CN115710311B

CN115710311B - Antibodies or antigen-binding fragments thereof to coronavirus

Info

Publication number: CN115710311B
Application number: CN202210890641.3A
Authority: CN
Inventors: 黄竞荷; 吴凡; 刘梅
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER; Super Extraordinary Shanghai Medical Technology Co ltd
Current assignee: Super Extraordinary Shanghai Medical Technology Co ltd
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2025-09-02
Anticipated expiration: 2040-09-30
Also published as: CN115710311A; CN116063464A; CN112159469A; CN116063464B; CN112159469B

Abstract

The invention relates to an antibody or antigen binding fragment thereof of coronavirus, a nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a vector containing the nucleic acid molecule, a host cell containing the vector, application of the antibody or antigen binding fragment thereof in preparing a medicament for treating or preventing diseases caused by coronavirus, and application in detecting products, wherein the inventor utilizes B cell in vitro monoclonal culture and high-throughput antibody screening technology to obtain a series of antibodies and antigen binding fragments thereof of coronavirus, the antibodies and antigen binding fragments thereof have strong binding capacity and neutralizing capacity for SARS-CoV-2 virus, and the antibodies and antigen binding fragments thereof can recognize and bind S1 protein of SARS-CoV-2 virus and RBD thereof, have very strong affinity, so that the antibodies and antigen binding fragments thereof can be presumed to have binding capacity and neutralizing capacity for other coronaviruses and coronaviruses possibly occurring in the future, and have good clinical application prospect in the future.

Description

Antibodies or antigen binding fragments thereof to coronaviruses

Technical Field

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

Background

Novel coronavirus pneumonia (2019-nCOV) is an acute respiratory infectious disease caused by the SARS-COV-2 novel coronavirus. The virus has extremely strong transmission capability and can be transmitted through multiple ways such as respiratory tract, contact and the like to bring 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. Wherein 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 new coronaries 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 new coronavirus rehabilitation person, which can effectively overcome the above problems and is one of the main directions of the current new coronavirus drug development.

Up to the present, 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 new coronal pneumonia healers, 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.

A Huang Jinghe doctor (one of the inventors) firstly initiated in 2013 uses an in-vitro monoclonal culture and high-throughput antibody screening technology (Huang J et al Nature Protocols 2013) to separate fully human monoclonal antibodies from peripheral blood of a new coronal pneumonia rehabilitation person, and the process comprises the steps of firstly detecting neutralizing antibodies of serum of the new coronal pneumonia rehabilitation person by using a SARS-CoV-2 and SARS-CoV pseudovirus neutralizing system, screening a rehabilitation person with higher neutralizing activity on the SARS-CoV-2 and the SARS-CoV, collecting peripheral blood lymphocytes of the rehabilitation person, sorting memory B lymphocytes by using flow cells, inoculating single B cells into a 384-well plate, adding cytokines and feeder cells for culture, and secreting the antibodies into supernatant after in-vitro amplification and differentiation of the cultured B cells. 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 because the SARS-CoV-2 virus is RNA virus, the genome sequence of the virus is easy to mutate in the process of transmission and 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 new antibodies that have binding and neutralizing capabilities for coronaviruses, including the SARS-CoV-2 virus.

Disclosure of Invention

To solve the above technical problems, 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 sequence general formula of the HCDR1 is GX ₁ TVSSNY, wherein X ₁ is any one amino acid of L, I or F;

The sequence general formula of the HCDR2 is X ₂YSGGSX₃, wherein X ₂ is any one of L or I amino acid, and X ₃ is any one of A or T amino acid.

Preferably, the sequence general formula of the HCDR3 is ARDLIX ₄ YGMDV, wherein X ₄ is any one of D or T amino acid;

the sequence of the LCDR1 is QGISSY, and the sequence of the LCDR2 is AAS;

The sequence general formula of the LCDR3 is QQLNSYPPX ₅ T, wherein X ₅ is any one of L or Y amino acid.

