Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody

A coronavirus and antibody technology, applied in the field of immunity, can solve problems such as drug resistance and reduced neutralization activity, and achieve the effect of avoiding biosafety risks

Active Publication Date: 2022-05-31
TSINGHUA UNIV +1
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AI-Extracted Technical Summary

Problems solved by technology

[0003] Most of the anti-SARS-CoV-2 neutralizing antibodies that are currently approved or in different research stages have no significant reduction or only partially reduce the...
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Method used

(2) sample preparation: the cell supernatant is filtered with a 0.45 μm filter membrane to reduce impurities, improve protein purification efficiency and prevent clogged columns.
(3) add equal volume solution PC (if the gel weighs 0.1 g, its volume can be regarded as 100 μl, then add 100 μl PC solution) to the gel block, place it in a water bath at 50°C for about 10 min, during which Gently invert the centrifuge tube up and down frequently to ensure that the gel is fully dissolved.
(7) ...
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Abstract

The invention relates to a broad-spectrum neutralizing antibody for resisting novel coronavirus and application of the broad-spectrum neutralizing antibody. The invention provides an anti-novel coronavirus broad-spectrum neutralizing antibody or an antigen binding fragment thereof, the broad-spectrum neutralizing antibody or the antigen binding fragment thereof has a heavy chain variable region containing VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region containing VLCDR1, VLCDR2 and VLCDR3, the VHCDR1, the VHCDR2 and the VHCDR3 respectively comprise amino acid sequences shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, and the VLCDR1, the VLCDR2 and the VLCDR3 respectively comprise amino acid sequences shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6. The finally screened antibody has efficient and broad-spectrum neutralizing activity, the IC50 of the antibody to a wild type of the novel coronavirus and 13 mutant strains is less than 0.05 g/ml, and the IC50 of the antibody to WT, Beta, Delta and Omicro BA.1 live viruses is less than 0.1 g/ml.

Application Domain

Immunoglobulins against virusesAntivirals +4

Technology Topic

AntigenMolecular biology +12

Image

  • Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody
  • Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody
  • Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody

Examples

  • Experimental program(4)
  • Effect test(1)

