A human monoclonal antibody neutralizing sars-cov-2 and use thereof

Abstract

The application discloses a human monoclonal antibody for neutralizing SARS-CoV-2 and application thereof, wherein the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region are respectively shown as SEQ ID NO:11, GKN and SEQ ID NO:15, the monoclonal antibody has strong neutralization activity on SARS-CoV-2, lays a foundation for diagnosis, treatment and development of related drugs of SARS-CoV-2 infection related diseases, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and in particular relates to a human monoclonal antibody neutralizing SARS-CoV-2 and application thereof. BACKGROUND

[0002] The pathogen SARS-CoV-2 of novel coronavirus infection is a linear positive strand RNA virus of the coronavirus family beta virus, and research has found that SARS-CoV-2 is adjacent to SARS-CoV and SARS-CoV-like groups in the position of the evolutionary tree. The novel coronavirus has four major structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein). The S protein contains two subunits, S1 and S2. The RBD domain on the S1 subunit completes the adsorption of the virus particle on the receptor cell through the interaction with the human respiratory epithelial cell membrane ACE2, while the S2 subunit mediates the fusion process of the virus membrane and the membrane structure of the susceptible cell. Therefore, both S1 and S2 subunits contain epitopes of neutralizing antibodies.

[0003] As a supplement to vaccines and chemotherapy, antibody-mediated prevention and treatment measures for viral infection have shown good effects, and their application prospects have been recognized by experts. Unlike the characteristics of S1 easy to mutate, as a typical membrane fusion protein, the S2 domain is more conservative in sequence and structure, and is more suitable as a target for screening broad-spectrum antibodies, and has become a recognized region for preparing protective vaccines and broad-spectrum neutralizing antibodies against SARS-CoV-2. At present, there is no therapeutic drug for SARS-CoV-2 infection, and the treatment plan for COVID-19 caused by SARS-CoV-2 infection is mainly to relieve symptoms, prevent secondary infection, reduce complications, and support organ function. Therefore, there is an urgent need in the art to develop coronavirus neutralizing antibodies, especially monoclonal antibodies, with good virus neutralizing effect. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a human monoclonal antibody S2 neutralizing SARS-CoV-2 and application thereof for the art. The present application adopts phage display technology to construct a large-capacity human immunotype phage antibody library, and uses SARS-CoV-2 virus S2 subunit as a target to screen human antibody single chain antibody fragments (ScFv), and obtains a ScFv antibody molecule S2. Further research shows that the monoclonal antibody has a strong neutralizing effect on SARS-CoV-2 virus.

[0005] The application achieves the above-mentioned application purposes by adopting the following technical solutions:

[0006] The first aspect of the application provides a human monoclonal antibody neutralizing SARS-CoV-2, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region;

[0007] The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are respectively shown in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6;

[0008] The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region are respectively shown in SEQ ID NO:11, GKN and SEQ ID NO:15.

[0009] Further, the amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 in the heavy chain variable region are respectively shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, or amino acid sequences having at least 70% homology with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7;

[0010] The amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 in the light chain variable region are respectively shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16, or amino acid sequences having at least 70% homology with SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0011] Further, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, or an amino acid sequence having at least 70% homology with SEQ ID NO:8;

[0012] Optionally, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:17, or an amino acid sequence having at least 70% homology with SEQ ID NO:17.

[0013] In some embodiments, the CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region, the CDR-L1, CDR-L2, CDR-L3 in the light chain variable region of the monoclonal antibody provided in the present application are not limited to the amino acid sequences as described above, and the corresponding amino acid sequences of the CDR1, CDR2, CDR3 in the heavy chain variable region and the light chain variable region of the monoclonal antibody provided in the present application defined by using any CDR numbering scheme are within the scope of the present application.

[0014] In some embodiments, the CDR numbering scheme includes but is not limited to any one or any combination (two or more) of the following: Chothia numbering scheme, Kabat numbering scheme, Contact numbering scheme, IMGT numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, Aho numbering scheme.