In a preferred embodiment of the present invention, the sequence of the HCDR1 is shown in SEQ ID NO.1, the sequence of the HCDR2 is shown in SEQ ID NO.2, the sequence of the HCDR3 is shown in SEQ ID NO.3, and the sequence of the LCDR1 is shown in SEQ ID NO.5, the sequence of the LCDR2 is shown in SEQ ID NO.6, and the sequence of the LCDR3 is shown in SEQ ID NO.7, or

The sequence of the HCDR1 is shown as SEQ ID NO.11, the sequence of the HCDR2 is shown as SEQ ID NO.12, 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.

In another preferred embodiment of the present invention, the heavy chain variable region has a sequence as shown in SEQ ID NO.4 or a sequence having 80% or more sequence homology with the sequence shown in SEQ ID NO.4, and the light chain variable region has a sequence as shown in SEQ ID NO.8 or a sequence having 80% or more sequence homology with the sequence shown in SEQ ID NO.8, or

The heavy chain variable region has a sequence shown as SEQ ID NO.14 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.14, and the light chain variable region has a sequence shown as SEQ ID NO.18 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO. 18.

In a preferred embodiment of the present invention, the sequence of HCDR1 is shown as SEQ ID NO.21, the sequence of HCDR2 is shown as SEQ ID NO.22, the sequence of HCDR3 is shown as SEQ ID NO.23, and 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

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

The sequence of the HCDR1 is shown as SEQ ID NO.41, the sequence of the HCDR2 is shown as SEQ ID NO.42, the sequence of the HCDR3 is shown as SEQ ID NO.43, the sequence of the LCDR1 is shown as SEQ ID NO.45, the sequence of the LCDR2 is shown as SEQ ID NO.46, the sequence of the LCDR3 is shown as SEQ ID NO.47, or

The sequence of the HCDR1 is shown as SEQ ID NO.51, the sequence of the HCDR2 is shown as SEQ ID NO.52, the sequence of the HCDR3 is shown as SEQ ID NO.53, the sequence of the LCDR1 is shown as SEQ ID NO.55, the sequence of the LCDR2 is shown as SEQ ID NO.56, the sequence of the LCDR3 is shown as SEQ ID NO.57, or

The sequence of the HCDR1 is shown as SEQ ID NO.61, the sequence of the HCDR2 is shown as SEQ ID NO.62, the sequence of the HCDR3 is shown as SEQ ID NO.63, the sequence of the LCDR1 is shown as SEQ ID NO.65, the sequence of the LCDR2 is shown as SEQ ID NO.66, and the sequence of the LCDR3 is shown as SEQ ID NO. 67.

In another preferred embodiment of the present invention, the heavy chain variable region has a sequence as shown in SEQ ID NO.24 or a sequence having 80% or more sequence homology with the sequence shown in SEQ ID NO.24, and the light chain variable region has a sequence as shown in SEQ ID NO.28 or a sequence having 80% or more sequence homology with the sequence shown in SEQ ID NO.28, or

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, or

The heavy chain variable region has a sequence shown as SEQ ID NO.44 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.44, and the light chain variable region has a sequence shown as SEQ ID NO.48 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.48, or

The heavy chain variable region has a sequence shown as SEQ ID NO.54 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.54, and the light chain variable region has a sequence shown as SEQ ID NO.58 or a sequence with more than 80% of sequence homology with the sequence shown as SEQ ID NO.58, or

The heavy chain variable region has a sequence shown as SEQ ID NO.64 or a sequence with more than 80% sequence homology with the sequence shown as SEQ ID NO.64, and the light chain variable region has a sequence shown as SEQ ID NO.68 or a sequence with more than 80% sequence homology with the sequence shown as SEQ ID NO. 68.