Example Embodiment

[0078] Example 1. Preparation of anti-novel coronavirus monoclonal antibodies
[0079] 1. Detection of plasma binding activity
[0080] 1. Sample: plasma of volunteers immunized twice with novel coronavirus inactivated vaccine (BBIBP-CorV).
[0081] 2. Detection of binding activity: The binding titers of 28 samples to the RBD protein of the novel coronavirus Omicron mutant strain (B.1.1.529) were detected. Methods as below:
[0082] (1) Coating: Dilute the above RBD protein with PBS to 0.5 µg/ml, add 100 µl/well to a 96-well ELISA plate, and coat overnight at 4°C.
[0083] (2) Blocking: discard the coating solution, add 200 µl blocking solution (PBST+3%BSA) to each well, and block at 37°C for 1 h.
[0084] (3) Washing: The blocking solution was discarded and washed three times with PBST.
[0085] (4) Sample: each plasma sample was diluted 100 times, serially diluted 3 times, and a negative control was added, and incubated at 37°C for 2 h.
[0086] (5) Washing: The sample was discarded and washed 5 times with PBST.
[0087] (6) Secondary antibody: Dilute goat anti-human IgG antibody with diluent (PBST+1%BSA) 1:50000, add 100 µl to each well, and incubate at 37°C for 30 min.
[0088] (7) Washing: The sample was discarded and washed 5 times with PBST.
[0089] (8) Color development: add 100 µl of color developing solution to each well, and incubate at room temperature for 15 minutes in the dark.
[0090] (9) Stop: Add 50 µl stop solution to each well.
[0091] (10) Reading: Read the absorbance at 450 nm and 630 nm with a microplate reader, and the result is the reading value at 450 nm minus the reading value at 630 nm.
[0092] (11) Calculate the binding titer of plasma: the mean value of OD450 of the plasma samples before immunization + 2×SD is taken as the cut-off value, and the maximum dilution higher than this value is the binding titer of the sample.
[0093] The results are shown in Table 1 below: After activity screening, sample No. 17 with a binding titer higher than 1000 can be used as a candidate sample for neutralizing antibody sorting.
[0094] Table 1. Test results for sample binding titers
[0095]
[0096] 2. Sorting of novel coronavirus-specific single memory B cells
[0097] (1) PBMC recovery: freeze the PBMC of the candidate sample No. 17 obtained in the above step 1 (about 1×10 7 A) were rapidly recovered in a 37°C water bath, resuspended in 10 ml of pre-warmed RPMI 1640 medium (+10% FBS) at 37°C, and centrifuged at 400 × g for 5 min.
[0098] (2) Labeling: discard the medium, resuspend the cells with 10 ml of pre-cooled cell staining buffer, centrifuge at 400×g for 5 min, discard the supernatant, and resuspend the cells in 100 μl of cell staining buffer; Add 0.4 µg biotin-labeled RBD protein of novel coronavirus Omicron mutant strain to the cells, mix well, incubate at 4°C for 1 h; wash twice with 2 ml of pre-cooled cell staining buffer, and add 95 µl cells Resuspend cells in staining buffer; fluorescein-labeled antibodies (CD3-Pacific Blue, CD8a-Pacific Blue, CD14-Pacific Blue, CD19-FITC, CD27-PerCP-Cy5.5, PE streptavidin, APC chain Mycovidin) was mixed into the cell suspension, incubated at 4°C for 30 min; washed twice with 2 ml of pre-cooled cell staining buffer, and resuspended in 500 μl of cell staining buffer to obtain labeled PBMC.
[0099] (3) Sorting: The labeled PBMCs were analyzed and sorted on a Sony MA900 Cell Sorter flow cytometer, and CD3 cells were sorted out. − CD8 − CD14 − CD19 + CD27 + RBDomicron + B cells (see figure 1 ), which are antigen-specific memory B cells, a total of 35 cells were obtained. Antigen-specific memory B cells were aliquoted individually into 96-well PCR plates containing 4 µl of lysis buffer (0.5×PBS, 10 mM DTT, 10 U RNase Inhibitor).
[0100] (4) Cryopreservation: quickly placed in dry ice, directly used for reverse transcription or moved to -80°C freezer for freezing.
[0101] 3. Single B cell gene cloning
[0102] 1. Reverse transcription
[0103] After thawing the above frozen antigen-specific memory B cells, according to the instructions of the High Capacity cDNA Reverse Transcription Kit (Thermo, 4368813), add reagents for reverse transcription, and the steps are as follows:
[0104] (1) Add to each hole
[0105] 2 μl 10× random hexamer,
[0106] 0.8 μl of 25× dNTP mix, 10 mM each,
[0107] 1 μl 10% v/v Igepal CA-630 (Sigma)
[0108] 8.95 μl Nuclease-free H 2 O.
[0109] (2) Heated at 65°C for 5 minutes, and placed on ice for at least 2 minutes.
[0110] (3) Add 2 μl 10× RT Buffer, 0.25 μl RNase inhibitor (40U/μl) and 1 μl MultiScribe™ reverse transcriptase.
[0111] (4) Reaction conditions: 10 min at 25°C, 120 min at 37°C, and then inactivation at 85°C for 5 min.
[0112] 2. Nest PCR
[0113] The cDNA obtained by reverse transcription in the above step 1 was used as the first-round PCR template, and the first-round PCR product was used as the second-round PCR template.
[0114] (1) heavy chain variable region (H) nested PCR
