Antibodies or antigen-binding fragments thereof against coronavirus

Abstract

The present application relates to antibodies or antigen-binding fragments thereof of coronavirus, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, vectors comprising the nucleic acid molecules, host cells comprising the vectors, and the antibodies or antigen-binding fragments thereof for preparing medicines for treating or preventing diseases caused by coronavirus, and for detecting products; the inventors have obtained a series of antibodies and antigen-binding fragments thereof of coronavirus by using B cell in vitro monoclonal culture and high-throughput antibody screening technology, which have strong binding and neutralizing abilities to SARS-CoV-2 virus, and can all recognize and bind to S1 protein and RBD of SARS-CoV-2 virus, and have very strong affinity, so it can be inferred that they also have binding and neutralizing abilities to other coronaviruses and future possible coronaviruses, and have good clinical application prospects in the future.

Description

Technical Field

[0001] The present invention relates to a coronavirus antibody or antigen-binding fragment thereof, 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 in preparing a medicament for treating or preventing diseases caused by coronavirus, as well as its use in detection products, and belongs to the field of biomedicine. Background Art

[0002] Novel coronavirus pneumonia (2019-nCOV) is an acute respiratory infectious disease caused by the new coronavirus SARS-COV-2.

[0003] The SARS-CoV-2 virus belongs to the Coronaviridae family and is a beta coronavirus, the same as the SARS coronavirus that broke out in 2003. The amino acid homology is as high as 77.2%. The main envelope protein of the SARS-CoV-2 virus is its spike protein (also known as Spike protein, referred to as S protein), which is hydrolyzed by intracellular proteases into two parts, S1 and S2, during the viral infection process. S2 is a transmembrane protein, and S1 has a receptor binding domain (RBD) that recognizes and binds to the cell receptor angiotensin-converting enzyme-2 (ACE-2). The spike protein composed of S1 and S2 is the viral receptor that the SARS-CoV-2 virus specifically recognizes, binds to the target cell receptor, and mediates viral infection. It is also the recognition target of the neutralizing antibodies to be developed.

[0004] Research has shown that the clinical use of virus-specific plasma from recovered patients can effectively neutralize the virus, prevent its spread throughout the body's organs, and play a significant role in patient outcomes. However, not only is the supply of polyclonal plasma limited, but its clinical application is also hampered by challenges such as difficulty in quality control, differences in donor and recipient blood types, and potential infectious agents. Isolating fully human monoclonal antibodies that neutralize SARS-CoV-2 from recovered COVID-19 patients could effectively overcome these challenges and is currently a key focus of COVID-19 drug development.

[0005] To date, multiple research teams domestically and internationally have reported the isolation of fully human monoclonal antibodies (mAbs) that bind to the SARS-CoV-2 S protein from the peripheral blood of recovered COVID-19 patients, such as BD-368-2 and B38. These antibodies are currently in the experimental development stage. The method employed by these research teams uses recombinantly expressed SARS-CoV-2 S protein or its receptor-binding domain (RBD) as bait. B cells (memory B cells) that bind to these proteins are then isolated from the peripheral blood of recovered patients. Cell-based or single-cell sequencing methods are then used to identify the heavy and light chain pairs of antibodies expressed by individual B cells. After the antibodies are recombinantly expressed in vitro, their ability to neutralize the virus is then validated. Because this method pre-selects and enriches B cells with a marker protein (the recombinantly expressed SARS-CoV-2 S protein or its RBD, referred to as bait) before antibody gene sequencing, only antibodies that specifically bind to the marker protein are screened.

[0006] Dr. Huang Jinghe (one of the inventors of this application) pioneered the human B cell in vitro monoclonal culture and high-throughput antibody screening technology in 2013 (Huang J et al. Nature Protocols 2013), which isolated fully human monoclonal antibodies from the peripheral blood of patients recovered from COVID-19. The process is as follows: first, the neutralizing antibodies in the serum of patients recovered from COVID-19 are detected using the SARS-CoV-2 and SARS-CoV pseudovirus neutralization system, and patients with high neutralizing activity against both SARS-CoV-2 and SARS-CoV are screened; then, peripheral blood lymphocytes are collected from the recovered patients, and memory B lymphocytes are sorted out by flow cytometry; single B cells are inoculated into 384-well plates, and cytokines and feeder cells are added for culture. After the cultured B cells are amplified and differentiated in vitro, they secrete antibodies into the supernatant. Then, an in vitro high-throughput neutralization experiment was used to detect the neutralizing ability of the antibodies in the supernatant against SARS-CoV-2 and SARS-CoV viruses, and positive clones that could simultaneously neutralize both viruses were screened. The heavy and light chain variable regions of the antibodies were cloned using RT-PCR, constructed into antibody heavy chain and light chain expression vectors, and then transfected into 293T cells to express and purify monoclonal antibodies.