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.9, the light chain amino acid sequence is shown in SEQ ID NO.10, or

The heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.19, the light chain amino acid sequence is shown as SEQ ID NO.20, or

The heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.29, the light chain amino acid sequence is shown as SEQ ID NO.30, or

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

The heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.49, the light chain amino acid sequence is shown as SEQ ID NO.50, or

The heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.59, the light chain amino acid sequence is shown as SEQ ID NO.60, or

The heavy chain amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO.69, and the light chain amino acid sequence is shown as SEQ ID NO. 70.

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 inhibits the biological function of the viral receptor protein, such as preventing the receptor protein from binding to its target cell receptor, and which specifically reduces the ability of the virus to infect target cells, and in the present application, neutralizing antibody or antigen-binding fragment 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, which 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 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, and such chemically modified variants are also within the scope of the invention.

In a preferred embodiment of the invention, the antibodies or antigen binding fragments thereof described above may be conjugated by chemical or genetic engineering means to other factors, for example, which may provide the effect or other properties of targeting the antibody to a desired functional site, for example, which may be one or more heterologous molecules, preferably, which 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 invention, the nucleic acid molecule has a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO.71, or a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO.72

The nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO.75, the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO.76, or

The nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO.79, the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO.80, or

The nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO.83, the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO.84, or

The nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO.87, the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO.88, or

The nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO.91, the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO.92, or

The nucleic acid sequence for encoding the heavy chain variable region is shown as SEQ ID NO.95, and the nucleic acid sequence for encoding the light chain variable region is shown as SEQ ID NO. 96.

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.73, the nucleic acid sequence for coding the light chain is shown as SEQ ID NO.74, or

The nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.77, the nucleic acid sequence for coding the light chain is shown as SEQ ID NO.78, or

The nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.81, the nucleic acid sequence for coding the light chain is shown as SEQ ID NO.82, or

The nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.85, the nucleic acid sequence for coding the light chain is shown as SEQ ID NO.86, or

The nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.89, the nucleic acid sequence for coding the light chain is shown as SEQ ID NO.90, or

The nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.93, the nucleic acid sequence for coding the light chain is shown as SEQ ID NO.94, or

The nucleic acid sequence for coding the heavy chain is shown as SEQ ID NO.97, and the nucleic acid sequence for coding the light chain is shown as SEQ ID NO. 98.

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 invention, the vector 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-virus (e.g., SV 40).

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

As the "host cell", there may be selected, but not limited to, prokaryotic cells such as E.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 a neutralizing 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 neutralizing 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 a 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 antibody or the antigen binding fragment thereof can be labeled by a chemical method or a genetic engineering method, the labeled antibody or the antigen binding fragment thereof can be used for detection, and the labeled antibody or the antigen binding fragment thereof falls within the protection scope of the invention.

Specific detection methods may employ the steps of 1) providing a sample, 2) contacting the sample with an antibody or antigen-binding fragment thereof of the coronavirus of the present invention as described above, and 3) detecting an immune reaction between the sample and the antibody or antigen-binding fragment thereof.

The inventor obtains a series of antibodies and antigen binding fragments thereof of coronaviruses by utilizing B cell in-vitro monoclonal culture and high-flux antibody screening technology, the antibodies and antigen binding fragments thereof have strong binding capacity and neutralization capacity for SARS-CoV-2 viruses, and can recognize and bind S1 protein of SARS-CoV-2 viruses and RBD thereof, and have very strong affinity, so that the antibodies and antigen binding fragments thereof of a series of coronaviruses of the invention can be presumed to have binding capacity and neutralization capacity for other coronaviruses, and coronaviruses possibly occurring in the future, and have good clinical application prospect in the future.