[0115] Primers are shown in Table 2.
[0116] Table 2. Heavy chain variable region nested PCR primers
[0117]
[0118] The first round of nested PCR reaction system for antibody heavy chain gene was: PrimerSTAR Max Premix (2×) 10 μl; primer mixture: 5′ primer 0.6 μl; 3′ Cγ CH1 0.6 μl; template 5 μl; sterilized distilled water 3.8 μl; Total volume 20 μl.
[0119] PCR reaction conditions: pre-denaturation at 98°C for 2 min; 35 cycles of 98°C for 10 s, 56°C for 5 s, 72°C for 10 s; final extension at 72°C for 5 min.
[0120] The second-round nested PCR reaction system for antibody heavy chain gene was: PrimerSTAR Max Premix (2×) 10 μl; primer mix: 5′ primer 0.6 μl; Primer mix: 3′ primer 0.6 μl; template 2 μl; sterilized distilled water 6.8 μl; total volume 20 μl.
[0121] PCR reaction conditions: pre-denaturation at 98°C for 2 min; 35 cycles of 98°C for 10 s, 58°C for 5 s, 72°C for 10 s; extension at 72°C for 5 min.
[0122] (2) Nested PCR of light chain (kappa, κ) variable region
[0123] Primers are shown in Table 3.
[0124] Table 3. Light chain (κ) variable region nested PCR primers
[0125]
[0126] The first-round nested PCR reaction system for antibody light chain (κ) gene was: PrimerSTAR Max Premix (2×) 10 μl; primer mix: 5′ primer 0.6 μl; 3′ Cκ 543 0.6 μl; template 5 μl; sterilized distilled water 3.8 μl; total volume 20 μl.
[0127] PCR reaction conditions: pre-denaturation at 98°C for 2 min; 35 cycles of 98°C for 10 s, 56°C for 5 s, 72°C for 10 s; final extension at 72°C for 5 min.
[0128] Antibody light chain (κ) gene second-round nested PCR reaction system: PrimerSTAR Max Premix (2×) 10 μl; primer mix: 5′ primer 0.6 μl; primer mix: 3′ primer 0.6 μl; template 2 μl; sterilization Distilled water 6.8 μl; total volume 20 μl.
[0129] PCR reaction conditions: pre-denaturation at 98°C for 2 min; 35 cycles of 98°C for 10 s, 58°C for 5 s, 72°C for 10 s; extension at 72°C for 5 min.
[0130] (3) Light chain (Lambda, λ) variable region nested PCR
[0131] Primers are shown in Table 4.
[0132] Table 4. Light chain (λ) variable region nested PCR primers
[0133]
[0134] The first-round nested PCR reaction system for antibody light chain (λ) gene was: PrimerSTAR Max Premix (2×) 10 μl; primer mix: 5′ primer 0.6 μl; 3′ Cλ 0.6 μl; template 5 μl; sterilized distilled water 3.8 μl ; The total volume is 20 μl.
[0135] PCR reaction conditions: pre-denaturation at 98°C for 2 min; 35 cycles of 98°C for 10 s, 58°C for 5 s, 72°C for 10 s; final extension at 72°C for 5 min.
[0136] The second-round nested PCR reaction system for antibody light chain (λ) gene was: PrimerSTAR Max Premix (2×) 10 μl; primer mix: 5’ primer 0.6 μl; 3’Cl 0.6 μl; template 2 μl; sterilized distilled water 6.8 μl ; total volume 20 μl.
[0137] PCR reaction conditions: pre-denaturation at 98°C for 2 min; 35 cycles of 98°C for 10 s, 60°C for 5 s, 72°C for 10 s; extension at 72°C for 5 min.
[0138] Analysis of the results: 1% agarose gel electrophoresis was used to detect the PCR products of the last round, and the results were as follows figure 2 As shown, wherein, each column is the same memory B cell to carry out the PCR product electrophoresis result of heavy chain variable region nested PCR (IgH) and light chain variable region nested PCR (Igκ, Igλ), the size is 300bp. PCR products with positive fragments at ~400bp and paired heavy and light chains were gel-recovered.
[0139] 3. Gel recovery and purification of PCR products
[0140] PCR product gel recovery and purification was performed using a common agarose gel DNA recovery kit (Tiangen, DP209).
[0141] The specific method is as follows:
[0142] (1) Column equilibration step: add 500 μl of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. in the tube.
[0143] (2) Cut a single target DNA band from the agarose gel, put it into a clean centrifuge tube, and weigh it.
[0144] (3) Add an equal volume of PC solution to the gel block (if the weight of the gel is 0.1 g, and its volume can be regarded as 100 µl, then add 100 µl PC solution), and place it in a water bath at 50 °C for about 10 minutes, during which time Invert the microcentrifuge tube to ensure that the gel block is fully dissolved.
[0145] (4) Add the solution obtained in the previous step to an adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.
[0146] (5) Add 600 μl of rinsing solution PW (absolute ethanol has been added) to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube; repeat the operation steps.
[0147] (6) Put the adsorption column CB2 into the collection tube, centrifuge at 12,000 rpm for 2 min, and remove the rinsing solution as much as possible. Leave the adsorption column at room temperature for a few minutes to dry thoroughly.
[0148] (7) Put the adsorption column CB2 into a clean centrifuge tube, drop an appropriate amount of preheated pure water into the middle of the adsorption membrane, and leave it at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min to collect the DNA solution; drop the collected DNA solution on the adsorption membrane and place at room temperature for 2 min; centrifuge at 12,000 rpm for 2 min, and collect the DNA solution again to increase the recovery of DNA.
