CN116023478A

CN116023478A - Neutralizing antibodies or antigen binding fragments thereof for coronaviruses

Info

Publication number: CN116023478A
Application number: CN202210842284.3A
Authority: CN
Inventors: 黄竞荷; 吴凡; 刘梅
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Current assignee: Super Extraordinary Shanghai Medical Technology Co ltd
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2023-04-28
Also published as: CN112175073A; CN112175073B

Abstract

The invention relates to a neutralizing antibody or antigen binding fragment thereof of coronavirus, a nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a vector comprising the nucleic acid molecule, a host cell comprising the vector, and the use of the antibody or antigen binding fragment thereof for preparing a medicament for treating or preventing diseases caused by coronavirus, and for detecting products; the coronavirus antibody and antigen binding fragment thereof have broad spectrum and strong neutralization capability for various coronaviruses including SARS-CoV-2, and have good clinical application prospect in the future.

Description

Neutralizing antibodies or antigen binding fragments thereof for coronaviruses

Technical Field

The invention relates to a neutralizing antibody or antigen binding fragment thereof of coronavirus, a nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a carrier containing the nucleic acid molecule, a host cell containing the carrier, application of the antibody or antigen binding fragment thereof in preparing medicines for treating or preventing diseases caused by coronavirus, and application in detecting products, and belongs to the field of biological medicines.

Background

Novel coronavirus infection (2019-nCOV) is an acute respiratory infectious disease caused by SARS-COV-2 virus. The virus has extremely strong transmission capability, can be transmitted through multiple ways such as respiratory tract, contact and the like, forms a world-wide pandemic, and brings serious challenges to public health safety worldwide.

SARS-CoV-2 virus belongs to coronaviridae, and has homology of 77.2% with SARS coronavirus in the same genus as the beta coronavirus in the fulminant of 2003. The major envelope protein of SARS-CoV-2 virus is its Spike protein (also called Spike protein, abbreviated as S protein) which is hydrolyzed into two parts S1 and S2 by intracellular proteases during viral infection. Where S2 is a transmembrane protein and S1 has a receptor binding domain (ReceptorBinding domain, abbreviated RBD) that recognizes and binds the cellular receptor angiotensin converting enzyme-2 (ACE-2). The spike protein formed by S1 and S2 is the virus receptor which specifically recognizes and binds to the target cell receptor of SARS-CoV-2 virus and mediates virus infection, and is also the recognition target of the neutralizing antibody to be developed.

So far, no effective medicine and vaccine for treating and preventing SARS-CoV-2 virus infection exists worldwide, and only supportive symptomatic treatment can be adopted for patients with novel coronavirus infection clinically. Research shows that the virus specific recovered human blood plasma is clinically used, can effectively neutralize viruses, prevent the viruses from diffusing in various organs in the body, and plays an important role in the prognosis of the disease course of patients. However, polyclonal plasma is not only of limited origin, but its clinical use is also limited by conditions such as difficulty in quality control, differential donor-acceptor blood types, potential infectious agents, and the like. The fully human monoclonal antibody capable of neutralizing SARS-CoV-2 virus is separated from the body of recovered person, and can effectively overcome the above-mentioned problems, so that it is one of main directions of development of new-type coronavirus medicine.

Up to now, several research teams at home and abroad report that fully human monoclonal antibodies capable of combining SARS-CoV-2 virus S protein, such as BD-368-2, B38, etc., are separated from peripheral blood of novel coronavirus infection convalescence patients, and are still in the experimental research and development stage at present. The technical method adopted by the research teams is to utilize S protein or S protein Receptor Binding Domain (RBD) of recombinant expressed SARS-CoV-2 virus as bait, screen and separate B cells (memory B cells) capable of binding the proteins from peripheral blood of recovered people, obtain heavy chain and light chain pairing genes of antibodies expressed by single B cells by using a cell sequencing or single cell sequencing method, express the antibodies by an in vitro recombination mode, and then verify the capability of neutralizing the viruses. Since the method uses a marker protein (the S protein or S protein receptor binding region of the above-described recombinant expressed SARS-CoV-2 virus called bait) to screen and enrich B cells in advance before performing antibody gene sequencing, only antibodies specifically binding to the marker protein can be screened.

Huang Jinghe doctor (one of the inventors of the present application) initiated in 2013 in vitro monoclonal culture and high throughput antibody screening technology (Huang J et al nature Protocols 2013) of human B cells, isolated fully human monoclonal antibodies from peripheral blood of new coronavirus infected rehabilitation persons, by the following procedure: firstly, detecting neutralizing antibodies of serum of a convalescence patient by utilizing a SARS-CoV-2 and SARS-CoV pseudovirus neutralizing system, and screening out a convalescence patient with higher neutralizing activity on the SARS-CoV-2 and the SARS-CoV simultaneously; then collecting peripheral blood lymphocytes of a rehabilitee, and sorting out memory B lymphocytes by using flow cells; single B cells were seeded in 384 well plates and cultured with the addition of cytokines and feeder cells, and the cultured B cells secreted antibodies into the supernatant after expansion and differentiation in vitro. Then, the neutralizing capacity of the antibody in the supernatant to SARS-CoV-2 and SARS-CoV viruses is detected by utilizing an in vitro high-flux neutralization experiment, positive clones capable of neutralizing the two viruses simultaneously are screened out, the heavy chain and light chain variable regions of the antibody are cloned out by utilizing an RT-PCR method, and the monoclonal antibody is obtained by constructing the heavy chain and light chain expression vectors of the antibody and then transfecting 293T cells for expression and purification.