Drawings

FIG. 1 shows the detection results of the monoclonal antibody 4L12 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 2 shows the detection results of the monoclonal antibody 12F5 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 3D13 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 10C2 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 5 shows the detection results of the monoclonal antibody 16L9 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 6 shows the detection results of the monoclonal antibody 20E21 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 7 shows the detection results of the monoclonal antibody 22H22 recognizing the S1 protein and RBD thereof of SARS-CoV-2 virus, and the S2 protein;

FIG. 8 shows the results of affinity detection of RBD of monoclonal antibody 4L12 binding to S1 protein of SARS-CoV-2 virus;

FIG. 9 shows the results of affinity detection of RBD of monoclonal antibody 12F5 binding to S1 protein of SARS-CoV-2 virus;

FIG. 10 shows the results of affinity detection of RBD of monoclonal antibody 3D13 binding to S1 protein of SARS-CoV-2 virus;

FIG. 11 shows the results of affinity detection of RBD of monoclonal antibody 10C2 binding to S1 protein of SARS-CoV-2 virus;

FIG. 12 shows the results of affinity detection of RBD of monoclonal antibody 16L9 binding to S1 protein of SARS-CoV-2 virus;

FIG. 13 shows the results of affinity detection of RBD of monoclonal antibody 20E21 binding to S1 protein of SARS-CoV-2 virus;

FIG. 14 shows the results of affinity detection of RBD of monoclonal antibody 22H22 binding to S1 protein of SARS-CoV-2 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 different forms and should not be construed as limited to the embodiments set forth herein, but rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the exemplary 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 the 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, compiled "molecular cloning Experimental guidelines" Huang Peitang et al) or according to the product specifications.

Example 1 screening and detection of antibodies to coronaviruses

The inventor carries out pseudo-virus neutralization experimental screening on the blood plasma of novel coronavirus pneumonia 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) Peripheral blood lymphocyte separation, namely, after the peripheral blood of the recovery period of the patient is extracted and mixed with the same amount of physiological saline, the peripheral blood lymphocyte is separated by using lymphocyte separation liquid Lymphoprep (Stemcell Technologies, product number 07851), and the operation process is described in the specification of the lymphocyte separation liquid.

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

2. Incubation of peripheral blood memory B cells

The CD19+ IgA-IgD-IgM-memory B cells selected above were resuspended in medium containing 10% FBS and 100U/ml IL-2, 50ng/ml IL-21 and irradiated 3T3-msCD L feeder cells, the memory B cells were seeded at a density of 4 cells/well in 384 well microtiter plates (final volume 50. Mu.l), incubated for 13 days, and growth factors IL-2 and IL-21 stimulated memory B cell division growth, secreting antibodies to the incubated medium. 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 Jinsrui 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 harvested, 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 assay was performed by mixing 20. Mu.l of culture supernatant with 20. Mu.l of the pseudovirus supernatant obtained by the above production in 384 well cell culture plates, incubating at room temperature for 30 minutes, adding 50. Mu.l of 5000 293T-ACE2 cells per well and continuing the culture in the 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 detection methods reference kit instructions. The chemiluminescent RLU values were detected per well using a multifunctional microplate reader (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 shown in reference Tiller, T.et al.J.Immunol Methods 2018,329:112-124, after the amplified antibody heavy chain and light chain variable region genes are purified and recovered by agarose gel electrophoresis, the amplified antibody heavy chain and light chain variable region genes are cloned into a PMD19-T vector by using a PMD19-T vector cloning kit (Takara 6013), the specific operation procedures are shown in the kit specification, and monoclonal antibodies are selected for gene sequencing.

6. Expression and purification of monoclonal antibodies

The method comprises the steps of respectively carrying out enzyme digestion on a correct sequencing antibody heavy chain variable region gene and a pCMV/R-10E8 heavy chain gene (NIHAIDS ReagentProgram Cat 12290) by Age I and Sal I, connecting a target fragment after gel purification and recovery, and transforming DH5 alpha competent cells to construct an antibody expression heavy chain plasmid, carrying out purification on the antibody Lambda or Kappa light chain variable region gene and the pCMV/R-10E8 Lambda light chain gene expression plasmid (NIHAIDS REAGENT Program Cat 12291) or the pCMV/R-N6 Kappa light chain gene expression plasmid (NIH AIDS REAGENT Program Cat 12966) by using a purification kit (see FIG. 1 for an expressed purified antibody SDS-PAGE detection result), and carrying out cotransfection on the plasmid by using an EZ Trans-cell transfection reagent (293 organism) in a T cell co-transfection ratio of 293 1. 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 1-6 parts, the present inventors obtained several IgG antibodies, 7 of which were disclosed (designations: 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 in this order).