[0007] While antibodies reported by other teams have demonstrated strong neutralizing activity against the tested SARS-CoV-2 strains, SARS-CoV-2 is an RNA virus, and its genome is susceptible to mutations during transmission. Mutations in the non-conserved regions recognized by these antibodies, resulting in new circulating strains, can lead to the antibodies losing their protective effect against the mutated virus.

[0008] Therefore, those skilled in the art still hope to develop new antibodies that have binding and neutralizing capabilities against coronaviruses including SARS-CoV-2 virus. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides, on one hand, a coronavirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three light chain complementary determining regions LCDR1, LCDR2, and LCDR3; wherein:

[0010] The general sequence formula of the HCDR1 is: GX1TVSSNY, wherein X1 is any one of L, I or F amino acids;

[0011] The general sequence formula of the HCDR2 is: X2YSGGSX3, wherein X2 is any one of L and I, and X3 is any one of A and T.

[0012] Preferably, the general sequence formula of the HCDR3 is: ARDLIX4YGMDV, wherein X4 is any amino acid of D or T;

[0013] The sequence of the LCDR1 is QGISSY, and the sequence of the LCDR2 is AAS;

[0014] The general sequence formula of the LCDR3 is: QQLNSYPPX5T, wherein X5 is any amino acid of L or Y.

[0015] In a preferred embodiment of the present invention, 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; and 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,

[0016] 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; and 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.

[0017] In another preferred embodiment of the present invention, the heavy chain variable region has the sequence shown in SEQ ID NO. 4 or a sequence having a sequence homology of more than 80% to the sequence shown in SEQ ID NO. 4, and the light chain variable region has the sequence shown in SEQ ID NO. 8 or a sequence having a sequence homology of more than 80% to the sequence shown in SEQ ID NO. 8; or,

[0018] The heavy chain variable region has a sequence as shown in SEQ ID NO. 14 or a sequence with more than 80% sequence homology to 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 with more than 80% sequence homology to the sequence shown in SEQ ID NO. 18.

[0019] In a preferred embodiment of the present invention, 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; and the sequence of the LCDR1 is shown as SEQ ID NO.25, the sequence of the LCDR2 is shown as SEQ ID NO.26, and the sequence of the LCDR3 is shown as SEQ ID NO.27; or,

[0020] The sequence of the HCDR1 is shown in SEQ ID NO.31, the sequence of the HCDR2 is shown in SEQ ID NO.32, and the sequence of the HCDR3 is shown in SEQ ID NO.33; and the sequence of the LCDR1 is shown in SEQ ID NO.35, the sequence of the LCDR2 is shown in SEQ ID NO.36, and the sequence of the LCDR3 is shown in SEQ ID NO.37; or,

[0021] The sequence of the HCDR1 is shown in SEQ ID NO.41, the sequence of the HCDR2 is shown in SEQ ID NO.42, and the sequence of the HCDR3 is shown in SEQ ID NO.43; and the sequence of the LCDR1 is shown in SEQ ID NO.45, the sequence of the LCDR2 is shown in SEQ ID NO.46, and the sequence of the LCDR3 is shown in SEQ ID NO.47; or,

[0022] the sequence of the HCDR1 is as set forth in SEQ ID NO. 61, the sequence of the HCDR2 is as set forth in SEQ ID NO. 62, the sequence of the HCDR3 is as set forth in SEQ ID NO. 63; and, the sequence of the LCDR1 is as set forth in SEQ ID NO. 65, the sequence of the LCDR2 is as set forth in SEQ ID NO. 66, the sequence of the LCDR3 is as set forth in SEQ ID NO. 67.

[0023] the sequence of the HCDR1 is as set forth in SEQ ID NO. 61, the sequence of the HCDR2 is as set forth in SEQ ID NO. 62, the sequence of the HCDR3 is as set forth in SEQ ID NO. 63; and, the sequence of the LCDR1 is as set forth in SEQ ID NO. 65, the sequence of the LCDR2 is as set forth in SEQ ID NO. 66, the sequence of the LCDR3 is as set forth in SEQ ID NO. 67.

[0024] In another preferred embodiment of the present application, the heavy chain variable region has a sequence as set forth in SEQ ID NO. 24 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 24, and the light chain variable region has a sequence as set forth in SEQ ID NO. 28 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 28; or,

[0025] the heavy chain variable region has a sequence as set forth in SEQ ID NO. 34 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 34, and the light chain variable region has a sequence as set forth in SEQ ID NO. 38 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 38; or,

[0026] the heavy chain variable region has a sequence as set forth in SEQ ID NO. 44 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 44, and the light chain variable region has a sequence as set forth in SEQ ID NO. 48 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 48; or,

[0027] the heavy chain variable region has a sequence as set forth in SEQ ID NO. 54 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 54, and the light chain variable region has a sequence as set forth in SEQ ID NO. 58 or a sequence with 80% or higher sequence homology to the sequence set forth in SEQ ID NO. 58; or,

[0028] The heavy chain variable region has a sequence as shown in SEQ ID NO. 64 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO. 64, and the light chain variable region has a sequence as shown in SEQ ID NO. 68 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO. 68.