The antibodies reported by other groups at present have better neutralizing capacity on the tested SARS-CoV-2 virus strain, but lack of binding and neutralizing capacity on other coronaviruses similar to the SARS-CoV-2 virus gene sequence, such as SARS-CoV, SARS-like virus and the like, so that the antibodies are specifically bound to non-conserved regions of the SARS-CoV-2 virus. Since SARS-CoV-2 virus is an RNA virus, the genomic sequence of the virus is prone to mutation during the course of the transmission epidemic. When mutations occur in the non-conserved region sites recognized by these antibodies, new epidemic strains are generated, resulting in the antibodies losing their protective effect against the mutant virus.

Thus, it would still be desirable for those skilled in the art to develop antibodies that have binding and neutralizing capabilities for a wide variety of coronaviruses, including SARS-CoV-2.

Disclosure of Invention

To solve the above technical problems, in one aspect, the present invention provides an antibody of coronavirus, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein, the liquid crystal display device comprises a liquid crystal display device,

the sequence of the HCDR1 is shown as SEQ ID NO.11, the sequence of the HCDR2 is shown as SEQ ID NO.12, and the sequence of the HCDR3 is shown as SEQ ID NO. 13; the sequence of the LCDR1 is shown as SEQ ID NO.15, the sequence of the LCDR2 is shown as SEQ ID NO.16, and the sequence of the LCDR3 is shown as SEQ ID NO. 17; or alternatively, the process may be performed,

the sequence of the HCDR1 is shown as SEQ ID NO.1, the sequence of the HCDR2 is shown as SEQ ID NO.2, and the sequence of the HCDR3 is shown as SEQ ID NO. 3; the sequence of the LCDR1 is shown as SEQ ID NO.5, the sequence of the LCDR2 is shown as SEQ ID NO.6, and the sequence of the LCDR3 is shown as SEQ ID NO. 7; or alternatively, the process may be performed,

the sequence of the HCDR1 is shown as SEQ ID NO.21, the sequence of the HCDR2 is shown as SEQ ID NO.22, and the sequence of the HCDR3 is shown as SEQ ID NO. 23; the sequence of LCDR1 is shown as SEQ ID NO.25, the sequence of LCDR2 is shown as SEQ ID NO.26, and the sequence of LCDR3 is shown as SEQ ID NO. 27; or alternatively, the process may be performed,

the sequence of the HCDR1 is shown as SEQ ID NO.31, the sequence of the HCDR2 is shown as SEQ ID NO.32, and the sequence of the HCDR3 is shown as SEQ ID NO. 33; and the sequence of LCDR1 is shown as SEQ ID NO.35, the sequence of LCDR2 is shown as SEQ ID NO.36, and the sequence of LCDR3 is shown as SEQ ID NO. 37.

In a preferred embodiment of the present invention, the heavy chain variable region has a sequence as shown in SEQ ID NO.14 or a sequence having 80% or more sequence homology with the sequence shown in SEQ ID NO.14, and the light chain variable region has a sequence as shown in SEQ ID NO.18 or a sequence having 80% or more sequence homology with the sequence shown in SEQ ID NO. 18; or alternatively, the process may be performed,

the heavy chain variable region has a sequence shown as SEQ ID NO.4 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.4, and the light chain variable region has a sequence shown as SEQ ID NO.8 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO. 8; or alternatively, the process may be performed,

the heavy chain variable region has a sequence shown as SEQ ID NO.24 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.24, and the light chain variable region has a sequence shown as SEQ ID NO.28 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO. 28; or alternatively, the process may be performed,

the heavy chain variable region has a sequence shown as SEQ ID NO.34 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.34, and the light chain variable region has a sequence shown as SEQ ID NO.38 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO. 38.

With respect to the percentage of "sequence homology," the percentage of sequence identity is generated by determining the number of amino acid residues present in two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the structure by 100.

In a specific embodiment of the invention, the heavy chain variable region may be increased or decreased in amino acid sequence based on the first amino acid sequence or the light chain variable region may be increased or decreased in amino acid sequence based on the second amino acid sequence, such as a similar amino acid substitution or a small amino acid substitution, particularly an amino acid increase or decrease in the conserved sequence portion, resulting in variants of the antibody having higher homology (80% homology or more) and retaining the original antibody function, i.e., the function and properties of specifically binding to coronaviruses, which variants are also within the scope of the invention.