[0149] The concentration of purified DNA fragments was determined using Nanodrop.
[0150] 4. Variable region gene ligation vector
[0151] 1. The cytomegalovirus expression vector CMV of the heavy chain and light chain is respectively digested with restriction endonucleases to obtain the digested product. The reaction system is as follows:
[0152] Double digestion reaction system of heavy chain expression vector: CMV-H 1 μl (1 μg); 10×NEBuffer 3 μl; AgeI-HF 1 μl; Sal I-HF 1 μl; deionized water 24 μl; total volume 30 μl .
[0153] Double digestion reaction system of light chain (κ) expression vector: CMV-κ 1 μl (1 μg); 10×NEBuffer 3 μl; Age I-HF 1 μl; BsiW I-HF 1 μl; deionized water 24 μl; Total volume 30 μl.
[0154] Double enzyme digestion reaction system of light chain (λ) expression vector: CMV-λ 1 μl (1 μg); 10×NEBuffer 3 μl; Age I-HF 1 μl; Xho I-HF 1 μl; deionized water 24 μl; Total volume 30 μl.
[0155] Reaction conditions: 37°C for 20min.
[0156] After enzyme digestion, agarose gel electrophoresis and gel recovery and purification were carried out in the same manner as above.
[0157] 2. Homologously recombine the PCR gel recovery products of the heavy chain and light chain in step 3 with the double-enzyme-digested expression vector to obtain a ligation product. The reaction system is as follows:
[0158] Reaction system of CMV-H ligation product: Homologous recombination reagent (2×) (vazyme, C115) 5 μl; CMV-H digestion product 1 μl (about 50 ng); H chain gene fragment 1 μl (about 10 ng) ; Nuclease-free water 3 μl; total volume 10 μl.
[0159] Reaction system for CMV-κ ligation product: Homologous recombination reagent (2×) 5 μl; CMV-κ digestion product 1 μl (about 50 ng); k-chain gene fragment 1 μl (about 10 ng); nuclease-free water 3 μl; total volume 10 μl.
[0160] Reaction system for CMV-λ ligation product: Homologous recombination reagent (2×) 5 μl; CMV-λ digestion product 1 μl (about 50 ng); λ chain gene fragment 1 μl (about 10 ng); nuclease-free water 3 μl; total volume 10 μl.
[0161] The above reaction solutions were mixed, and ligated at 50 °C for 20 min to obtain CMV-H, CMV-κ and CMV-λ ligated products.
[0162] 3. The ligation product was transformed into competent cells DH5α
[0163] (1) Thaw the competent cells DH5α (100 μl) on an ice bath, add 10 μl of CMV-H, CMV-κ or CMV-λ ligation products respectively, mix gently, and let stand on ice for 30 min.
[0164] (2) Heat shock in a water bath with a temperature of 42 °C for 45 s, quickly transfer to an ice bath, and let stand for 2 min.
[0165] (3) Add 500 μl of sterile medium LB without antibiotics to the centrifuge tube, mix well, and recover in a shaker with a temperature of 37°C and a speed of 200 rpm for 1 h.
[0166] (4) The revived bacterial liquid was evenly spread on the LB medium containing ampicillin, and after the bacterial liquid was absorbed, the plate was placed upside down in a 37° C. incubator for overnight cultivation.
[0167] 4. Inoculation
[0168] Pick a single colony grown on the above LB plate, add it to 3 ml of ampicillin-resistant liquid LB medium, and culture it in a shaker at a temperature of 37 °C and a speed of 220 rpm for 6 h to 7 h. Pick 2 colonies per plate.
[0169] 5. Identification of Positive Clones
[0170] The bacterial liquid was sent to a biotechnology company for sequencing and identification.
[0171] The result is as image 3 As shown, 20 pairs of antibody sequences were obtained. In the figure, A is the proportion of germline genes in the variable region of the heavy chain, the proportion of germline genes in the variable region of the light chain kappa chain, and the proportion of germline genes in the variable region of the light chain λ chain. In the figure, B is the statistical result of the heavy chain CDR3 length and light chain CDR3 length in turn, and C in the figure is the mutation statistical result of the heavy chain variable region and the light chain variable region. The heavy chain variable regions are derived from 16 different germline genes, IGHV3-23, IGHV3-7, IGHV4-39 and IGHV5-51 all account for 10%, and the rest are 5%; The mutation frequency of the chain variable region was between 0% and 12.2%; the length analysis of the CDR3 loop of the complementarity determining region showed that the heavy chain variable region had 9aa-22aa. The light chain is mostly κ chain, which is 13, and λ chain is 7, mainly IGKV3-15 and IGLV2-23; compared with germline genes, the mutation frequency of light chain variable region is 0%-5.7% Between; the length analysis of the CDR3 loop of the complementarity determining region shows that the light chain variable region has 8aa-12aa.
[0172] DNA fragments of the above sequences can also be prepared by artificial synthesis.