[0029] With respect to percentages of "sequence homology," the number of matching positions is determined by determining the number of amino acid residues present in the two sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying that number by 100 to yield the percent identity of the sequences.

[0030] In a specific embodiment of the present invention, the heavy chain variable region can be subjected to amino acid addition, deletion or substitution based on the first amino acid sequence, and the light chain variable region can be subjected to amino acid addition, deletion or substitution based on the second amino acid sequence, such as substitution of similar amino acids or addition or subtraction of a small number of amino acids, especially addition, subtraction or substitution of amino acids in the conserved sequence portion. The obtained antibody variants have a high homology (more than 80% homology) and retain the original antibody function, i.e., the function and property of specific binding to coronavirus. These variants also fall within the scope of protection of the present invention.

[0031] In a preferred embodiment of the present invention, the heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.9, and the light chain amino acid sequence is shown in SEQ ID NO.10; or,

[0032] 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,

[0033] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.29, and the light chain amino acid sequence is shown in SEQ ID NO.30; or,

[0034] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.39, and the light chain amino acid sequence is shown in SEQ ID NO.40; or,

[0035] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.49, and the light chain amino acid sequence is shown in SEQ ID NO.50; or,

[0036] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.59, and the light chain amino acid sequence is shown in SEQ ID NO.60; or,

[0037] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.69, and the light chain amino acid sequence is shown in SEQ ID NO.70.

[0038] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof against coronavirus.

[0039] The term "neutralizing antibody" refers to an antibody or antigen-binding fragment that specifically binds to a viral receptor protein. This specific binding can inhibit the biological function of the viral receptor protein, such as preventing the receptor protein from binding to its target cell receptor, and specifically reducing the ability of the virus to infect target cells. In this application, a neutralizing antibody or antigen-binding fragment thereof for coronavirus refers to an antibody or antigen-binding fragment thereof that binds to the S protein of the coronavirus.

[0040] The term "antibody" is used herein in the broadest sense to cover natural antibodies 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, etc. that exhibit the desired antigen-binding activity.

[0041] In a preferred embodiment of the present invention, the antibody is a monoclonal antibody.

[0042] In a more preferred embodiment of the present invention, the antibody is a fully human monoclonal antibody.

[0043] In a preferred embodiment of the present invention, the Fc domain of the antibody is derived from the Fc domain of an immunoglobulin, including a native sequence Fc domain or a variant Fc domain.

[0044] In a preferred embodiment of the present invention, the antibody is any one of IgG1, IgG2, IgG3 or IgG4, or a combination of several of them.

[0045] Preferably, the antibody may be a complete antibody selected from IgG1, IgG2, IgG3 or IgG4.

[0046] In a preferred embodiment of the present invention, the antigen-binding fragment is Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or a single-chain antibody.

[0047] In a preferred embodiment of the present application, the antibody or antigen-binding fragment thereof can 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 can be different epitopes of the same antigen or different antigens.

[0048] In a preferred embodiment of the present application, the antibody or antigen-binding fragment thereof described above can be further chemically modified, for example, one or more chemical groups can be attached to the antibody to increase one or more functional properties of the antibody. For example, common chemical modifications include glycosylation modification and polyethylene glycol modification, etc. For example, glycosylation modification can be performed on the heavy chain or light chain variable region to increase one or more glycosylation sites to improve some functions of the antibody, such as enhancing the immunogenicity of the antibody or improving the pharmacokinetics of the antibody, etc. For example, under suitable conditions, acylation reaction or alkylation reaction of the antibody or antigen-binding fragment thereof with active polyethylene glycol (such as active ester or aldehyde derivative of polyethylene glycol) is performed to achieve polyethylene glycol modification to improve some functions of the antibody, such as increasing the biological (such as serum) half-life of the antibody, etc. The above-mentioned chemical modifications do not significantly change the basic functions and properties of the antibody or antigen-binding fragment thereof of the present application, i.e., the functions and properties of specific binding to the coronavirus; these chemically modified variants also fall within the scope of protection of the present application.

[0049] In a preferred embodiment of the present application, the antibody or antigen-binding fragment thereof described above can be conjugated with other factors by chemical methods or genetic engineering methods; for example, these factors can provide the effect of targeting the antibody to the desired functional site or other properties; for example, these factors can be one or more heterologous molecules, preferably, the heterologous molecule is a cytotoxic agent. The complex formed by conjugating the above-mentioned antibody or antigen-binding fragment thereof with other factors falls within the scope of protection of the present application.