In a preferred embodiment of the invention, the heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown in SEQ ID NO.19 and the light chain amino acid sequence is shown in SEQ ID NO. 20; or alternatively, the process may be performed,

the heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.9, and the light chain amino acid sequence is shown as SEQ ID NO. 10; or alternatively, the process may be performed,

the heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.29, and the light chain amino acid sequence is shown as SEQ ID NO. 30; or alternatively, the process may be performed,

the heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.39, and the light chain amino acid sequence is shown as SEQ ID NO. 40.

In a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof of a coronavirus.

The term "neutralizing antibody" is an antibody or antigen binding fragment that specifically binds to a viral receptor protein, which specifically binds to a protein that inhibits a biological function of the viral receptor protein, e.g., prevents the receptor protein from binding to its target cell receptor, which specifically reduces the ability of the virus to infect a target cell; in the present application, neutralizing antibody or antigen-binding fragment thereof of coronavirus refers to an antibody or antigen-binding fragment thereof that binds to the S protein of coronavirus.

The term "antibody" is used herein in its broadest sense to encompass natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, and antibody fragments, antigen-binding proteins, fusion proteins, and the like, that exhibit the desired antigen-binding activity.

In a preferred embodiment of the invention, the antibody is a monoclonal antibody.

In a more preferred embodiment of the invention, the antibody is a fully human monoclonal antibody.

In a preferred embodiment of the invention, the Fc domain of the antibody is derived from an Fc domain of an immunoglobulin, including an Fc domain of a native sequence or a variant Fc domain.

In a preferred embodiment of the invention, the antibody is any one or a combination of several of IgG1, igG2, igG3 or IgG 4.

Preferably, the antibody may be an intact antibody selected from the group consisting of IgG1, igG2, igG3, or IgG 4.

In a preferred embodiment of the invention, the antigen binding fragment is Fv, fab, F (ab ') 2, fab', dsFv, scFv, sc (Fv) 2 or a single chain antibody.

In a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof may be combined with other antibodies or antigen-binding fragments thereof to form an antibody or antigen-binding fragment thereof having at least two antigen-binding sites, i.e., a multispecific antibody or antigen-binding fragment thereof; the two antigen binding sites may be different epitopes of the same antigen or different epitopes of different antigens.

In a preferred embodiment of the invention, the above-described antibodies, or antigen binding fragments thereof, may be further chemically modified, e.g., one or more chemical groups may be attached to the antibody to increase one or more functional properties of the antibody. For example, common chemical modifications are glycosylation modifications, polyethylene glycol modifications, and the like. Among other things, glycosylation modifications can be made, for example, in the heavy or light chain variable regions, adding one or more glycosylation sites, to improve a portion of the function of an antibody, e.g., to enhance the immunogenicity of an antibody or to improve the pharmacokinetics of an antibody, etc. For example, the antibody or antigen-binding fragment thereof is subjected to an acylation reaction or an alkylation reaction with an active polyethylene glycol (e.g., an active ester or aldehyde derivative of polyethylene glycol) under suitable conditions to effect polyethylene glycol modification, to improve a portion of the function of the antibody, e.g., to increase the biological (e.g., serum) half-life of the antibody, etc. The above-described chemical modifications do not significantly alter the basic function and properties of the antibodies or antigen binding fragments thereof of the invention, i.e., the function and properties of binding specifically to coronaviruses; such chemically modified variants are also within the scope of the present invention.

In a preferred embodiment of the invention, the above-described antibodies or antigen-binding fragments thereof may be conjugated to other factors by chemical means or by genetic engineering means; for example, these factors may provide the effect of targeting the antibody to a desired functional site or other properties; for example, these factors may be one or more heterologous molecules, preferably, the heterologous molecules are cytotoxic agents. The above antibodies, or antigen binding fragments thereof, are conjugated to other factors to form complexes, which fall within the scope of the present invention.

In another aspect, the invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody as described above, or an antigen binding fragment thereof.

In a preferred embodiment of the present invention, in the nucleic acid molecule, the nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO.45 and the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO. 46; or alternatively, the process may be performed,

the nucleic acid sequence for encoding the heavy chain variable region is shown as SEQ ID NO.41, and the nucleic acid sequence for encoding the light chain variable region is shown as SEQ ID NO. 42; or alternatively, the process may be performed,

the nucleic acid sequence for encoding the heavy chain variable region is shown as SEQ ID NO.49, and the nucleic acid sequence for encoding the light chain variable region is shown as SEQ ID NO. 50; or alternatively, the process may be performed,

the nucleic acid sequence for encoding the heavy chain variable region is shown as SEQ ID NO.53, and the nucleic acid sequence for encoding the light chain variable region is shown as SEQ ID NO. 54.