Amino acid sequence numbering information for 7 antibodies is given in table 1 below:

TABLE 1

Nucleotide sequence numbering information for 7 antibodies is given in table 2 below:

TABLE 2

7. Detection of neutralizing Activity of 7 MAbs (4L 12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H 22) of the application on SARS-CoV-2 coronavirus

Different concentrations of mab 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 assay method comprises 1) inoculating Huh-7 cells to 96-well cell plates, inoculating 1X10 ⁴ cells per well, culturing at 37 ℃ for 24 hours in a 5% CO ₂ cell incubator, 2) diluting the monoclonal antibodies to different concentrations with a cell culture medium, mixing with an equal volume of pseudovirus diluent containing 100TCID50, incubating at 37 ℃ for 1 hour, 3) discarding the cell culture medium, adding 50 μl of virus antibody complex per well, setting a multiplex well, setting no antibody group, no virus group and a positive serum control group, 4) culturing for 12 hours, adding 150 μl of maintenance solution per well, culturing at 37 ℃ for 48 hours, 5) lysing the cells by using a Luciferase assay kit (Luciferase ASSAY SYSTEM, promega Cat. #E1500), and detecting the Luciferase activity of each well, the specific assay method being referred to the kit instructions, detecting the chemiluminescent RLU value per well by using a multifunctional enzyme-marker (PERKIN ELMER), 6) calculating the percentage of inhibition of the monoclonal antibodies against viruses in different concentrations and the percent of the PRIC (Pad50) by using the software calculated percent of inhibition of the monoclonal antibodies against the virus in the phRLU value after culturing for 12 hours.

The results are shown in Table 3 below.

TABLE 3 Table 3

	IC50(ng/mL)
		4L12	4.5
12F5	11.1
		3D13	16.1
10C2	30.1
		16L9	4.1
20E21	2.3
		22H22	60.6

As can be seen from Table 3, 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21, 22H22 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 7 antibodies of 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 have better clinical application prospect.

8. Detection of S1 protein and RBD protein of 7 monoclonal antibodies (4L 12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H 22) for recognizing SARS-CoV-2 virus

The recognition of S1 and RBD proteins of SARS-CoV-2 virus by the 7 monoclonal antibodies obtained by the purification is sequentially detected by an enzyme-linked immunosorbent assay (ELISA).

The assay was performed by coating 1. Mu.g/ml of antigen protein (Yinqiao China) in 96-well ELISA plates at 4℃overnight. The plates were 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-T, 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 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 lABTS 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 FIG. 1, monoclonal antibody 4L12 recognizes the S1 protein of SARS-CoV-2 virus and its RBD, and the detection result of S2 protein;

referring to FIG. 2, monoclonal antibody 12F5 recognizes the S1 protein of SARS-CoV-2 virus and its RBD, and the detection result of the S2 protein;

Referring to FIG. 3, the detection results of the monoclonal antibody 3D13 recognizing the S1 protein of SARS-CoV-2 virus and its RBD, and the S2 protein;

referring to FIG. 4, the detection results of the monoclonal antibody 10C2 recognizing the S1 protein of SARS-CoV-2 virus and its RBD, and the S2 protein;

referring to FIG. 5, the detection results of the monoclonal antibody 16L9 recognizing the S1 protein of SARS-CoV-2 virus and its RBD, and the S2 protein;

referring to FIG. 6, the detection results of the monoclonal antibody 20E21 recognizing the S1 protein of SARS-CoV-2 virus and its RBD, and the S2 protein;

Referring to FIG. 7, monoclonal antibody 22H22 recognizes the S1 protein of SARS-CoV-2 virus and its RBD, and the detection result of the S2 protein.