Example Embodiment

[0173] Example 2. Preparation of monoclonal antibodies
[0174] 1. Antibody expression in different cell lines
[0175] 1. Antibody expression in 293T cell system
[0176] Transient transfection of successfully paired antibody heavy and light chain gene expression vectors into 293T cells:
[0177] (1) 24 hours before transfection in a 12-well plate, each well was plated with 5 × 10 5 293T cells.
[0178] (2) Observe the degree of confluence of cells on the day of transfection, preferably 70% to 80%.
[0179] (3) Take 1 μg of each plasmid DNA extracted by the plasmid mini-extraction kit (Tiangen, DP103) and dilute it with 60 μl of Opti-MEM medium.
[0180] (4) Dilute 4 μl Lipofectamine® 2000 Reagent with 60 μl Opti-MEM medium.
[0181] (5) Mix the above-diluted DNA and Lipofectamine® 2000 at a volume of 1:1 to obtain a mixed solution, and incubate at room temperature for 5 min.
[0182] (6) Gently add 120 μl of the mixture to 293T cells, 5% CO 2 Incubate at 37°C.
[0183] (7) After 48 h of culture, collect the cells and supernatant, centrifuge at 12,000 rpm for 2 min, collect the cell culture supernatant and cell pellet, and store the cell culture supernatant at -20°C. Add 1 ml of pre-cooled PBS to the centrifuge tube containing the cell pellet, mix the cell pellet by pipetting, centrifuge at 12,000 rpm for 2 min, discard the supernatant, wash twice, and then add 100 μl of cell lysis buffer (Promega, E1531), In an ice bath for 10 min, centrifuge at 12,000 rpm for 5 min, and harvest the supernatant. The cell culture supernatant and the supernatant harvested after lysis and precipitation were detected by Western blotting respectively.
[0184] The result is as Figure 4 As shown, most antibodies were expressed and secreted into the cell supernatant.
[0185] 2. Preliminary Screening of Antibody Binding Activity
[0186] The expressed antibody was screened for binding activity, and the process was as follows:
[0187] (1) Coating: The new coronavirus wild-type RBD protein, Omicron mutant RBD protein, S1 protein of Beta mutant strain and S1 protein of Delta mutant strain were diluted with PBS to 0.5 µg/ml. Add 100 µl/well to a 96-well ELISA plate and coat overnight at 4°C.
[0188] (2) Blocking: discard the coating solution, add 200 µl blocking solution (PBST+3%BSA) to each well, and block at 37°C for 1 h.
[0189] (3) Washing: The blocking solution was discarded and washed three times with PBST.
[0190] (4) Sample: Dilute the antibody expression supernatant sample by 2-fold and 10-fold, add positive and negative controls, and incubate at 37°C for 2 h.
[0191] (5) Washing: The sample was discarded and washed 5 times with PBST.
[0192] (6) Secondary antibody: Dilute goat anti-human IgG antibody with diluent (PBST+1%BSA) 1:50000, add 100 µl to each well, and incubate at 37°C for 30 min.
[0193] (7) Washing: The sample was discarded and washed 5 times with PBST.
[0194] (8) Color development: add 100 µl of color developing solution to each well, and incubate at room temperature for 15 minutes in the dark.
[0195] (9) Stop: Add 50 µl stop solution to each well.
[0196] (10) Reading: Read the absorbance at 450 nm and 630 nm with a microplate reader, and the result is the reading value at 450 nm minus the reading value at 630 nm.
[0197] Take the negative control OD 450 2.5 times and greater than 0.1 is the cut-off value, the results refer to Figure 5 , when the dilution factor is 10 times, 10 of the total 20 antibodies can bind at least one of the four antigens.
[0198] 3. Primary Screening of Antibody Neutralizing Activity
[0199] The 10 antibodies with binding activity screened out were initially screened for neutralizing activity. The specific process is as follows:
[0200] (1) In a 96-well cell culture plate, add 150 µl GM (DMEM+10% FBS + 1% double antibody) for cell control, add 130 µl GM to the initial dilution well of the sample, and add 100 µl GM to the remaining wells.
[0201] (2) Add 22 µl of the antibody expression supernatant to the initial dilution hole of the sample, the initial dilution is 10, and the serial dilution is 3 times, with a total of 4 gradients.
[0202] (3) Mix the antibody thoroughly, transfer 50 µl to the next column of processing wells, and dilute it 3-fold serially.
[0203] (4) Thaw the novel coronavirus pseudovirus and dilute it with GM to 4000 TCID 50 /ml, take 50 µl (200TCID 50 ) to join columns 2-12.
[0204] (5) Incubate at 37°C for 1 h.
[0205] (6) Digest Hela-hACE2 cells, resuspend with GM, and the cell concentration is 1.3×10 5 pcs/ml.
[0206] (7) Take 100 µl of cell suspension (i.e. per 1.3 × 10 4 cells) were added to the cell plate, 37°C 5 %CO 2 Culture for 48h.
[0207] (8) Reading: discard the cell culture medium after 48 hours, wash once with 200 μl PBS, and pat dry; add 100 μl Bright-Lite to each well TM The detection reagent was placed at room temperature for 2 min, and the plate was read with a microplate reader.
[0208] (9) Calculate the 50% inhibitory concentration (ID) of the expression supernatant 50 ): inhibition rate=(V-T)/(V-C), T is the microplate reader reading of the antibody to be tested, C is the cell control affinity, and V is the virus control affinity; the log(inhibitor) vs. response (can be Variable slope) (log(inhibitor) vs. response -- Variable slope) Calculate ID 50.
[0209] The result is as Image 6 shown, Image 6 A in the middle is the neutralization rate (inhibition rate) of different antibodies to Omicron BA.1 mutants under different dilution conditions. It can be seen from the figure that 10-5B under the same concentration conditions has a negative effect on Omicron BA.1. The neutralizing activity of the Omicron mutant was significantly higher than that of the other antibodies screened. Image 6 The B in it is the neutralization activity result of antibody 10-5B on pseudoviruses of different mutant strains. It can be seen from this that 10-5B can effectively neutralize the wild-type, Alpha, Beta, Delta and Omicron BA.1. Detect fake virus.
[0210] 4. Antibody expression in 293F cell system
[0211] The primary screened antibody 10-5B with neutralizing activity was transfected into 293F cells for mass expression and purification.
[0212] (1) One day before transfection, 293F cells in logarithmic growth phase with viability higher than 90% were treated with 1.5×10 6 The density of cells/ml was inoculated into fresh medium and placed at 37°C, 5% CO. 2 , 150 rpm rotating speed incubator shaker (125 ml shake flask).
[0213] (2) On the day of transfection, samples were taken to count the cell density and viability. The cell density is 2×10 6 -3×10 6 /ml, the viability rate is higher than 90%. Adjust the cell density to 2 x 10 6 cells/ml, and the volume of each bottle of cell fluid is 30 ml.
[0214] (3) Preparation of transfection solution: Dilute 60 µg of DNA extracted with endotoxin-free plasmid extraction kit (Tiangen, DP117-T) with 150 mM NaCl solution (heavy chain and light chain molar ratio 1:1) To a total volume of 0.75 ml, mix gently; dilute 120 µl Sinofection transfection reagent with 150 mM NaCl to a total volume of 0.75 ml, mix gently; let the diluted DNA and transfection reagent stand alone for about 5 minutes Mix gently, the total volume is 1.5 ml, and then stand at room temperature for 10 minutes to obtain the transfection solution.
[0215] (4) Add the transfection solution dropwise to the cell solution obtained in step (2), gently shake the culture flask while adding dropwise, and then put it back on the shaker to continue culturing.