In a more preferred embodiment of the invention, in the nucleic acid molecule,

the nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.47, and the nucleic acid sequence for coding the light chain is shown as SEQ ID NO. 48; or alternatively, the process may be performed,

the nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.43, and the nucleic acid sequence for coding the light chain is shown as SEQ ID NO. 44; or alternatively, the process may be performed,

the nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.51, and the nucleic acid sequence for coding the light chain is shown as SEQ ID NO. 52; or alternatively, the process may be performed,

the nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.55, and the nucleic acid sequence for coding the light chain is shown as SEQ ID NO. 56.

In a further aspect the invention provides a vector comprising a nucleic acid molecule as described above.

In a preferred embodiment of the invention, the vector further comprises an expression control sequence linked to the nucleic acid molecule described above.

The term "vector" refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to allow expression of the genetic material elements carried thereby within the host cell. The vector may contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes, etc. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these. In embodiments of the present invention, the carrier may be selected from, but is not limited to: plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosome YAC, bacterial artificial chromosome BAC, or P1-derived artificial chromosome PAC), phages (e.g., lambda phage or M13 phage), and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma viruses (e.g., SV 40).

In a further aspect the invention provides a host cell comprising the vector described above.

With respect to "host cells," one can choose, but is not limited to: prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cell models such as fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 cells, etc.

Preferably, the host cell is a HEK293 cell.

In a further aspect, the invention provides a method of producing an antibody, or antigen-binding fragment thereof, as described above, wherein the host cell described above is cultured to produce the antibody, or antigen-binding fragment thereof.

In a further aspect, the invention provides a pharmaceutical composition comprising an antibody, or antigen-binding fragment thereof, as described above.

In a preferred embodiment of the invention, the pharmaceutical composition comprises a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent. One skilled in the art can use a suitable pharmaceutical carrier or diluent in combination with a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, for administration to a patient for the treatment or prevention of a disease caused by a coronavirus.

In a further aspect, the invention provides the use of an antibody, or antigen-binding fragment thereof, as defined above, or a pharmaceutical composition as defined above, in the manufacture of a medicament for the treatment or prophylaxis of a disease caused by a coronavirus.

In a preferred embodiment of the invention, the use refers to the use in the manufacture of a medicament for the treatment or prophylaxis of a disease caused by SARS-CoV-2, SARS-CoV or SARS-like coronavirus.

In one aspect, the invention also provides a method of treating or preventing a disease caused by a coronavirus by administering to a patient a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, as described above; or administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, as described above. Preferably, the disease caused by coronavirus is a disease caused by SARS-CoV-2, SARS-CoV or SARS-like coronavirus.

In a further aspect the invention provides an assay product, wherein the assay product comprises an antibody as described above, or an antigen binding fragment thereof.

The detection product is used to detect the presence or level of coronavirus in a sample.

In one embodiment of the invention, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or test paper, and the like.

The neutralizing antibody or the antigen binding fragment thereof of the present invention may be labeled by a chemical method or a genetic engineering method, and the labeled antibody or the antigen binding fragment thereof may be used for detection; the labeled antibodies or antigen binding fragments thereof fall within the scope of the invention.

The specific detection method can adopt the following steps, 1) providing a sample; 2) Contacting the sample with a neutralizing antibody or antigen binding fragment thereof of the coronavirus of the invention described above; 3) An immune response between the sample and the neutralizing antibody or antigen binding fragment thereof is detected.

The inventor of the invention obtains a series of antibodies and antigen binding fragments thereof of coronaviruses by utilizing B cell in vitro monoclonal culture and high-throughput antibody screening technology, the antibodies and antigen binding fragments thereof have strong binding capacity and neutralization capacity for SARS-CoV-2 virus, and can recognize and bind S1 protein and RBD thereof of SARS-CoV-2 virus, and have very strong affinity; in particular, the compound has binding capacity and neutralization capacity for various coronaviruses including SARS-CoV-2, has good broad spectrum and has good clinical application prospect in the future.

Drawings

FIG. 1 shows the detection results of monoclonal antibody 6I18 recognizing S1 protein and RBD thereof of SARS-CoV-2 virus, and S2 protein;

FIG. 2 shows the detection results of the monoclonal antibody 6M6 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 3 shows the detection results of the monoclonal antibody 7C17 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 4 shows the detection results of the monoclonal antibody 7F3 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 5 shows the results of detection of RBD of monoclonal antibody 6I18 recognizing S1 protein of SARS-CoV virus;

FIG. 6 shows the results of affinity detection of RBD of monoclonal antibody 6I18 binding to S1 protein of SARS-CoV-2 virus;

FIG. 7 shows the results of affinity detection of RBD of monoclonal antibody 6M6 binding S1 protein of SARS-CoV-2 virus;

FIG. 8 shows the results of affinity detection of RBD of monoclonal antibody 7C17 binding to S1 protein of SARS-CoV-2 virus;

FIG. 9 shows the results of affinity detection of RBD of monoclonal antibody 7F3 binding S1 protein of SARS-CoV-2 virus;

FIG. 10 shows the results of affinity detection of RBD of monoclonal antibody 6I18 binding to S1 protein of SARS-CoV virus.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments shown in the drawings. These embodiments are not intended to limit the invention and structural, methodological, or functional modifications of these embodiments that may be made by one of ordinary skill in the art are included within the scope of the invention.

