CN115448986A - Bispecific antibody of novel coronavirus SARS-CoV-2 and application thereof - Google Patents
Bispecific antibody of novel coronavirus SARS-CoV-2 and application thereof Download PDFInfo
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Abstract
The invention discloses a bispecific antibody of a novel coronavirus SARS-CoV-2 and application thereof, belonging to the technical field of antibody research. The invention designs a bispecific antibody by combining non-overlapping specificities, and provides the bispecific antibody capable of effectively inhibiting SARS-CoV-2 infection, wherein the antibody comprises a first heavy chain and a second heavy chain, and the amino acid sequence of the variable region of the first heavy chain is SEQ ID NO:1 and the variable region amino acid sequence of the second heavy chain is SEQ ID NO:3. the antibody can be applied to the preparation of reagents or medicines for diagnosing or treating diseases caused by the novel coronavirus.
Description
Technical Field
The invention belongs to the technical field of antibody research, and particularly relates to a bispecific antibody of a novel coronavirus SARS-CoV-2 and application thereof.
Background
The phage antibody library technology (phage antibody library) is a new molecular biology technology developed in recent years, and more researchers in recent years use the technology to obtain target antibodies to be applied to various fields such as medical pharmacy, safety detection and the like. The development and application of phage antibody library technology brings great changes to the technical field of antibodies, and greatly promotes the development and application of various genetic engineering antibodies with excellent performance. Obtaining large antibody libraries is a prerequisite for screening to obtain specific antibodies.
The new coronavirus is named severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) by the world health organization. The new coronavirus is a novel coronavirus of beta genus, and has envelope, round or elliptical particle, usually polymorphism, and diameter of 60-140nm. The whole genome comparison finds that the homology of the novel coronavirus and severe acute respiratory syndrome virus (SARS-CoV) reaches 70%, and the sequence difference is mainly reflected in a key spike gene (coding S-protein) which acts with a host cell.
The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variation threatens the effectiveness of existing vaccines and therapeutic antibodies and emphasizes the need for additional antibody-based tools to effectively neutralize the variant strain by targeting multiple sites of the spike protein. We isolated multiple monoclonal antibodies against SARS-CoV-2 from mononuclear cells (PBMC) from patients with COVID-19. A bispecific antibody is a specific antibody that is made by man and can bind to two different antigens or different sites of the same antigen simultaneously. The natural antibodies targeting two sites on SARS-CoV-2 virus are connected together to prepare a bispecific antibody simultaneously targeting two independent virus sites, which can embody the advantages of cocktail in a single molecule, and more effectively target the treatment resistance caused by the virus variant and block the infection of new coronavirus to human cells, thereby solving the problem.
Disclosure of Invention
The invention designs the bispecific antibody by combining non-overlapping specificities, provides the bispecific antibody which can effectively inhibit SARS-CoV-2 infection, and can be applied to the preparation of reagents or medicaments for diagnosing or treating diseases caused by novel coronaviruses.
In order to achieve the purpose, the invention adopts the technical scheme that:
a bispecific antibody of novel coronavirus SARS-CoV-2, comprising a first heavy chain and a second heavy chain, wherein the variable region amino acid sequence of the first heavy chain is SEQ ID NO:1 and the variable region amino acid sequence of the second heavy chain is SEQ ID NO:3.
further, the bispecific antibody of the novel coronavirus SARS-CoV-2 further comprises a first light chain and a second light chain, wherein the variable region amino acid sequence of the first light chain is SEQ ID NO:2 and the variable region amino acid sequence of the second light chain is SEQ ID NO:4.
further, the variable region of the first heavy chain of the bispecific antibody of the novel coronavirus SARS-CoV-2 recognizes one of amino acid positions Ser447, glu484, val445, gly446 and Lys444 of the RBD domain of the S protein, and the variable region of the second heavy chain recognizes one of amino acid positions Thr333, leu335, glu340, thr345 and Asn440 of the RBD domain of the S protein.
Further, the bispecific antibody of the novel coronavirus SARS-CoV-2 can specifically bind to novel coronavirus SARS-CoV-2S protein.
The application of bispecific antibody of new coronavirus SARS-CoV-2 in preparing reagent or medicine for diagnosing or treating diseases caused by new coronavirus.
Compared with the prior art, the invention has the following beneficial effects:
the invention designs the bispecific antibody by combining non-overlapping specificity, provides the bispecific antibody which can effectively inhibit SARS-CoV-2 infection, has high affinity, can be specifically combined with the novel coronavirus SARS-CoV-2S protein, and can be applied to the preparation of reagents or medicaments for diagnosing or treating diseases caused by the novel coronavirus.
Drawings
FIG. 1 is a graph showing the inhibition curve of specific neutralizing antibodies against SARS-CoV-2, a novel coronavirus of the present invention.
Detailed Description
The technical solutions and effects of the present invention will be further described with reference to the drawings and specific embodiments, but the scope of the present invention is not limited thereto.