[0050] Another aspect of the present application provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof described above.

[0051] In a preferred embodiment of the present application, in the nucleic acid molecule, the nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO. 71, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO. 72; or,

[0052] the nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO. 75, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO. 76; or,

[0053] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.79, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.80; or,

[0054] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.83, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.84; or,

[0055] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.87, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.88; or,

[0056] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.91, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.92; or,

[0057] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.95, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.96.

[0058] In a more preferred embodiment of the present invention, in the nucleic acid molecule,

[0059] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.73, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.74; or,

[0060] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.77, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.78; or,

[0061] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.81, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.82; or,

[0062] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.85, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.86; or,

[0063] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.89, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.90; or,

[0064] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.93, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.94; or,

[0065] The nucleic acid sequence encoding the heavy chain is shown as SEQ ID NO. 97, and the nucleic acid sequence encoding the light chain is shown as SEQ ID NO. 98.

[0066] The present application also provides a vector comprising the nucleic acid molecule as described above.

[0067] In a preferred embodiment of the present application, the vector further comprises an expression control sequence linked to the nucleic acid molecule as described above.

[0068] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed in a host cell. The vector can be transformed, transduced or transfected into a host cell, so that the genetic material elements carried by the vector can be expressed in the host cell. The vector can comprise various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector can also contain a replication initiation site. The vector can also include components that facilitate its entry into a cell, such as viral particles, liposomes or protein coats, but not only these. In the embodiments of the present application, the vector can be selected from, but not limited to: plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes YAC, bacterial artificial chromosomes BAC or P1-derived artificial chromosomes PAC), bacteriophages (such as lambda phage or M13 phage), and animal viruses used as vectors, such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40).

[0069] The present application also provides a host cell comprising the vector as described above.

[0070] As for the "host cell", prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 fruit fly cells or Sf9, or animal cell models such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc. can be selected, but are not limited to.

[0071] Preferably, the host cell is a HEK293 cell.

[0072] The present application also provides a method for producing an antibody, or an antigen-binding fragment thereof, as described above, wherein the host cell as described above is cultured to produce the antibody, or the antigen-binding fragment thereof.

[0073] The present application also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the antibody, or the antigen-binding fragment thereof, as described above.

[0074] In a preferred embodiment of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of a neutralizing antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent. Those skilled in the art can use an appropriate pharmaceutical carrier or diluent in combination with a therapeutically effective amount of the neutralizing antibody, or an antigen-binding fragment thereof, and administer the composition to a patient for the treatment or prevention of a disease caused by a coronavirus.

[0075] In another aspect, the present invention provides the use of the above-mentioned antibody, or its antigen-binding fragment, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating or preventing diseases caused by coronavirus.

[0076] In a preferred embodiment of the present invention, the use refers to use in the preparation of drugs for treating or preventing diseases caused by SARS-CoV-2, SARS-CoV or SARS-like coronaviruses.

[0077] In one aspect, the present invention also provides a method for treating or preventing a disease caused by a coronavirus, comprising administering to a patient a therapeutically effective amount of the aforementioned antibody, or an antigen-binding fragment thereof; or administering to a patient a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned antibody, or an antigen-binding fragment thereof. Preferably, the disease caused by the coronavirus is a disease caused by SARS-CoV-2, SARS-CoV, or a SARS-like coronavirus.

[0078] In another aspect, the present invention provides a detection product, wherein the detection product comprises the above-mentioned antibody, or an antigen-binding fragment thereof.

[0079] The detection product is used to detect the presence or level of coronavirus in a sample.

[0080] In a specific embodiment of the present invention, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or a test paper, etc.

[0081] The above-mentioned antibodies or antigen-binding fragments thereof of the present invention can be labeled by chemical methods or genetic engineering methods, and the labeled antibodies or antigen-binding fragments thereof can be used for detection; the labeled antibodies or antigen-binding fragments thereof fall within the scope of protection of the present invention.

[0082] The specific detection method can adopt the following steps: 1) providing a sample; 2) contacting the sample with the above-mentioned coronavirus antibody or antigen-binding fragment thereof of the present invention; 3) detecting the immune reaction between the sample and the antibody or antigen-binding fragment thereof.