[0015] In some embodiments, the antibody sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the corresponding amino acid sequence of the heavy chain variable region, the light chain variable region, the CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region, or the CDR-L1, CDR-L2, CDR-L3 in the light chain variable region of the monoclonal antibody provided in the present application is within the scope of the present application.

[0016] In some embodiments, the SARS-CoV-2 includes but is not limited to wild-type SARS-CoV-2, SARS-CoV-2 variant XBB.1.16, SARS-CoV-2 variant XBB.2.3, SARS-CoV-2 variant JN.1S, SARS-CoV-2 variant BQ.1.1, SARS-CoV-2 Alpha (Alpha) variant, SARS-CoV-2 Beta (Beta) variant, SARS-CoV-2 Gamma (Gamma) variant, SARS-CoV-2 Delta (Delta) variant, SARS-CoV-2 Omicron (Omicron) variant.

[0017] The second aspect of the present application provides a nucleic acid molecule encoding the monoclonal antibody of the first aspect of the present application.

[0018] Optionally, the nucleotide sequence of the monoclonal antibody heavy chain variable region is as shown in SEQ ID NO: 9 or a nucleotide sequence having at least 70% homology with SEQ ID NO: 9.

[0019] Optionally, the nucleotide sequence of the monoclonal antibody light chain variable region is as shown in SEQ ID NO: 18 or a nucleotide sequence having at least 70% homology with SEQ ID NO: 18.

[0020] In the present application, the nucleic acid molecule refers to a polymerized form of nucleotides, and includes RNA, cDNA, sense strands and antisense strands of genomic DNA, as well as synthetic forms and mixed polymers of the above. In particular embodiments, the nucleotides refer to ribonucleotides, deoxyribonucleotides or modified forms of either nucleotide type, and combinations thereof. The nucleic acid molecule also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, a polynucleotide, e.g., a cDNA or mRNA, can include any or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds.

[0021] In some embodiments, the nucleic acid molecule can be chemically or biochemically modified, or can contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, amino phosphonates, phosphoramidates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc).

[0022] A third aspect of the present application provides an expression vector comprising the nucleic acid molecule of the second aspect of the present application;

[0023] Optionally, the vector is a DNA vector, an RNA vector or a viral-derived vector.

[0024] Optionally, the viral-derived vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, an adenoviral vector, a poxviral vector or a herpesviral vector.

[0025] In the present application, the expression vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The expression vector includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked.

[0026] In some embodiments, the expression vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), as well as vectors derived from bacteriophage or plant or animal (including human) viruses. Vectors can include origins of replication recognized by host cells, and in the case of expression vectors, include promoters and other regulatory sequences recognized by the host cells. In particular embodiments, the vectors include nucleic acid molecules encoding the monoclonal antibodies of the application as previously described operably linked to a promoter and optional other regulatory elements. Certain vectors are capable of autonomous replication in the host in which they are introduced (e.g., vectors having a bacterial origin of replication can replicate in bacteria). Other vectors can integrate into the genome of the host and thereby be replicated with the host genome upon introduction into the host. Vectors include, but are not limited to, those suitable for recombinant production of the monoclonal antibodies of the application as previously described, as well as those suitable for introduction into a desired subject or cell thereof to provide the monoclonal antibodies of the application as previously described to the subject.

[0027] In some embodiments, the choice of particular vector depends on the recombination procedure followed and the host used. Introduction of the vector into the host cell can be effected by calcium phosphate transfection, viral infection, DEAE-Dextran-mediated transfection, lipofection, or electroporation, among others. Vectors can replicate autonomously or can replicate together with the chromosome into which they have been integrated. In certain embodiments, the vectors comprise one or more selectable markers. The choice of marker can depend on the host cell selected. These include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, bleomycin, the thymidine kinase gene from Herpes simplex virus, and the dihydrofolate reductase gene from mouse.