Example embodiments will now be described more fully with reference to the accompanying drawings. However, the exemplary embodiments can be embodied in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the example embodiments to those skilled in the art.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, third edition, scientific Press, et al, translation of molecular cloning Experimental guidelines, huang Peitang, et al) or according to the product specifications.

Example 1: screening and detection of neutralizing antibodies to coronaviruses

The inventor carries out pseudo-virus neutralization experimental screening on the blood plasma of novel coronavirus infected patients (follow-up visit after two weeks of recovery discharge) collected at the unit of the inventor (Shanghai city public health clinical center) from the 1 st month of 2020 to the 2 nd month of 2020, finds that the blood serum of three light patients has strong neutralization activity on SARS-CoV-2 pseudo-virus, and extracts the peripheral blood for research through the written consent of the unit ethics committee of the inventor and the patient.

1. Sorting of peripheral blood memory B cells

1) Isolation of peripheral blood lymphocytes: peripheral blood lymphocytes of the patient in the recovery period are isolated by mixing the peripheral blood with an equal amount of physiological saline and using lymphocyte separation liquid Lymphoprep (Stemcell Technologies, cat# 07851), and the operation process is described in the specification of lymphocyte separation liquid.

2) Sorting peripheral blood memory B cells: staining the peripheral blood lymphocytes isolated in step 1) above with an antibody mixture at 4 ℃ and in the dark for 30min, wherein the antibody mixture is a mixture of anti-CD 19-PE-Cy7 (BD Bioscience), igA-APC (Jackson Immunoresearch), igD-FITC (BD Bioscience) and IgM-PE (Jackson Immunoresearch); after staining, washing with 10ml PBS-BSA buffer and re-suspending in 500. Mu.l PBS-BSA; finally, CD19+ IgA-IgD-IgM-memory B cells were sorted using a FACSaria III cell sorter (Becton Dickinson).

2. Incubation of peripheral blood memory B cells

Resuspending the selected CD19+ IgA-IgD-IgM-memory B cells in a medium containing 10% FBS and 100U/ml IL-2, 50ng/ml IL-21 and irradiated 3T3-msCD40L feeder cells; memory B cells were seeded at a density of 4 cells/well in 384 well microtiter plates (final volume 50 μl) and incubated for 13 days; the growth factors IL-2 and IL-21 stimulate memory B cells to divide and grow, and secrete antibodies into the culture medium after incubation. Specific culturing methods are described in reference Huang J et al Nature Protocols 2013,8 (10): 1907-15.

3. Production of SARS-CoV-2 and SARS-CoV pseudoviruses

SARS-CoV-2 and SARS-CoV pseudoviruses are non-replication defective retroviral particles having SARS-CoV-2 and SARS-CoV Spike membrane proteins (Spike, S) on their surfaces and carrying a luciferase reporter gene, which can mimic the infection process of SARS-CoV-2 and SARS-CoV viruses on host cells (e.g., human hepatoma cell line Huh-7, 293T cell line 293T-ACE2 stably expressing human ACE2 receptor), respectively, and express the luciferase reporter gene in the infected cells. Since pseudovirus infection does not produce mature viral particles, the relevant procedures can be safely performed in biosafety secondary laboratories.

SARS-CoV-2 and SARS-CoV pseudoviruses are obtained by cotransfection of 293T cells with respective S protein expression plasmids and HIV Env-deficient backbone plasmid (pNL 4-3.Luc. R-E-) with luciferase reporter gene, respectively. The S gene sequences of SARS-CoV-2 and SARS-CoV are designed according to NCBI GenBank sequences NC_045512 and ABD72979.1, and the gene sequences are synthesized by Nanjing Jinsrey company after codon optimization and are connected to pcDNA3 1 eukaryotic expression vectors to construct SARS-CoV-2 and SARS-CoV S protein expression plasmids. The pNL4-3.Luc. R-E-backbone plasmid was derived from U.S. NIH AIDS ReagentProgram. All plasmids were expanded by transformation of DH 5. Alpha. Competent cells and purified using the plasmid purification kit from Meiy organisms, the purification procedure being as described in the kit instructions.

293T cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco) and plated onto 10cm cell plates prior to transfection. After 24 hours of incubation, 293T cells were co-transfected with the backbone plasmid (pNL 4-3.Luc. R-E-) and the SARS-CoV or SARS-CoV-2 expressing plasmid at a ratio of 3:1 using EZ Trans cell transfection reagent (Lissajous organism), see instructions for use of EZ Trans cell transfection reagent for detailed transfection methods. After 48 hours of transfection, the supernatant containing pseudoviruses was collected, centrifuged at 1500 rpm for 10 minutes to remove cell debris and sub-packaged for detection of neutralizing antibodies by freezing in a-80℃freezer.

