safety cabinet and placed on ice for operation. The RT-PCR reaction system and reaction conditions were as follows:

PCR reaction system:

Table 2

Element	system	Final concentration
RNase-free water	-	-
5 x RT-PCR buffer	10.0μl	1×
dNTP mix	2.0μl	400μM each dNTP
A primer	-	0.6μM
B primer	-	0.6μM
mixed enzyme	2.0μl
RNase inhibitor	-	5-10 units
template RNA	-	1pg–2μg
Overall system	50.0μl	-

PCR reaction conditions:

Nested PCR Amplification of Single B Cell Antibody Light and Heavy Chain Variable Region Genes

Design nested PCR primers (SEQ ID NO.26-38), as shown in Table 3:

table 3

Using the RT-PCR product as a template, three PCR reaction systems were established to amplify the variable region genes of the H chain, κ chain and λ chain of the antibody respectively. Each reaction system used the corresponding mixed primers, and the nested PCR reaction The system and reaction conditions are as follows:

PCR reaction system:

Table 4

PCR reagents	Volume per sample (μl) (25μl system)	final concentration
Taq DNA polymerase	0.25	50U ml -1
10x buffer	2.5	1×
dNTPs (10mM)	0.5	200μM
Forward primers: VH3a and VH3b or PanVκ	0.5	1.2μM
Reverse primer: PW-Cgamma or CK494-516	0.5	1.2μM
RNase-free water	17.25-19.25 (fill the system to 24μl)	-
template	1.0	-

PCR reaction conditions:

Example 3: Experimental Results

This application obtains a total of 5 light chain variable region sequences and 3 heavy chain variable region sequences from B cells of recovered patients that can bind to the new coronavirus spike protein. The five light chain variable region sequences and the three heavy chain variable region sequences can be freely combined to form an antibody, which is a neutralizing antibody of the new coronavirus SARS-COV-2.

Among them, the 5 light chain variable regions are named A5K VL (SEQ ID NO.1), B5K VL (SEQ ID NO.2), D4K VL (SEQ ID NO.3), D5K VL (SEQ ID NO.4) ) and D7K VL (SEQ ID NO.5), the three heavy chain variable regions were named as HD5K VH (SEQ ID NO.6), HF4L VH (SEQ ID NO.7) and HC5K VH (SEQ ID NO.8 ).

Antibody expression and purification were performed as follows:

In this example, 9 antibodies were constructed, namely: HC5K VH/B5K VL, HD5K VH/D4K VL, HF4L VH/A5K VL, HF4L VH/B5K VL, HD5K VH/B5K VL, HC5K VH/D4K VL, HF4L VH /D5K VL, HC5K VH/D5K VL and HD5K VH/A5K VL.

1. Vector construction

Primers were designed according to the corresponding antibody light and heavy chains to amplify the corresponding VH and VL sequences. Using recombinase (ClonExpress II One Step Cloning Kit, C112-01/02, Vazyme), the amplified VH and VL sequences were recombined into the antibody expression vector backbone plasmid, and the bacterial solution was sent to sequencing for verification.

2. Plasmid extraction

The cloned bacteria that were verified to be correct were inoculated into LB (Amp 100ug/ml) medium, cultured at 37°C overnight, and the plasmid was extracted with a plasmid extraction kit the next day, and the concentration was determined.

3. Transient expression of cells

Transient the extracted plasmid into 293F cells, as follows:

1) Count 293F in logarithmic growth phase, resuspend the cells with fresh 293 medium to a density of 2*10 ⁶ /mL, and the total volume is 100 mL.

2) Dilute 100ug of plasmid with medium.

3) Dilute PEI (1 μg/μL) with medium, and add the diluted PEI to the diluted plasmid DNA. Mix by swirling and/or inverting the tube or pipetting gently 2 to 3 times. The complexes were incubated at room temperature for about 20 minutes.

4) The plasmid/PEI mixture was added to the cell suspension, and the flask was shaken gently during the addition, and placed at 37° C., 8% CO ₂ , and cultured at 125 rpm.

5) Add 5% (v/v) feed to the shake flask 16-22 hours after transfection, shake the shake flask gently during the addition, and place the shake flask back into the 37°C incubator.

6) Harvest the supernatant on day 6.

4. Antibody purification

1) Harvest supernatant

The supernatant was harvested by centrifugation. The supernatant was filtered with a 0.4um membrane filter and docked to a purification column.

2) Protein purification

The Protein A affinity filler Mabselect SuReTM from GE was selected to remove the medium components and enrich the target protein.

5. The purified protein was analyzed by SDS-PAGE

The purified antibody was analyzed by SDS-PAGE electrophoresis. The separating gel concentration used during the analysis was 4-20%. The sample was first reduced, then the reduced sample was added to the Loading Buffer, boiled at 70°C for 10 min, and then loaded for analysis. The results are shown in Figure 2.

Example 4: Pseudovirus neutralization experiment

In this experiment, the pseudovirus carrying the 2019-nCoV spike protein was used to evaluate the neutralizing antibody of 2019-nCoV. The virus system uses HIV-1 carrying a luciferase reporter gene as the viral backbone, and at the same time expresses the new coronavirus Spike protein on the virus shell, and the formed pseudovirion infects exogenous cell lines with high expression of ACE2, which highly simulates the passage of the new coronavirus through the virus. In the invasion process of Spike-ACE2 to target cells, the degree of infection of target cells by the pseudovirus particles was positively correlated with the luminescence value of luciferase, and negatively correlated with the neutralizing activity of antibodies.