[0083] The inventors used B cell in vitro monoclonal culture and high-throughput antibody screening technology to obtain a series of coronavirus antibodies and antigen-binding fragments thereof. These antibodies and antigen-binding fragments thereof have strong binding and neutralizing abilities for the SARS-CoV-2 virus, and are all able to recognize and bind to the S1 protein and RBD of the SARS-CoV-2 virus with very strong affinity. It can be inferred that the series of coronavirus antibodies and antigen-binding fragments thereof of the present invention may also have binding and neutralizing abilities for other coronaviruses, as well as coronaviruses that may appear in the future, and have good prospects for clinical application in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The results of the monoclonal antibody 4L12 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0085] Figure 2 The results of the monoclonal antibody 12F5 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0086] Figure 3 The results of the monoclonal antibody 3D13 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0087] Figure 4 The test results of monoclonal antibody 10C2 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0088] Figure 5 The results of the monoclonal antibody 16L9 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0089] Figure 6 The detection results of monoclonal antibody 20E21 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0090] Figure 7 The results of the monoclonal antibody 22H22 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0091] Figure 8 This is the affinity test result of monoclonal antibody 4L12 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0092] Figure 9 The results of affinity test of monoclonal antibody 12F5 binding to the RBD of the S1 protein of SARS-CoV-2 virus;

[0093] Figure 10 The results of affinity test of monoclonal antibody 3D13 binding to the RBD of the S1 protein of SARS-CoV-2 virus;

[0094] Figure 11 The results of affinity test of monoclonal antibody 10C2 binding to the RBD of the S1 protein of SARS-CoV-2 virus;

[0095] Figure 12 This is the affinity test result of monoclonal antibody 16L9 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0096] Figure 13 This is the affinity test result of monoclonal antibody 20E21 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0097] Figure 14 This is the affinity test result of monoclonal antibody 22H22 binding to the RBD of the S1 protein of the SARS-CoV-2 virus. DETAILED DESCRIPTION

[0098] The present invention will be described in detail below with reference to the specific embodiments shown in the accompanying drawings. However, these embodiments do not limit the present invention, and any structural, methodological, or functional changes made by those skilled in the art based on these embodiments are all within the scope of protection of the present invention.

[0099] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example 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.

[0100] Unless otherwise specified, the materials and reagents used in the following examples were commercially available. Where specific techniques or conditions are not specified in the examples, the experiments were performed according to those described in the literature in the field (e.g., Molecular Cloning: A Laboratory Manual, 3rd edition, by J. Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions.

[0101] Example 1: Screening and detection of coronavirus antibodies

[0102] The inventors conducted pseudovirus neutralization experiment screening on the plasma of patients with novel coronavirus pneumonia who were admitted to the inventor's unit (Shanghai Public Health Clinical Center) from January 20, 2020 to February 26, 2020 (followed up two weeks after recovery and discharge). It was found that the serum of three mild patients had strong neutralizing activity against SARS-CoV-2 pseudovirus. With the written consent of the ethics committee of the inventor's unit and the patients themselves, their peripheral blood was drawn for research.

[0103] 1. Isolation of peripheral blood memory B cells

[0104] 1) Isolation of peripheral blood lymphocytes: Peripheral blood was drawn from the patients in the convalescent phase and mixed with an equal amount of normal saline. Peripheral blood lymphocytes were isolated using Lymphoprep (Stemcell Technologies, Catalog No. 07851), a lymphocyte separation medium. For the procedure, refer to the instructions for the lymphocyte separation medium.

[0105] 2) Isolation of Peripheral Blood Memory B Cells: The peripheral blood lymphocytes isolated in step 1) above were stained with an antibody mixture consisting of anti-CD19-PE-Cy7 (BD Bioscience), IgA-APC (Jackson Immunoresearch), IgD-FITC (BD Bioscience), and IgM-PE (Jackson Immunoresearch) at 4°C in the dark for 30 min. After staining, the cells were washed with 10 ml of PBS-BSA buffer and resuspended in 500 μl of PBS-BSA. Finally, CD19+IgA-IgD-IgM- memory B cells were sorted using a FACSAria III cell sorter (Becton Dickinson).

[0106] 2. Incubation of peripheral blood memory B cells

[0107] The sorted CD19+IgA-IgD-IgM- memory B cells were resuspended in culture medium containing 10% FBS, 100 U / ml IL-2, 50 ng / ml IL-21, and irradiated 3T3-msCD40L feeder cells. Memory B cells were seeded at a density of 4 cells / well in a 384-well microtiter plate (final volume of 50 μl) and incubated for 13 days. Growth factors IL-2 and IL-21 stimulated the memory B cells to divide and grow, secreting antibodies into the incubation medium. For detailed culture methods, see Huang J et al. Nature Protocols 2013, 8(10):1907-15.

[0108] 3. Production of SARS-CoV-2 and SARS-CoV pseudoviruses

[0109] SARS-CoV-2 and SARS-CoV pseudoviruses are non-replication-defective retroviral particles that bear the SARS-CoV-2 and SARS-CoV spike (S) proteins on their surfaces, respectively, and carry a luciferase reporter gene. They can mimic the infection process of SARS-CoV-2 and SARS-CoV viruses in host cells (such as the human hepatoma cell line Huh-7 and the 293T cell line 293T-ACE2, which stably expresses the human ACE2 receptor) and express the luciferase reporter gene in infected cells. Because pseudovirus infection cannot produce mature viral particles, related operations can be safely performed in a biosafety level 2 laboratory.