4. Neutralization screening

After 13 days of in vitro culture of peripheral blood memory B cells, 40. Mu.l of culture supernatant was collected per well for detection of SARS-CoV-2 and neutralizing antibodies to SARS-CoV. The detection method comprises the following steps: mu.l of the culture supernatant was mixed with 20. Mu.l of the pseudovirus supernatant obtained by the above production in 384-well cell culture plates, and after incubation at room temperature for 30 minutes, 50. Mu.l of 5000 293T-ACE2 cells were added to each well and the culture was continued in a cell culture incubator. After 48 hours, cells were lysed using a luciferase assay kit (Luciferase Assay System, promega cat.#e1500) and luciferase activity was measured per well, for specific assay methods, see kit instructions. The chemiluminescent RLU values per well were measured using a multifunctional enzyme-labeled instrument (Perkin Elmer). And calculating the neutralization inhibition percentage of the culture supernatant to the pseudoviruses according to the ratio of the culture supernatant to the virus control RLU value, and screening out holes with the inhibition percentage of more than 90 percent as virus neutralization positive holes.

5. RT-PCR amplification of heavy and light chain genes

Virus neutralizes B cells of the positive well and variable regions of heavy and light chains of immunoglobulin genes are amplified using RT-PCR. Primer design and specific operation procedures of RT-PCR are disclosed in reference Tiller, T.et al.J.Immunol Methods 2018, 329:112-124. the amplified antibody heavy chain and light chain variable region genes are purified and recovered by agarose gel electrophoresis, and then cloned into a PMD19-T vector by using a PMD19-T vector cloning kit (Takara 6013), and the specific operation process is referred to a kit instruction, and monoclonal is selected for gene sequencing.

6. Expression and purification of monoclonal antibodies

The heavy chain variable region gene of the antibody and the pCMV/R-10E8 heavy chain gene (NIHAIDS ReagentProgram Cat 12290) which are sequenced correctly are respectively subjected to enzyme digestion by Age I and Sal I, and then the target fragment after purification and recovery of the connecting gel is connected and DH5 alpha competent cells are transformed to construct an antibody expression heavy chain plasmid; sequencing correct antibody Lambda or Kappa light chain variable region gene and pCMV/R-10E8 Lambda light chain gene expression plasmid (NIHAIDS Reagent Program Cat 12291) or pCMV/R-N6 Kappa light chain gene expression plasmid (NIH AIDS Reagent Program Cat 12966) respectively through enzyme digestion of Age I and Xho I or Age I and BsiwI, connecting glue purification and recovery of target fragment and transformation of DH5 alpha competent cells to construct antibody expression light chain plasmid; antibody heavy and light chain plasmids were purified by a plasmid purification kit (Meiy organism) (see FIG. 1 for SDS-PAGE detection of expressed purified antibodies) and co-transfected with 293T cell expression using EZ Trans cell transfection reagent (Lissajous organism) at a 1:1 ratio. After 72 hours, the cell transfected supernatant was collected and antibody IgG was purified from the supernatant using protein-G column (Tiandi and Biotech, changzhou) according to the instructions for use of protein-G column. The antibody IgG obtained by purification was measured for absorbance at 280nm using Nanodrop 2000 (Thermo Fisher) and the antibody concentration was calculated.

Through the above sections 1-6, the inventors of the present application obtained several IgG antibodies, 4 of which were disclosed herein (designations: 6I18, 6M6, 7C17, and 7F3, in order).

Amino acid sequence numbering information for the 4 antibodies is given in table 1 below:

TABLE 1

Nucleotide sequence numbering information for the 4 antibodies is given in table 2 below:

TABLE 2

7. Detection of neutralizing Activity of 4 monoclonal antibodies (6I 18, 6M6, 7C17 and 7F 3) of the present application against coronaviruses

Different concentrations of mab (6I 18, 6M6, 7C17 and 7F 3) were tested on 96-well cell plates to inhibit pseudovirus infection of Huh-7 cells to test the neutralizing capacity of mab against SARS-CoV-2 coronavirus.