The neutralizing antibody binds to the S protein of the virus coat in vitro, blocking the site on the S protein that binds to ACE2, so that the S protein cannot bind to ACE2, and the virus cannot invade cells; on the contrary, antibodies without neutralizing activity cannot interfere with the S protein. Binding to ACE2 on the cell surface, the virus entering the cell will express the Fluc protein, and after reacting with the luminescent substrate, its luminescence value is detected by a microplate reader.

Experimental steps:

1. Sample preparation: transfer the pseudovirus from -80°C to 4°C refrigerator or thaw on ice in advance, dilute the virus to 1-2×10 ⁴ TCID ₅₀ /mL with DMEM medium containing 10% FBS before use If the transfected ACE2 overexpressing cell line was not purchased by our company, the optimal TCID50 needs to be explored by yourself).

2. Take a new 96-well cell culture plate for sample dilution.

3. Add 135 μL of the sample to be tested to the second column of the 96-well cell culture plate.

4. Add 90μl of serum-free DMEM to columns 3-10, and then use a drain gun to take 45μl of dilution from column 2 and add it to column 3 for doubling dilution until it reaches column 9, and the last column has an excess of 45μL of liquid. discard. Another 90 μl of the diluted pseudovirus solution was added to each well. 90 μL of pseudovirus was added to the virus control group (VC), and 180 μL of serum-containing DMEM medium was added to the cell control group (CC), and the 96-well cell culture plate was incubated in a 37°C incubator for 1 h.

5. The sample-pseudovirus mixture in the 96-well cell culture plate and the two groups of controls were divided into three and transferred to a 96-well white plate, 50 μL/well (3 replicates).

6. Immediately spread 50 μl of 293-ACE2 cells at a density of 0.4×10 ⁶ cells/ml (2×10 ⁴ cells/well) into a 96-well white plate (no need to change the medium), and place it in a 37°C incubator incubate.

7. After 20-24 hours of incubation, add 25 μL of DMEM medium containing 10% FBS pre-warmed at 37°C to each well.

8. After culturing for 48 hours, take out the 96-well white plate, equilibrate to room temperature, add 125 μl of room temperature-equilibrated Bio-Lite reporter gene detection reagent to each well, shake the plate for 2 minutes, and then use a microplate reader to detect the chemiluminescence value after standing at room temperature for 5 minutes. (RLU).

9. Data processing: The log value of the antibody concentration and the corresponding RLU are brought into the graph pad software, and the IC50 value is calculated and compared.

The experimental results are shown in Table 5 and Figures 3 to 8: the neutralizing antibodies of the present invention all have a certain ability to neutralize the new coronavirus. However, the neutralizing ability of each antibody against the new coronavirus varies. The lower the IC50 value in the virus neutralization experiment, the better, indicating that very little antibody is required to neutralize the virus. The reported IC50 values of neutralizing antibodies against 2019-nCoV in virus neutralization experiments are mostly in the range of 0.5 μg/mL to 0.0012 μg/mL. Among the new coronavirus neutralizing antibodies of the present invention, some antibodies such as HD5K VH/B5K VL and HC5K VH/D4K VL have IC50s of 0.00033 μg/mL and 0.00084 μg/mL in the virus neutralization experiment, which are larger than Most SARS-CoV-2 neutralizing antibodies are stronger at neutralizing the virus.

Table 5 Virus neutralizing ability of neutralizing antibodies

Antibody	Virus neutralization assay IC 50 (μg/mL)
HC5K VH/B5K VL	0.1273
HD5K VH/D4K VL	0.1996
HF4L VH/A5K VL	0.6049
HF4L VH/B5K VL	0.6077
HD5K VH/B5K VL	0.00033
HC5K VH/D4K VL	0.00084
HF4L VH/D5K VL	0.0046
HC5K VH/D5K VL	0.0066
HD5K VH/A5K VL	0.0017

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention, such as base substitution within 3% of the heavy chain or light chain sequence of the antibody variable region, or substitution of the heavy chain or light chain of the antibody variable region Amino acid substitution within 3% of the amino acid sequence of the antibody part expressed by the sequence is within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims (7)

1. A neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2, characterized in that it comprises: a light chain variable region DNA sequence, and a heavy chain variable region DNA sequence;

   The nucleotide sequence of the light chain variable region DNA sequence is one or more combinations in SEQ ID NO.1-5;

   The nucleotide sequence of the heavy chain variable region DNA sequence is one or more of SEQ ID NO.6-8.
2. The neutralizing antibody according to claim 1, wherein the neutralizing antibody is a complete antibody comprising a constant region and a variable region, a partial antibody comprising only the variable region, or a chimeric antibody comprising only the variable region Antibody.
3. A gene encoding the neutralizing antibody of claim 1 or 2.
4. An expression vector carrying the gene of claim 3.
5. A recombinant cell expressing the neutralizing antibody of claim 1 or 2.
6. The application of the neutralizing antibody of claim 1 or 2 in the preparation of a medicine for the treatment of pneumonia COVID-19.
7. A kit for treating pneumonia COVID-19, characterized in that the kit contains the neutralizing antibody of claim 1 or 2.

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