Example 1
A bispecific antibody of novel coronavirus SARS-CoV-2, comprising a first heavy chain and a second heavy chain, wherein the variable region amino acid sequence of the first heavy chain is SEQ ID NO:1,
EVQLVQSGGGLVQPGGSLRLSCAASGITVNSNYMSWVRQAPGKGLEWVSIIYSGGSTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYGDYYFDYWGQGTLVTVSS；
the variable region amino acid sequence of the second heavy chain is SEQ ID NO:3,
QITLKESGGGLVQTGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGSSWNSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIRQKYYDISTGYYKYGEQDYGMDVWGQGTLVTVSS。
further, the bispecific antibody of the novel coronavirus SARS-CoV-2 further comprises a first light chain and a second light chain, wherein the variable region amino acid sequence of the first light chain is SEQ ID NO:2,
DIQMTQSPSSLSASVGDRVTITCQASQDIRNYLNWYQQKPGRAPDLLIFDASDLETGVPSRFSGSGSGTDFTFTISSLQPEDIGTYYCQQYGNLPLTFGGGTKVEIK；
the variable region amino acid sequence of the second light chain is SEQ ID NO:4,
QAVLTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQHHPGKAPELMIYDVTKRPAGLPNRFSGSKSGNTASLTISGLQAEDEASYYCSSYTTSSTVIFGGGTKLTVL。
the first heavy chain amino acid sequence of the bispecific antibody Fab of the novel coronavirus SARS-CoV-2 is SEQ ID NO:5,
EVQLVQSGGGLVQPGGSLRLSCAASGITVNSNYMSWVRQAPGKGLEWVSIIYSGGSTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYGDYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT；
the Fab first light chain amino acid sequence is SEQ ID NO:6,
DIQMTQSPSSLSASVGDRVTITCQASQDIRNYLNWYQQKPGRAPDLLIFDASDLETGVPSRFSGSGSGTDFTFTISSLQPEDIGTYYCQQYGNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
the second heavy chain amino acid sequence of the Fab is SEQ ID NO:7,
QITLKESGGGLVQTGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGSSWNSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIRQKYYDISTGYYKYGEQDYGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT；
the second light chain amino acid sequence of the Fab is SEQ ID NO:8,
QAVLTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQHHPGKAPELMIYDVTKRPAGLPNRFSGSKSGNTASLTISGLQAEDEASYYCSSYTTSSTVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS。
further, the variable region of the first heavy chain of the bispecific antibody of the novel coronavirus SARS-CoV-2 recognizes one of amino acid positions Ser447, glu484, val445, gly446 and Lys444 of the RBD domain of the S protein, and the variable region of the second heavy chain recognizes one of amino acid positions Thr333, leu335, glu340, thr345 and Asn440 of the RBD domain of the S protein.
Application of bispecific antibody of novel coronavirus SARS-CoV-2 in preparing reagent or medicine for diagnosing or treating diseases caused by novel coronavirus is provided.
Example 2
The preparation method of the bispecific antibody of the novel coronavirus SARS-CoV-2 comprises the following steps:
1. obtaining total RNA/mRNA of immune material
Total RNA was extracted from peripheral blood lymphocytes of convalescent patients infected with the novel coronavirus, and mRNA was further isolated from the RNA (Takara Cat # Z652N/636592, see product Specification for specific experimental procedures).
2. Design of synthetic primers
The primers for the human complete set of antibodies are designed according to the gene sequence information of the human complete set of antibodies provided in the V-BASE website (http:// vbase. Mrc-cpe.cam. Ac.uk /), and are introducedThe material was synthesized by Jinwei Zhi Co. Primers in H 2 Dissolve in O at 100 pmol/. Mu.l and store at-20 ℃.
3. Synthesis of cDNA first chain and amplification of complete set of IgG1 light chain full-length gene and heavy chain variable region gene
Amplifying a component antibody gene library by a two-step method: 1. using the obtained mRNA as a template, performing reverse transcription by using MMLV of Promega to synthesize cDNA (according to the instruction of Promega company, wherein the primer is ThermoCat # N8080127, and the reverse transcriptase is Promega Cat # M1701); 2. the KLC, LLC and VH gene libraries of the module antibodies were amplified by PCR (Takara Cat # RR900A, according to the instructions of the company products) using the cDNA obtained in the first step as template and the primers designed in 2. After gel electrophoresis purification and recovery (using a gel recovery kit of the leveng according to the record operation in the molecular cloning experimental manual), PCR products, namely a KLC fragment, an LLC fragment and a VH fragment are respectively obtained.
4. Construction of Fab phage display libraries
And connecting the target fragment with a vector through T4 ligase to construct a Fab phage display library. Calculating the original library capacity of the constructed Fab phage display phage library to be 1.44 multiplied by 10 according to the number of bacterial clones growing on the LB plate with ampicillin resistance 8 . The results of randomly selecting 10 monoclonals from antibody Fab phage library for sequence determination show that there are 6 different light and heavy chain gene coding sequence combinations with diversity of 60%, i.e. 8.64X 10 7 。
5. Specific antibody screening
Screening antigen specific Fab by liquid phase screening method of SARS-CoV-2 S1 antigen marked by biotin combined with avidin marked magnetic bead adsorption. The antigen concentration of the four rounds of liquid phase screening is 10ug/ml, 1ug/ml and 1ug/ml respectively. Phage collected from the first round of screening elution were only amplified for the second round of screening. Phage collected in the second and third screening elution are not amplified and are directly used in the subsequent screening process.