[0216](5) Add 2% by volume SMS 293-SUPI feed solution (SinoBiological, M293-SUPI) at the 24th hour after transfection, and then add feed solution (2% by volume) every 48 hours after transfection. day to receive samples.
[0217] The result is as Figure 7 Shown in A is the result of Western blotting of the antibody expressed in 293F cells, from which the melted heavy and light chains can be clearly seen.
[0218] 2. Antibody purification
[0219] 1. Purification of Antibodies by Affinity Chromatography
[0220] (1) Preparation of buffer: the water and buffer used were filtered through a 0.45 μm filter, and the binding/washing buffer (0.15M NaCl, 20 mM Na 2 HPO 4 , pH 7.0), elution buffer (0.1 M glycine, pH 3.0), neutralization solution (1 M Tris-HCl, pH 8.5).
[0221] (2) Sample preparation: The cell supernatant is filtered with a 0.45 μm filter to reduce impurities, improve protein purification efficiency and prevent column clogging.
[0222] (3) Sample purification
[0223] 1) Mix rProtein G Beads (Solarbio, R8300) with the collected cell supernatant and incubate for 2 h with gentle shaking on a shaker.
[0224] 2) Load an appropriate amount of rProtein G Beads into the chromatography column, and equilibrate the chromatography with a binding buffer of 5 times the column volume.
[0225] 3) Add the incubated cell supernatant to the equilibrated rProtein G Beads, and collect the effluent; add the effluent back to the chromatography column, and collect the effluent.
[0226] 4) Wash with 10 column volumes of impurity wash buffer to remove non-specifically adsorbed impurity proteins.
[0227] 5) Use 10 column volumes of elution buffer to collect the eluate, that is, the target protein fraction.
[0228] 6) Use 3 times the column volume of the binding buffer and 5 times the column volume of deionized water to equilibrate the packing in turn, and finally equilibrate with 5 times the column volume of 20% ethanol, and then store it in an equal volume of 20% ethanol, Store at 4°C.
[0229] 7) Protein quantification of purified antibody using BCA method. The quantitative results showed that the concentration of purified antibody was 1 mg/ml-2 mg/ml. The concentration of antibody 10-5B was 1.34 mg/ml.
[0230] 8) The purified antibody was detected by electrophoresis by SDS-PAGE, and the results were as follows Figure 7 As shown in B, it can be seen from the figure that the protein purity after affinity chromatography is relatively high, suggesting that the target antibody can be used for in vitro activity analysis.
[0231] The sequence of monoclonal antibody 10-5B is as follows:
[0232] The amino acid sequence of the heavy chain variable region (VH) is:
[0233] EVQLVESGGGLIQPGGSLRLSCAVSGFTVSRMSWVRQAPGKGLECVSVIYTGGNTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCVRGSGGIHDAFDIWGQGTMVTVSS (SEQ ID No. 7);
[0234] Wherein, VHCDR1 is GFTVSR (positions 26-31 in SEQ ID No.7, namely SEQ ID No.1), VHCDR2 is IYTGGNT (positions 49-55 in SEQ ID No.7, namely SEQ ID No.2); VHCDR3 is CVRGSGGIHDAFDI (positions 94-106 in SEQ ID No. 7, namely SEQ ID No. 3); the nucleotide sequence of VH is shown in SEQ ID No. 9.
[0235] The amino acid sequence of the light chain variable region (VL) is:
[0236] DIQMTQSPSSVSASVGDRVTITCRASQGISTWLAWYQQKPGKAPKVLINAASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSFPPTFGPGTKLEIK (SEQ ID No. 8);
[0237] Wherein, VLCDR1 is QGISTW (positions 27-32 in SEQ ID No.8, namely SEQ ID No.4); VLCDR2 is AAS (positions 50-52 in SEQ ID No.8, namely SEQ ID No.5); VLCDR3 is QQAHSFPPT (positions 89 to 97 in SEQ ID No. 8, namely SEQ ID No. 6); the nucleotide sequence of VL is shown in SEQ ID No. 10.
[0238] The amino acid sequence of the heavy chain constant region (CH) is:
[0239] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No.11);
[0240] The amino acid sequence of the light chain constant region (CL) is:
[0241] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID No. 12).