From FIGS. 1-7, it can be seen that each of the monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 is capable of recognizing and binding to the S1 protein of SARS-CoV-2 virus and its RBD (conserved region), whereas the RBD of the S1 protein of coronavirus is an ACE2 receptor binding region with high conservation, it can be presumed that the monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application may have binding and neutralizing capabilities for other coronaviruses in addition to the strong binding and neutralizing capabilities for SARS-CoV-2 virus, and coronaviruses that may occur in the future.

8. Biological membrane layer interference technique for detecting binding capacity of 7 monoclonal antibodies (4L 12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H 22) of the application and RBD of S1 protein of SARS-CoV-2 virus

In order to detect the interaction between 7 monoclonal antibodies of the application and RBD of S1 protein of SARS-CoV-2 virus, the binding kinetics between them is detected by using a biological membrane layer interference technique, and the detection process is performed on an OctetRED96 (Fortebio) instrument.

The detection method comprises the following steps of soaking an AHC probe in sterile water for 10 minutes in advance to balance, and carrying out all detection processes under the reaction condition of 30 ℃, wherein the detection processes can be divided into five steps of 1) zeroing, namely, immersing the probe in sterile water for 60 seconds to obtain a detection baseline, 2) capturing antibody, namely, immersing the probe in 10 mug/ml monoclonal antibody solution for 200 seconds to capture antibody, 3) zeroing again, namely, immersing the probe in buffer solution (PBS solution added with 0.02% Tween 20) for 120 seconds to remove unbound antibody, 4) immersing the probe in RBD protein solution with initial concentration of 100nM and 3 times of gradient dilution, and carrying out the action for 300 seconds to obtain a dynamic curve of the monoclonal antibody binding with the RBD, and 5) dissociating the binding, namely, immersing the probe in the 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 and monoclonal antibody binding dissociation of the application is detected. And subtracting the data of the buffer solution control hole from the data of the sample hole during data analysis, deducting the nonspecific interference of the buffer solution, and adopting a 1:1 binding model to perform integral curve fitting on the binding with the monoclonal antibody under different RBD dilution concentrations to obtain the average binding constant K _on, the dissociation constant K _off and the affinity constant K _D.

The detection results are shown in FIGS. 8-14, which are sequentially the affinity detection results of RBD of monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 binding to S1 protein of SARS-CoV-2 virus, and each graph has five curves representing dynamic binding dissociation curves of the monoclonal antibodies with five RBDs with different concentrations.

As can be seen from FIGS. 8-14, the 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the application all have concentration gradient dependence on RBD binding of S1 protein of SARS-CoV-2 virus, dissociation is carried out after binding, the dissociated RBD is very few, and the K _D values of the 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 are (1.49+ -0.06) nM, (2.22+ -0.07) nM, (4.17+ -0.15) nM, (3.36+ -0.18) nM, (1.21+ -0.06) nM, (2.3+ -0.07) nM and (5.07+ -0.2) nM, which show very strong affinity of the 7 monoclonal antibodies of the application with RBD conserved regions of S1 protein of SARS-CoV-2. From this it can be deduced that the 7 mabs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 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 7 mabs of the present application to RBD conserved regions of S1 protein of SARS-CoV-2 virus. From a combination of the results in Table 1 and FIGS. 1-14, it was further verified that 7 mabs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 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 having strong binding and neutralizing capabilities for SARS-CoV-2.

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 SARS-CoV-2 virus 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, characterized in that:

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

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

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.

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

The antibody is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody is a fully human monoclonal antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is any one or a combination of a plurality of IgG1, igG2, igG3, or IgG 4.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antigen-binding fragment is Fv, fab, F (ab ') 2, fab', dsFv, scFv or sc (Fv) 2.

7. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the vector of claim 8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

11. An assay product comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

12. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the host cell according to claim 9 is cultured to produce the antibody or antigen-binding fragment thereof.

13. 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 10 for the manufacture of a medicament for the treatment or prophylaxis of a disease caused by SARS-CoV-2 virus.