[0110] SARS-CoV-2 and SARS-CoV pseudoviruses were generated by co-transfecting 293T cells with their respective S protein expression plasmids and an HIV Env-deficient backbone plasmid (pNL4-3.Luc.RE-) carrying a luciferase reporter gene. The S gene sequences of SARS-CoV-2 and SARS-CoV were designed based on NCBI GenBank sequences NC_045512 and ABD72979.1, respectively. After codon optimization, the gene sequences were synthesized by Nanjing GenScript and ligated into the pcDNA3.1 eukaryotic expression vector to construct SARS-CoV-2 and SARS-CoV S protein expression plasmids. The pNL4-3.Luc.RE- backbone plasmid was obtained from the NIH AIDS Reagent Program. All plasmids were amplified by transformation into DH5α competent cells and purified using a plasmid purification kit manufactured by Meiji Biotechnology. Purification procedures were performed according to the kit instructions.

[0111] 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and seeded into 10 cm dishes before transfection. After 24 hours of culture, the backbone plasmid (pNL4-3.Luc.RE-) and a plasmid expressing SARS-CoV or SARS-CoV-2 were co-transfected into the 293T cells at a 3:1 ratio using EZ Trans Cell Transfection Reagent (Liji Biotechnology). For detailed transfection procedures, refer to the EZ Trans Cell Transfection Reagent user manual. Forty-eight hours after transfection, the pseudovirus-containing supernatant was harvested, centrifuged at 1500 rpm for 10 minutes to remove cell debris, and aliquots were stored frozen at -80°C for neutralizing antibody testing.

[0112] 4. Neutralization screening

[0113] After 13 days of in vitro culture of peripheral blood memory B cells, 40 μl of culture supernatant was collected from each well for testing for neutralizing antibodies against SARS-CoV-2 and SARS-CoV. The assay was performed as follows: 20 μl of culture supernatant was mixed with 20 μl of pseudovirus supernatant produced above in a 384-well cell culture plate. After incubation at room temperature for 30 minutes, 5000 293T-ACE2 cells (50 μl) were added to each well and cultured 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 in each well. The specific assay method is described in the kit instructions. Chemiluminescence (RLU) values ​​were measured in each well using a multi-function microplate reader (Perkin Elmer). The percentage of neutralization inhibition of the pseudovirus by the culture supernatant was calculated based on the ratio of the RLU values ​​of the culture supernatant to the virus control. Wells with an inhibition percentage greater than 90% were selected as virus neutralization-positive wells.

[0114] 5. RT-PCR amplification of heavy and light chain genes

[0115] B cells from virus neutralization-positive wells were used to amplify the variable regions of the heavy and light chains of immunoglobulin genes using RT-PCR. For primer design and detailed RT-PCR procedures, see Tiller, T. et al. J. Immunol Methods 2018, 329:112–124. The amplified heavy and light chain variable region genes were purified by agarose gel electrophoresis and cloned into the PMD19-T vector using the PMD19-T vector cloning kit (Takara 6013). Detailed procedures are described in the kit instructions. Single clones were then selected for gene sequencing.

[0116] 6. Expression and purification of monoclonal antibodies

[0117] The antibody heavy chain variable region gene correctly sequenced was digested with Age I and Sal I, respectively, and then connected to the target fragment purified from the gel after ligation and transformed into DH5a competent cells to construct an antibody expression heavy chain plasmid; the antibody Lambda or Kappa light chain variable region gene correctly sequenced was digested with Age I and Xho I or Age I and Bsiw I, respectively, and then connected to the target fragment purified from the gel after ligation and transformed into DH5a competent cells to construct an antibody expression light chain plasmid; the antibody heavy chain and light chain plasmids were purified by a plasmid purification kit (MegaBioservices) (see Figure 1 The expression of the purified antibody was detected by SDS-PAGE, and the 293T cells were co-transfected with the antibody heavy chain and light chain plasmids at a ratio of 1:1 using the EZ Trans cell transfection reagent (RiBio) to express the antibody. After 72 hours, the cell transfection supernatant was collected, and the antibody IgG in the supernatant was purified by a protein-G column (Tiandihuan Biotech, Changzhou), and the purification method was in accordance with the instructions of the protein-G column. The antibody IgG obtained by purification was determined by the Nanodrop 2000 (Thermo Fisher) at 280 nm absorbance, and the antibody concentration was calculated.