3. Four rounds of screening, elution of the collected phages, infection of TG1 bacteria, ampicillin plating, 37 ℃ culture overnight. The following day, monoclonal cultures were expanded from each antigen-specific plate and induced overnight with IPTG (final concentration of 1 nM). The culture broth was analyzed for antigen-specific Fab by ELISA. Clones with ELISA readings greater than twice the lowest ELISA reading were scored as positive clones. And selecting a proper number of positive clones for sequencing analysis to obtain a unique amino acid sequence with a high ELISA reading.
6. Screening by neutralization test to obtain SARS-CoV-2 specific neutralizing antibody
The test antibody sample and positive control antibody (Kisrey) were pre-incubated with HRP-RBD to allow binding of neutralizing antibody to HRP-RBD. The mixture was then added to capture plates pre-coated with hACE2 protein. Unbound HRP-RBD and HRP-RBD bound to non-neutralizing antibody will be captured on the plate, while HRP-RBD-neutralizing antibody complex bound to neutralizing antibody is in the supernatant and washed away during the wash. After the washing step was completed, TMB solution was added to turn blue. The stop solution was added and the reaction quenched and turned yellow in color. The plate was read at 450nm and the absorbance of the sample was inversely proportional to the titer of neutralizing antibody against SARS-CoV-2.
Finally, 2 SARS-CoV-2 specific neutralizing antibodies were obtained. Cut-off value (cut-off): signal value inhibition =20% calculation, antibody S82 signal value inhibition 71.4%, EC50 0.03ug/ml; the inhibition rate of the antibody S126 signal value is 58 percent, and the EC50 is 0.02ug/ml.
7. Bispecific antibody expression
Using plasmid containing monoclonal antibody as template, using correspondent restriction enzyme to make enzyme digestion to obtain light chain and heavy chain variable region molecular fragment of antibody, adding N-terminal signal peptide and C-terminal 6 × His Tag by means of conventional molecular biological method, and inserting the molecular fragment into mammalian expression vector pcDNA3.1 (+). Bispecific antibody molecules were generated by transfection of Expi 293F-cell lines, 6 days after transfection, cell supernatants were collected and purified by HiLoad Superdex columns to recover the antibody. All antibodies were subjected to quality control and characterization analysis to assess stability and reproducibility.
8. Neutralization activity was verified in a pseudovirus neutralization assay
A plasmid with a Luciferase (LUC) or Green Fluorescent Protein (GFP) reporter gene and over-expression of ACE2 is constructed, and a 293T cell strain is stably transfected. The antibody was incubated with SARS-CoV-2 pseudovirus at 4-fold serial dilutions for 1 hour at 37 ℃. The mixture was then incubated with 293T cells for 48h; after washing twice with PBS, the cells were lysed. Luciferase activity in the lysates was measured using a luciferase detection system.
The inhibition curve of the bispecific neutralizing antibody of the novel coronavirus SARS-CoV-2 of the invention is shown in FIG. 1. The results show that the half inhibitory concentrations (IC 50) of bispecific antibody against the novel coronavirus WT, variant strains Alpha and Delta are 0.4505 ng/ml, 0.1822 ng/ml and 0.2349 ng/ml, respectively.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A bispecific antibody of novel coronavirus SARS-CoV-2, which comprises a first heavy chain and a second heavy chain, wherein the variable region amino acid sequence of the first heavy chain is SEQ ID NO:1 and the variable region amino acid sequence of the second heavy chain is SEQ ID NO:3.
2. the bispecific antibody against the novel coronavirus SARS-CoV-2 of claim 1, wherein the bispecific antibody against the novel coronavirus SARS-CoV-2 further comprises a first light chain and a second light chain, wherein the variable region amino acid sequence of the first light chain is SEQ ID NO:2 and the variable region amino acid sequence of the second light chain is SEQ ID NO:4.
3. the bispecific antibody against SARS-CoV-2 according to claim 2, wherein the variable region of the first heavy chain of said bispecific antibody against SARS-CoV-2 recognizes one of amino acid positions Ser447, glu484, val445, gly446, lys444 of the RBD domain of the S protein, and the variable region of the second heavy chain recognizes one of amino acid positions Thr333, leu335, glu340, thr345, asn440 of the RBD domain of the S protein.
4. The bispecific antibody against novel coronavirus SARS-CoV-2 according to claim 2, wherein the bispecific antibody against novel coronavirus SARS-CoV-2 is capable of specifically binding to novel coronavirus SARS-CoV-2S protein.
5. Use of a specific neutralizing antibody against the novel coronavirus SARS-CoV-2 according to any one of claims 1 to 4 for the preparation of a reagent or a medicament for the diagnosis or treatment of a disease caused by the novel coronavirus.
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| CN117603358A (en) * | 2023-02-24 | 2024-02-27 | 中国科学院微生物研究所 | Bispecific antibody of broad-spectrum novel coronavirus |
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