[0028] A fourth aspect of the application provides a recombinant host cell comprising the expression vector of the third aspect of the application.

[0029] In some embodiments, the host cells include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; NSO cells; yeast, and the like. Particular eukaryotic host cells are selected based on their ability to perform the desired post-translational modifications to the heavy and / or light chains of the monoclonal antibodies of the application as previously described.

[0030] In some embodiments, the nucleic acid molecules of the application as previously described can be introduced into the desired host cell by any method, including but not limited to, DEAE-Dextran-mediated transfection, calcium phosphate precipitation, cationic lipid-mediated transfection, electroporation, transduction, infection, and the like. In the present application, nucleic acids can be transiently or stably transfected into the desired host cell according to any suitable method.

[0031] A fifth aspect of the present application provides any one of the following products:

[0032] (1) an antibody derivative, which is a complex of the monoclonal antibody according to the first aspect of the present application directly or indirectly coupled to a detectable label;

[0033] (2) a detection reagent comprising the monoclonal antibody according to the first aspect of the present application, the recombinant host cell according to the fourth aspect of the present application, and / or the antibody derivative;

[0034] (3) a detection kit comprising the monoclonal antibody according to the first aspect of the present application, the antibody derivative, and / or the detection reagent;

[0035] (4) a detection test strip comprising the monoclonal antibody according to the first aspect of the present application, the antibody derivative, and / or the detection reagent;

[0036] (5) a therapeutic conjugate comprising the monoclonal antibody according to the first aspect of the present application;

[0037] (6) a pharmaceutical composition comprising the monoclonal antibody according to the first aspect of the present application and / or the therapeutic conjugate;

[0038] (7) a pharmaceutical preparation comprising the monoclonal antibody according to the first aspect of the present application, the therapeutic conjugate, and / or the pharmaceutical composition.

[0039] In some embodiments, the monoclonal antibody, the recombinant host cell, the therapeutic conjugate, the pharmaceutical composition, or the pharmaceutical preparation of the present application as previously described can be administered in vivo by various routes, including but not limited to intravenous, subcutaneous, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal, and intrathecal, or in other ways, for example, by implantation.

[0040] In some embodiments, the pharmaceutical composition or the pharmaceutical preparation can be formulated into a formulation in solid, semi-solid, liquid, or gaseous form; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.

[0041] In some embodiments, the pharmaceutical composition or pharmaceutical preparation can further comprise a variety of pharmaceutically acceptable carriers. A variety of pharmaceutically acceptable carriers can be used, including but not limited to: vehicles, adjuvants, and diluents. In addition, a variety of pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like can also be present. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0042] In some embodiments, the antibodies of the present application as described previously can be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying the antibodies of the present application as described previously in a aqueous or non-aqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, adding conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

[0043] In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present application as described previously can be formulated for inhalation, for example, using a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0044] In some embodiments, the pharmaceutical composition or pharmaceutical preparation as described previously can also be formulated into sustained release microcapsules with biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable formulations include polylactic-glycolic acid (PLGA) polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. The sustained release microcapsules can be prepared using certain methods well known to those skilled in the art for preparing such formulations.

[0045] The sixth aspect of the present application provides any one of the following methods:

[0046] (1) A method for producing the monoclonal antibody of the first aspect of the present application, the method comprising: culturing the recombinant host cell of the fourth aspect of the present application, and isolating the monoclonal antibody of the first aspect of the present application from the recombinant host cell culture product;

[0047] (2) A method for preparing the recombinant host cell of the fourth aspect of the present application, the method comprising: introducing the nucleic acid molecule of the second aspect of the present application or the expression vector of the third aspect of the present application into a host cell to obtain the recombinant host cell;

[0048] (3) A method for detecting SARS-CoV-2 S protein for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the monoclonal antibody of the first aspect of the present application, the antibody derivative of the fifth aspect of the present application, or the detection reagent with a sample to be tested, and detecting the formation of an immunocomplex of the monoclonal antibody and SARS-CoV-2 S protein.