[0118] Through the above Part 1-6, the present inventors obtained several IgG antibodies, and the present application discloses seven antibodies (the names are 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22, respectively).

[0119] The amino acid sequence number information of the seven antibodies is shown in Table 1 below:

[0120] Table 1

[0121]

[0122]

[0123] The nucleotide sequence number information of the seven antibodies is shown in Table 2 below:

[0124] Table 2

[0125]

[0126] 7. Detection of the neutralizing activity of the seven monoclonal antibodies (4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22) against SARS-CoV-2 coronavirus

[0127] Different concentrations of monoclonal antibodies were tested in 96-well cell plates to inhibit pseudovirus infection of Huh-7 cells to detect the neutralizing ability of monoclonal antibodies against SARS-CoV-2 coronavirus.

[0128] The detection method is as follows: 1) Huh-7 cells were seeded in a 96-well cell plate, with 1×10 4 1) The cells were incubated at 37°C in a 5% CO2 cell culture incubator for 24 hours; 2) the monoclonal antibody was diluted to different concentrations in cell culture medium, mixed with an equal volume of pseudovirus dilution containing 100 TCID50, and incubated at 37°C for 1 hour; 3) the cell culture medium was discarded, 50 μl of the virus-antibody complex was added to each well, and duplicate wells were set up. A no-antibody group, a no-virus group, and a positive serum control group were also set up; 4) after 12 hours of incubation, 150 μl of maintenance solution was added to each well, and the cells were incubated at 37°C for another 48 hours; 5) the cells were lysed using a luciferase assay kit (Luciferase Assay System, Promega Cat. #E1500) and the luciferase activity in each well was measured; the specific assay method was referred to the kit instructions; the chemiluminescence (RLU) value of each well was measured using a multi-function microplate reader (Perkin Elmer); 6) the percentage of neutralization inhibition of the pseudovirus by different concentrations of the monoclonal antibody was calculated based on the ratio of the RLU values ​​of the monoclonal antibody to the virus control, and the half-maximal inhibitory dose (IC50) of the monoclonal antibody for viral inhibition was calculated using PRISM7 software (GraphPad).

[0129] The results are shown in Table 3 below.

[0130] Table 3

[0131] IC50 (ng / mL) 4L12 4.5 12F5 11.1 3D13 16.1 10C2 30.1 16L9 4.1 20E21 2.3 22H22 60.6

[0132] As shown in Table 3, seven monoclonal antibodies (MAbs)—4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22—can effectively neutralize SARS-CoV-2 at concentrations in the ng / mL range, demonstrating exceptionally strong neutralizing activity. Stronger neutralizing activity requires less antibody, leading to lower costs. Therefore, these seven antibodies, 4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22, have promising clinical application prospects.

[0133] 8. Detection of the seven monoclonal antibodies (4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22) of this application for recognition of the S1 protein and RBD protein of the SARS-CoV-2 virus

[0134] The recognition of the S1 and RBD proteins of the SARS-CoV-2 virus by the seven purified monoclonal antibodies was detected in turn by enzyme-linked immunosorbent assay (ELISA).

[0135] The assay method is as follows: 1 μg / ml antigen protein (Sino Biological) was coated onto a 96-well ELISA plate overnight at 4°C. The plate was washed five times with PBS-T (0.2% Tween-20) and blocked with 300 μl of blocking buffer (PBS, 1% FBS, 5% milk) per well for 1 hour at room temperature. The plate was washed three times with PBS-T. Monoclonal antibody was serially diluted five-fold in PBS (PBS, 5% FBS, 2% BSA, 1% Tween-20). 100 μl of the sample was added to the ELISA plate and incubated at 37°C for 1 hour. The plate was washed five times with PBS-T and 100 μl of horseradish peroxidase-conjugated goat anti-human IgG antibody (Jackson Immunoresearch) diluted 1:2500 in PBS was added to each well and incubated for 1 hour at room temperature. The plate was washed five times with PBS-T, 150 μl of ABTS color development substrate (Thermo Fisher) was added, and color was developed at room temperature in the dark for 30 minutes. The absorbance at a wavelength of 405 nm was read using a microplate reader.

[0136] See also Figure 1 , the detection results of monoclonal antibody 4L12 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0137] See also Figure 2 , the detection results of monoclonal antibody 12F5 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0138] See also Figure 3 , the detection results of monoclonal antibody 3D13 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0139] See also Figure 4 , the detection results of monoclonal antibody 10C2 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0140] See also Figure 5 , the detection results of monoclonal antibody 16L9 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0141] See Figure 6 , the detection results of mAb 20E21 recognizing S1 protein and its RBD of SARS-CoV-2 virus, and S2 protein;

[0142] See Figure 7 , the detection results of mAb 22H22 recognizing S1 protein and its RBD of SARS-CoV-2 virus, and S2 protein.