Example Embodiment

[0242] Example 3. Antibody 10-5B Affinity and Neutralizing Activity Analysis
[0243] 1. Affinity analysis of monoclonal antibodies
[0244] 1. BLI detects the binding ability of monoclonal antibody 10-5B to 2019-nCoV RBD
[0245] (1) Ligand coupling: Biotin-labeled 10 µg/ml wild-type RBD protein (acrobiosystems, SPD-C82E9) and Omicron (Omicron) were mutated on Octet RED 384, a high-throughput intermolecular interaction instrument. The RBD protein of the strain (Sino Biological, 40592-V49H7-B) was coupled to the SA sensor (SARTORIUS, 18-0009).
[0246] (2) Antibody 10-5B was serially diluted 2-fold in PBS from 160 nM in 6 gradients.
[0247] (3) Set the injection analysis from low concentration to high concentration according to Table 5 below.
[0248] Table 5. Low to High Injection Settings
[0249]
[0250] (4) Data analysis: The following Table 6 settings are performed in the analysis software, and curve fitting is performed.
[0251] Table 6 Analysis software settings
[0252]
[0253] The result is as Figure 8 As shown, the graphs show the binding and dissociation curves of 10-5B with wild-type (WT) RBD protein at different concentrations. As can be seen from the figure, antibody 10-5B has a high affinity for wild-type RBD, K D was 2.68nM.
[0254] 2. Analysis of Neutralizing Activity of Monoclonal Antibodies
[0255] 1. Neutralization assay to detect the neutralizing activity of monoclonal antibodies
[0256] (1) In a 96-well cell culture plate, add 150 µl GM (DMEM+10% FBS+1% double antibody) to the cell control, and add 100 µl GM to the remaining wells.
[0257] (2) Add antibody to the initial dilution well of the sample to make the initial concentration 10 µg/ml, and add GM to make the total volume 150 µl.
[0258] (3) Mix the antibody thoroughly, transfer 50 µl into the treated wells in the last row, and dilute it 3-fold serially.
[0259] (4) Thaw the novel coronavirus pseudovirus and dilute it with GM to 4000 TCID 50 /ml, take 50 µl (200TCID 50 ) to join columns 2-12.
[0260] (5) Incubate at 37°C for 1 h.
[0261] (6) Digest Hela-hACE2 cells, resuspend with GM, and the cell concentration is 1.3×10 5 pcs/ml.
[0262] (7) Take 100 µl of cell suspension (i.e. per 1.3 × 10 4 cells) were added to the cell plate, 37°C 5% CO 2 Culture for 48h.
[0263] (8) Reading: discard the cell culture medium after 48 hours, wash once with 200 μl PBS, and pat dry; add 100 μl Bright-Lite to each well TM The detection reagent was placed at room temperature for 2 min, and the plate was read with a microplate reader.
[0264] (9) Calculate the 50% inhibitory concentration (IC) of the antibody 50 ): inhibition rate=(V-T)/(V-C), T is the microplate reader reading of the antibody to be tested, C is the cell control affinity, and V is the virus control affinity; log(inhibitor) vs. response by GraphPad Prism (can be variable slope) to calculate IC 50.
[0265] The result is as Figure 9 As shown in Table 7, it can be seen from the table that the antibody 10-5B provided in the examples of this application can efficiently neutralize the wild-type strain and 13 mutant strains of the novel coronavirus, IC 50All were less than 0.05 µg/ml. Among them, the ICs of the three VOC variants of Alpha, Gamma, and Delta except Beta 50 All below 0.02 µg/ml, IC for Omicron BA.1 and BA.2 50 It can reach 0.005 µg/ml and 0.016 µg/ml. It has broad-spectrum neutralizing activity against the wild-type and various mutant pseudoviruses of the new coronavirus.
[0266] Table 7. Antibody 10-5B Neutralizing Activity (IC 50 , µg/ml)
[0267]

PUM

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