[0049] (4) A method for inhibiting the activity of SARS-CoV-2 S protein in vitro, the method comprising: contacting the monoclonal antibody of the first aspect of the present application, the antibody derivative or detection reagent of the fifth aspect of the present application with a system in need thereof;

[0050] Optionally, the SARS-CoV-2 S protein is SARS-CoV-2 S protein, SARS-CoV-2 variant XBB S protein or SARS-CoV-2 variant JN.1 S protein.

[0051] In the present application, the sample refers to a material obtained or derived from a subject of interest, which contains cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological properties. For example, the sample to be tested and its variants refer to any sample obtained from a subject of interest, which is expected or known to contain cells and / or molecular entities to be characterized.

[0052] In some embodiments, the system in need includes, but is not limited to: a cell system, a subcellular system, a tissue system, an organoid system or an organ system.

[0053] The seventh aspect of the present application provides any one of the following uses:

[0054] (1) The use of the monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application or the recombinant host cell of the fourth aspect of the present application in the preparation of a detection reagent for detecting SARS-CoV-2 S protein;

[0055] (2) The use of the monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the recombinant host cell of the fourth aspect of the present application, the antibody derivative or detection reagent of the fifth aspect of the present application in the preparation of a detection kit or detection test strip for detecting SARS-CoV-2 S protein;

[0056] (3) The use of the monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the recombinant host cell of the fourth aspect of the present application, the antibody derivative or detection reagent of the fifth aspect of the present application in the preparation of a diagnostic product for diagnosing and / or assisting in the diagnosis of SARS-CoV-2 infection disease;

[0057] (4) The monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the recombinant host cell of the fourth aspect of the present application, or the antibody derivative or detection reagent of the fifth aspect of the present application for detecting SARS-CoV-2 S protein for non-diagnostic and non-therapeutic purposes.

[0058] (5) The monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, or the recombinant host cell of the fourth aspect of the present application for use in the preparation of a pharmaceutical composition for treating and / or preventing SARS-CoV-2 infection diseases.

[0059] (6) The monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the recombinant host cell of the fourth aspect of the present application, or the pharmaceutical composition of the fifth aspect of the present application for use in the preparation of a pharmaceutical preparation for treating and / or preventing SARS-CoV-2 infection diseases.

[0060] Further, the SARS-CoV-2 S protein is SARS-CoV-2 S protein, SARS-CoV-2 variant XBB S protein or SARS-CoV-2 variant JN.1 S protein.

[0061] Alternatively, the SARS-CoV-2 is SARS-CoV-2, SARS-CoV-2 variant XBB or SARS-CoV-2 variant JN.1 S.

[0062] The present application also provides a method for diagnosing and / or assisting in the diagnosis of SARS-CoV-2 infection diseases, comprising: contacting the monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the recombinant host cell of the fourth aspect of the present application, the antibody derivative of the fifth aspect of the present application, the detection reagent, the detection kit or the detection test strip with the test sample to be tested from the subject, and detecting the formation of the immunocomplex of the monoclonal antibody and SARS-CoV-2 S protein to diagnose and / or assist in the diagnosis of whether the subject has SARS-CoV-2 infection diseases or the risk of the subject suffering from SARS-CoV-2 infection diseases.

[0063] The present application also provides a method for treating and / or preventing a SARS-CoV-2 infection disease, comprising: administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of the monoclonal antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the recombinant host cell of the fourth aspect of the present application, the pharmaceutical composition or the pharmaceutical preparation of the fifth aspect of the present application.

[0064] In the present application, the effective amount means an amount of the monoclonal antibody, the recombinant host cell, the pharmaceutical composition or the pharmaceutical preparation (i.e. active ingredient) of the present application as previously described, which is sufficient to produce the desired activity when administered to a subject in need thereof. When a combination of active ingredients is administered, the effective amount of the combination can or can not include the amount of each ingredient that would be effective when administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug(s) being used, its mode of administration and the like.