[0143] From Figure 1-7 It can be seen that mAbs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 can all recognize and bind to S1 protein and its RBD (conserved region) of SARS-CoV-2 virus; since the RBD of S1 protein of coronavirus is the region of ACE2 receptor binding, it has high conservation, so it can be inferred that mAbs 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application have strong binding ability and neutralization ability not only to SARS-CoV-2 virus, but also to other coronaviruses, and future coronaviruses that may appear.

[0144] 8. Bio-layer interferometry detection of the binding ability of the 7 mAbs (4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22) of the present application to the RBD of S1 protein of SARS-CoV-2 virus

[0145] In order to detect the interaction between the 7 mAbs of the present application and the RBD of S1 protein of SARS-CoV-2 virus, the binding kinetics between them was detected by bio-layer interferometry, and the detection process was carried out on an Octet RED96 (Fortebio) instrument.

[0146] The detection method is as follows: the AHC probe is pre-equilibrated by soaking it in sterile water for 10 minutes. The entire detection process is performed at 30°C and can be divided into the following five steps: 1) Zeroing: Immerse the probe in sterile water for 60 seconds to obtain a detection baseline; 2) Antibody Capture: Immerse the probe in a 10 μg / ml monoclonal antibody solution for 200 seconds to capture the antibody; 3) Re-zeroing: Immerse the probe in a buffer solution (PBS with 0.02% Tween 20) for 120 seconds to remove unbound antibody; 4) RBD Binding: Immerse the probe in a 3-fold serial dilution of RBD protein starting at 100 nM for 300 seconds to obtain a dynamic curve of monoclonal antibody binding to RBD; 5) Binding and Dissociation: Immerse the probe in buffer for 300 seconds. Protein binding causes a change in the biofilm thickness, resulting in a relative shift of the interfering light waves, which is detected by the spectrometer and forms an interference spectrum, displayed as the real-time displacement (nm) of the interference pattern. This was used to detect the dynamic curve of the binding and dissociation of RBD with the monoclonal antibody of the present application. During data analysis, the data of the sample wells were subtracted from the data of the buffer control wells to deduct the nonspecific interference of the buffer solution. A 1:1 binding model was used to fit the overall curve of the binding of RBD with the monoclonal antibody at different dilution concentrations to obtain the average binding constant K. on , dissociation constant K off and the affinity constant K D value.

[0147] Test results see Figure 8-14 , which are the affinity test results of monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 binding to the RBD of the S1 protein of the SARS-CoV-2 virus; Each figure has five curves, which represent the dynamic binding and dissociation curves of the monoclonal antibody with five different concentrations of RBD.

[0148] from Figure 8-14 It can be seen that the seven monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application all bind to the RBD of the S1 protein of the SARS-CoV-2 virus in a concentration gradient-dependent manner; after binding, they dissociate, and the dissociated RBD is very small; the K values ​​of the seven monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 are DThe values ​​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, (5.07±0.2)nM, respectively; it shows that the 7 monoclonal antibodies of the present application have very strong affinity with the RBD conserved region of the S1 protein of SARS-CoV-2. It can be inferred from this that the 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application demonstrated in Section 7 above have strong neutralizing activity against the RBD of the S1 protein of the SARS-CoV-2 virus. This is because the 7 monoclonal antibodies of the present application have very strong affinity for the RBD conserved region of the S1 protein of the SARS-CoV-2 virus. Comprehensive Table 1 and Figure 1-14 The results further verified that the seven monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application not only have strong binding and neutralizing abilities against SARS-CoV-2, but may also have binding and neutralizing abilities against other coronaviruses and coronaviruses that may appear in the future.

[0149] It should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation method can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0150] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against SARS-CoV-2 virus, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3); characterized in that: The sequence of the HCDR1 is shown as SEQ ID NO.41, the sequence of the HCDR2 is shown as SEQ ID NO.42, and the sequence of the HCDR3 is shown as SEQ ID NO.43; in addition, the sequence of the LCDR1 is shown as SEQ ID NO.45, the sequence of the LCDR2 is AAS, and the sequence of the LCDR3 is shown as SEQ ID NO.

47.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region has the sequence shown in SEQ ID NO.44, and the light chain variable region has the sequence shown in SEQ ID NO.

48.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The antibody is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, 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 of IgG1, IgG2, IgG3 or IgG4, or a combination of several of them.

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

8. A vector comprising the nucleic acid molecule according to claim 7.

9. A host cell comprising the vector according to claim 8.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

11. A product for detecting SARS-COV-2 virus, characterized by: The product comprises the 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, characterized in that: Cultivate the host cell of claim 9 to produce the antibody or antigen-binding fragment thereof.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating or preventing diseases caused by SARS-COV-2 virus.

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