The detection method comprises the following steps: 1) Huh-7 cells were seeded in 96-well cell plates at 1X10 per well ⁴ And 37 ℃,5% CO ₂ Culturing the cells in a cell culture box for 24 hours; 2) Diluting monoclonal antibody with cell culture medium to different concentrations, and diluting with equal volume of pseudovirus containing 100TCID50Mixing the solutions, and incubating at 37 ℃ for 1 hour; 3) Discarding the cell culture solution, adding 50 μl of virus antibody complex into each well, arranging multiple wells, and simultaneously arranging an antibody-free group, a virus-free group and a positive serum control group; 4) After 12 hours of cultivation, 150 μl of maintenance solution was added to each well, and cultivation was continued for 48 hours at 37deg.C; 5) Cells were lysed using a luciferase assay kit (Luciferase Assay System, promega cat.#e1500) and luciferase activity per well was measured, for specific assay methods reference kit instructions; detecting the chemiluminescent RLU value of each hole by using a multifunctional enzyme-labeled instrument (Perkin Elmer); 6) The percent neutralization inhibition of pseudoviruses by different concentrations of mab was calculated from the ratio of mab to virus control RLU values, and the median inhibition dose IC50 of mab against viruses was calculated using PRISM7 software (GraphPad).

In addition, the neutralizing ability of mab 6I18 against SARS-CoV, bats-type SARS coronavirus (bat-SL-CoV-WIV 1) and RS3367 virus was tested on 96-well cell plates by the same method as described above, with varying concentrations of mab 6I18 inhibiting pseudovirus infection of Huh-7 cells.

The results are shown in tables 3 and 4 below.

TABLE 3 Table 3

	IC50(ng/mL)
		6M6	15.9
6I18	61.7
		7C17	223.9
7F3	4.5

TABLE 4 Table 4

As can be seen from tables 3 and 4, the 4 monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 can well neutralize SARS-CoV-2 virus at ng/ml concentration, and the neutralization activity is very strong. The stronger the neutralizing activity, the less the amount of antibody used, and the lower the cost. Therefore, the 4 monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 have better clinical application prospect; in addition, as can be seen from Table 4, the monoclonal antibody 6I18 not only can well neutralize SARS-CoV-2 virus, but also has good neutralization activity on SARS coronavirus, bat class SARS coronavirus (bat-SL-CoV-WIV 1) and RS3367 virus; this demonstrates that the mab 6I18 of the present application has a strong broad-spectrum neutralizing capacity against coronaviruses.

8. Detection of S1 proteins and RBDs thereof for recognizing SARS-CoV-2 and SARS-CoV viruses by 4 monoclonal antibodies (6I 18, 6M6, 7C17 and 7F 3) of the application

The S1 proteins and RBD thereof of the SARS-CoV-2 and SARS-CoV viruses are identified by the 4 monoclonal antibodies obtained by the purification, and are sequentially detected by an enzyme-linked immunosorbent assay (ELISA) method.

The detection method comprises the following steps: 1 μg/ml of antigen protein (Yinqiao Shenzhou) was coated in 96-well ELISA plates at 4℃overnight. The plate was washed 5 times with PBS-T solution (0.2% Tween-20) and blocked for 1 hour at room temperature by adding 300. Mu.l blocking solution (PBS, 1%FBS,5%milk) to each well. After washing the plate 3 times with PBS, the monoclonal antibody was serially diluted 5-fold with PBS diluent (PBS, 5%FBS,2%BSA,1%Tween-20), 100. Mu.l of the sample was added to the ELISA plate and incubated at 37℃for 1 hour. PBS-T plates were washed 5 times, 100 μl horseradish peroxidase-labeled goat anti-human IgG antibody (Jackson Immunoresearch) diluted 1:2500 in PBS diluent was added to each well, and incubated for 1 hour at room temperature. PBS-T was washed 5 times, 150. Mu.l of ABTS chromogenic substrate (Thermo Fisher) was added, and after development for 30 minutes at room temperature in the dark, the absorbance at 405nm was read by an ELISA reader.

Referring to FIGS. 1-4, the detection results of the S1 protein and RBD thereof, and the S2 protein of the SARS-CoV-2 virus identified by the monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 are shown in sequence;

referring to FIG. 5, the detection result of RBD for recognizing S1 protein of SARS-CoV virus for monoclonal antibody 6I 18;

as can be seen from fig. 1 and 5, the monoclonal antibody 6I18 is capable of recognizing and binding to RBD (conserved region) of S1 proteins of SARS-CoV-2 and SARS-CoV viruses, thereby verifying that the monoclonal antibody 6I18 has a certain broad spectrum for coronaviruses; 1-4, monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 are all capable of recognizing and binding to SARS-CoV-2 virus S1 protein and RBD thereof; given that the RBD of the S1 protein of coronavirus is a region of ACE2 receptor binding, which is highly conserved, it is speculated that the monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 of the present application may have binding and neutralizing capabilities for other coronaviruses, as well as coronaviruses that may occur in the future, in addition to the strong binding and neutralizing capabilities for SARS-CoV-2 virus.

9. Biological membrane layer interference technology for detecting binding capacity of 4 monoclonal antibodies (6I 18, 6M6, 7C17 and 7F 3) and RBD of S1 protein of SARS-CoV-2 virus

In order to detect the interaction between the 4 monoclonal antibodies of the present application and the RBD of the S1 protein of SARS-CoV-2 virus, the binding kinetics between them were detected using a biological membrane layer interference technique, the detection procedure being performed on an OctetRED96 (Fortebio) instrument.