[0065] In the present application, treatment refers to alleviating or abating at least one symptom associated with such condition, or slowing or reversing the progression of such condition. In addition, the treatment also means halting, delaying the onset (i.e. the period prior to clinical manifestation of the disease) and / or reducing the risk of developing or worsening the disease. Furthermore, the treatment can also include: (1) preventing or delaying the appearance of at least one clinical or subclinical symptom of the state, disorder or condition in a subject who can be predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e. arresting, reducing or delaying the development of the disease or its recurrence (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) relieving the disease, i.e. causing the regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. In the present application, the prevention refers to preventing the spread of infection in a subject exposed to the virus, for example, preventing the virus from entering the cells of the subject.

[0066] In some embodiments, non-limiting examples of the symptoms of the SARS-CoV-2 infection disease include, but are not limited to: fever, cough, shortness of breath, pneumonia, acute respiratory distress syndrome (ARDS), acute lung syndrome, loss of smell, loss of taste, sore throat, nasal secretion, gastrointestinal symptoms (e.g. diarrhea), organ failure (e.g. kidney failure and kidney dysfunction), septic shock and death.

[0067] In the present application, the subject refers to human, veterinary animals (for example, cat, dog, cow, horse, sheep, pig, etc.), veterinary birds and experimental animal models of diseases (for example, mouse, rat, ferret, monkey, etc.). In a preferred embodiment, the subject of the present application is human.

[0068] Compared with the prior art, the present application has the advantages and beneficial effects as follows:

[0069] The present application discloses a brand-new human monoclonal antibody S2 for neutralizing SARS-CoV-2, and derivatives comprising the human monoclonal antibody S2, including nucleic acid molecules encoding the antibody, expression vectors comprising the nucleic acid molecules, host cells comprising the expression vectors, pharmaceutical compositions comprising the antibody or host cells, detection reagents, detection kits, etc. The human monoclonal antibody S2 provided by the present application has strong neutralizing activity on SARS-CoV-2, lays a foundation for the diagnosis, treatment and related drug development of SARS-CoV-2 infection related diseases, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 The results of the micro-neutralization experiment are shown in the graph. DETAILED DESCRIPTION

[0071] The present application will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present application, and should not be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents. The reagents and raw materials used in the present application are easily obtained by those skilled in the art, and can be obtained from commercial channels if not otherwise specified, and the experimental methods of the present application are generally implemented according to conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present application, and should not limit the scope of the present application in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present application, and should not and will not limit the present application as described in detail in the claims.

[0072] Screening of human monoclonal antibody S2 for neutralizing SARS-CoV-2 and verification of its function in the examples

[0073] 1. Virus culture (all operations were performed in a BSL-3 laboratory): SARS-CoV-2 Wild type strain (BetaCoV / JS02 / Human / 2019), Omicron XBB.1.19 strain (hCoV-19 / Jiangsu / JS04 / 2023) and Omicron JN.1 strain (hCoV-19 / Jiangsu / JS01 / 2024) were isolated by the applicant from respiratory samples of three COVID-19 patients in Jiangsu in 2020, 2023 and 2024, respectively. After inoculating Vero-E6 cells with the viruses, the cells were cultured at 37°C, 5% CO2 for 5 days, and the supernatant was collected and determined for 50% tissue culture infectious dose (TCID50).

[0074] 2. Recombinant S2 protein, purchased from a commercial company.

[0075] 3. Construction of ScFv human antibody library and screening of anti-SARS-CoV-2 S2 protein single-chain antibody

[0076] 3.1 Materials

[0077] Primers: Design specific light chain (Vκ and Vλ), IgG heavy chain (VH) and overlap-PCR primers (Table 1), in which Vκ12 pairs, Vλ24 pairs, VH 6 pairs, overlap-PCR 1 pair.

[0078] Table 1 Primers for constructing human single-chain antibody library

[0079]

[0080]

[0081] 3.2 Methods

[0082] 3.2.1 Isolation of peripheral blood lymphocytes and extraction of total RNA

[0083] The peripheral blood of 10 COVID-19 patients in the recovery stage was mixed with an equal amount of normal saline, and then single-nucleated cells were aspirated according to the lymphocyte separation medium instruction. After washing with normal saline for three times, the RNA was extracted according to the total RNA extraction kit instruction.

[0084] 3.2.2 PCR amplification of antibody variable region genes

[0085] The extracted 2 parts of total RNA were mixed and reverse transcribed into cDNA first strand, and the reverse transcription conditions were as follows: 55℃ for 30min, 85℃ for 5min, and 4℃ for 30min. Then, the human-derived antibody Vκ, Vλ and VH genes were amplified by PCR using cDNA as the template, and the PCR reaction conditions were as follows: 94℃ for 10min for pre-denaturation, then 94℃ for 20s, 57℃ for 45s, 72℃ for 1min for 25 cycles, and finally 72℃ for 20min for extension, gel electrophoresis and gel purification recovery.

[0086] 3.2.3 Splicing of ScFv gene

[0087] The purified Vκ gene fragment and Vλ gene fragment were mixed in equal molar amounts, and then mixed with the VH gene fragment in equal amounts, and the scFv gene was spliced by overlap-PCR, and the overlap-PCR reaction conditions were as follows: 94℃ for 10min for pre-denaturation, then 94℃ for 20s, 57℃ for 45s, 72℃ for 1min for 25 cycles, and finally 72℃ for 20min for extension, gel electrophoresis and gel purification recovery.

[0088] 3.2.4 Construction and quality identification of phage single-chain antibody library

[0089] The purified scFv gene and pComb3XSS plasmid were respectively digested with sfiI enzyme, and the target fragments were recovered by gel purification and ligation, and then transferred into competent E. coli XL1-Blue, and then added into 20mL 2YT culture solution and cultured at 37℃ for 45min, then centrifuged, and the precipitate was coated on 2YT plate and cultured at 30℃ overnight. The next day, the bacterial lawn grown on the plate was collected in 2YT culture medium, and cultured at 37℃ until the OD600 was 0.8. Then, the helper phage VCSM13 was added at a final concentration of 1×10 9 PFU / mL, and cultured at 37℃ for 1h. Then, kanamycin was added at a final concentration of 50μg / mL, and the culture was continued at 37℃ for 8h, then centrifuged at 900g for 20min, and the precipitate was discarded. Then, 5×PEG / NaCl was added to the supernatant, mixed well, and then placed on ice for 6h, then centrifuged at 900g for 45min, and the precipitate was resuspended in 3mL PBS, and then filtered through a 0.22μm filter membrane, and the filtrate was the human-derived phage single-chain antibody library.

[0090] 3.2.5 Screening of anti-SARS-CoV-2 S2 protein specific single-chain antibody

[0091] Take 200 μL of the amplified phage library and incubate with solid-phase coated SARS-CoV-2-S protein, and perform 4 rounds of "adsorption-elution-amplification" affinity screening. After the fourth round of elution, infect the logarithmic growth phase of E. coli XL1-Blue, and then spread 2xYT plates at 37°C overnight. Randomly pick 200 single colonies and inoculate 96-well deep plates (containing 100 μg / mL ampicillin, 12.5 μg / mL tetracycline, and 1 g / mL glucose). Incubate at 37°C overnight with shaking. The next day, inoculate 1:10 into new 96-well deep plates (containing 100 μg / mL ampicillin and 30 μg / mL tetracycline) at 37°C and shake for 6 h. Add helper phage VCSM13 (final concentration 1x10 9 PFU / mL) and incubate at 37°C for 1 h. Add kanamycin (final concentration 50 μg / mL) and shake overnight at 30°C to prepare phage single-chain antibodies. Coat the enzyme-labeled plate with 0.1 μg / well SARS-CoV-2 S2 protein. The secondary antibody is HRP-labeled anti-M13 antibody diluted 1:2000 in PBS buffer (containing 5 g / mL skim milk powder). Perform Phage-ELISA identification and measure OD450 values. When Positive / Negative≥2.1, it is considered positive. The bacterial solution of the positive clone is sent to a commercial company for sequencing.