The detection method comprises the following steps: immersing the AHC probe in sterile water for 10 minutes in advance for balancing, and carrying out the detection process under the reaction condition of 30 ℃ in advance, wherein the detection process can be divided into the following five steps of 1) zeroing: immersing the probe in sterile water for 60 seconds to obtain a detection baseline; 2) Capture antibody: immersing the probe into a monoclonal antibody solution with the concentration of 10 mug/ml for 200 seconds to act as a capture antibody; 3) Zeroing again: immersing the probe in buffer solution (PBS solution added with 0.02% Tween 20) for 120 seconds to remove unbound antibody; 4) Combining with RBD: immersing the probe into RBD protein solution with initial concentration of 100nM and 3 times of gradient dilution, and reacting for 300 seconds to obtain a dynamic curve of the combination of the monoclonal antibody and the RBD; 5) Binding dissociation: probe is used forPlacing into buffer solution for 300 seconds. The binding of the protein causes a change in the thickness of the biological membrane, resulting in a relative shift in the interference light wave, which is detected by the spectrometer, forming an interference spectrum, which is displayed as a real-time shift (nm) of the interference spectrum. The dynamic curve of RBD binding dissociation with the monoclonal antibodies of the application is detected. Subtracting the data of the buffer control wells from the data of the sample wells at the time of data analysis, subtracting the non-specific interference of the buffer solution, using 1:1, carrying out integral curve fitting on the combination of the monoclonal antibody and the RBD dilution concentration to obtain an average combination constant K _on Dissociation constant K _off Affinity constant K _D Values.

6-9, the detection results are shown as the detection results of the affinity of RBD of the S1 protein of the monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 combined with SARS-CoV-2 virus; FIG. 10 shows the results of affinity detection of RBD of monoclonal antibody 6I18 binding to S1 protein of SARS-CoV virus; five curves representing the dynamic binding dissociation curves of the mab with five different concentrations of RBD are shown in each figure.

As can be seen from fig. 6-9, the RBD binding of the S1 protein of SARS-CoV-2 virus by 4 mabs 6I18, 6M6, 7C17 and 7F3 of the present application is concentration gradient dependent; after combination, dissociation is carried out, and the number of dissociated RBDs is very small; k of 4 MAbs 6I18, 6M6, 7C17 and 7F3 _D Values were (0.43+ -0.01) nM, (0.47+ -0.02) nM, (5.58+ -0.23) nM, (0.69+ -0.03) nM; the 4 monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 of the present application are shown to have very strong affinity with the RBD conserved region of the S1 protein of SARS-CoV-2 virus. From this it can be deduced that the 4 mabs 6I18, 6M6, 7C17 and 7F3 of the present application demonstrated in section 7 above have strong neutralizing activity against RBD of S1 protein of SARS-CoV-2 virus, as a result of the very strong affinity of the 4 mabs of the present application to RBD conserved region of S1 protein of SARS-CoV-2 virus. From a combination of the results in tables 3-4 and FIGS. 1-4, 6-9, it was further verified that the 4 monoclonal antibodies 6I18, 6M6, 7C17 and 7F3 of the present application may have binding and neutralizing capabilities for other coronaviruses, as well as coronaviruses that may occur in the future, in addition to the strong binding and neutralizing capabilities for SARS-CoV-2.

Further, from the figure10, it can be seen that the monoclonal antibody 6I18 also has concentration gradient dependence on RBD binding of S1 protein of SARS-CoV virus, K _D The value is (5.3+/-0.32) nM, and the monoclonal antibody 6I18 also has strong affinity to RBD conserved region of S1 protein of SARS-CoV virus, so that it further verifies that the monoclonal antibody 6I18 has a certain broad spectrum for coronavirus and has broad spectrum and strong neutralization capability for various coronaviruses including SARS-CoV-2 and SARS-CoV.

It should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is for clarity only, and that the skilled artisan should recognize that the embodiments may be combined as appropriate to form other embodiments that will be understood by those skilled in the art.

The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof to a coronavirus comprising a heavy chain variable region comprising three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3; the method is characterized in that:

2. The antibody or antigen-binding fragment thereof of claim 1, wherein:

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein:

the antibody is a monoclonal antibody; preferably, the antibody is a fully human monoclonal antibody; preferably, the antibody is any one or a combination of a plurality of IgG1, igG2, igG3 or IgG 4; the antigen binding fragment is Fv, fab, F (ab ') 2, fab', dsFv, scFv or sc (Fv) 2.

4. A nucleic acid molecule characterized in that: the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the vector of claim 5.

7. A pharmaceutical composition characterized by: the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.

8. A test product, characterized by: the detection product comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.

9. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: culturing the host cell of claim 6 to produce the antibody or antigen-binding fragment thereof.

10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 7 for the manufacture of a medicament for the treatment or prophylaxis of a disease caused by coronavirus.