[0092] 3.3 Results

[0093] Screening of anti-SARS-CoV-2 S2 protein single-chain antibodies: SARS-CoV-2-S protein was used as an antigen for 4 rounds of affinity screening of human single-chain antibody library. Anti-SARS-CoV-2 S2 protein specific single-chain antibodies were selectively enriched, and the output / input ratio increased by nearly 50 times. Randomly pick 200 phage monoclonals for Phage-ELISA test and measure OD450 values. The results show that one single-chain antibody can specifically bind to SARS-CoV-2 S2 protein and has high affinity, named S2. The sequence information of S2 is shown in Table 2.

[0094] Table 2 Sequence information of human monoclonal antibody S2 against SARS-CoV-2

[0095]

[0096]

[0097] 4. Micro-neutralization experiment of a S2 specific monoclonal antibody clone (all operations were performed in a BSL-3 laboratory)

[0098] 4.1 Preparation of antibodies

[0099] The antibody clone fragment scfv screened by phage library was sequenced and given to a commercial company for whole molecule expression. The heavy chain isotype is γ4 and the light chain isotype is κ.

[0100] 4.2 Micro-neutralization experiment operation

[0101] (1) Vero-E6 cells were inoculated in 96-well plates and cultured at 37°C, 5% CO2 until the single cell density was ≥80%.

[0102] (2) The test antibody was diluted in a two-fold series of 8 concentrations, i.e. 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.56 μg / mL.

[0103] (3) 100 TCID50 virus was mixed with an equal volume of sterile treated antibody and incubated at 37°C for 1 h.

[0104] (4) 100 μL of the incubated antigen-antibody complex was added to the Vero-E6 cell culture well, and duplicate wells were set up. Positive control group (antibody with known neutralization titer), negative control group (normal human serum), virus control group and normal cell control group were also set up.

[0105] (5) The cell culture was incubated at 37°C, 5% CO2 for 3-7 days, and the cytopathic effect (CPE) was observed under an inverted microscope. When the CPE in the virus control cell well for neutralization test reached 76-100%, the experimental results were observed and recorded. Monoclonal antibodies that can inhibit more than 50% CPE are considered to have neutralization inhibition effect.

[0106] 4.3 Results

[0107] The results of the micro-neutralization experiment showed that IgG-S2 had neutralization inhibition effect on SARS-CoV-2 WT, XBB and JN.1 three mutant strains (as shown in Figure 1 ).

Claims

1. A human-derived monoclonal antibody that neutralizes SARS-CoV-2, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region are respectively as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6; the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 in the light chain variable region are respectively as shown in SEQ ID NO: 11, GKN, SEQ ID NO:

15.

2. The monoclonal antibody according to claim 1, characterized in that, the amino acid sequences of FR-H1, FR-H2, FR-H3, FR-H4 in the heavy chain variable region are respectively as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or an amino acid sequence having at least 70% homology with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7; the amino acid sequences of FR-L1, FR-L2, FR-L3, FR-L4 in the light chain variable region are respectively as shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or an amino acid sequence having at least 70% homology with SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:

16.

3. The monoclonal antibody according to claim 1, characterized in that, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8 or an amino acid sequence having at least 70% homology with SEQ ID NO: 8; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 17 or an amino acid sequence having at least 70% homology with SEQ ID NO:

17.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody of any one of claims 1-3.

5. The nucleic acid molecule of claim 4, wherein, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 9 or a nucleotide sequence having at least 70% homology with SEQ ID NO: 9; the nucleotide sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 18 or a nucleotide sequence having at least 70% homology with SEQ ID NO:

18.

6. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule of claim 4 or 5.

7. The expression vector of claim 6, wherein, The vector is a DNA vector or an RNA vector.

8. The expression vector of claim 6, wherein, The vector is a vector of viral origin.

9. The expression vector of claim 8, wherein, The vector of viral origin is a lentiviral vector.

10. The expression vector of claim 8, wherein, The vector of viral origin is a retroviral vector, an adeno-associated viral vector, an adenoviral vector, a poxviral vector or a herpesviral vector.

11. A recombinant host cell, characterized in that, The host cell comprises the expression vector of any one of claims 6-10.

12. An antibody derivative, characterized in that The antibody derivative is a complex formed by directly or indirectly coupling the monoclonal antibody of any one of claims 1-3 to a detectable marker.

13. A test reagent, characterized by, The detection reagent comprises the monoclonal antibody of any one of claims 1-3, the recombinant host cell of claim 11, and / or the antibody derivative of claim 12.

14. A test kit comprising, The detection kit comprises the monoclonal antibody of any one of claims 1-3, the antibody derivative of claim 12, and / or the detection reagent of claim 13.

15. A test strip, characterized in that The detection test strip comprises the monoclonal antibody of any one of claims 1-3, the antibody derivative of claim 12, and / or the detection reagent of claim 13.

16. A conjugate for therapeutic use, characterized in that, The therapeutic conjugate comprises the monoclonal antibody of any one of claims 1-3.

17. A pharmaceutical composition comprising, The pharmaceutical composition comprises the monoclonal antibody of any one of claims 1-3 and / or the therapeutic conjugate of claim 16.

18. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the monoclonal antibody of any one of claims 1-3, the therapeutic conjugate of claim 16, and / or the pharmaceutical composition of claim 17.

19. A method of producing the monoclonal antibody of any one of claims 1-3, comprising, The method comprises culturing the recombinant host cell of claim 11 and isolating the monoclonal antibody of any one of claims 1-3 from the recombinant host cell culture product.

20. A method of making the recombinant host cell of claim 11, wherein, The method comprises introducing the nucleic acid molecule of claim 4 or 5 or the expression vector of any one of claims 6-10 into a host cell to obtain the recombinant host cell.

21. A method of detecting SARS-CoV-2 S protein for non-diagnostic and non- therapeutic purposes, characterized in that, The method comprises contacting the monoclonal antibody of any one of claims 1-3, the antibody derivative of claim 12, or the detection reagent of claim 13 with a sample to be tested and detecting the formation of an immunocomplex of the monoclonal antibody and the SARS-CoV-2 S protein.

22. The method of claim 21, wherein, The SARS-CoV-2 S protein is a SARS-CoV-2 variant XBB S protein or a SARS-CoV-2 variant JN.1 S protein.

23. A method of inhibiting SARS-CoV-2 S protein activity in vitro, comprising contacting the SARS-CoV-2 S protein with a compound of any one of claims 1-22. The method comprises contacting the monoclonal antibody of any one of claims 1-3, the antibody derivative of claim 12, or the detection reagent of claim 13 with a system in need thereof.

24. The method of claim 23, wherein, The SARS-CoV-2 S protein is a SARS-CoV-2 variant XBB S protein or a SARS-CoV-2 variant JN.1 S protein.

25. Use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the expression vector of any one of claims 6-10, or the recombinant host cell of claim 11 in the preparation of a detection reagent for detecting a SARS-CoV-2 S protein.

26. The use according to claim 25, characterized in that, The SARS-CoV-2 S protein is a SARS-CoV-2 variant XBB S protein or a SARS-CoV-2 variant JN.1 S protein.

27. Use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the expression vector of any one of claims 6-10, the recombinant host cell of claim 11, the antibody derivative of claim 12, or the detection reagent of claim 13 in the manufacture of a detection kit for detecting a SARS-CoV-2 S protein.

28. The use according to claim 27, characterized in that, The SARS-CoV-2 S protein is a SARS-CoV-2 variant XBB S protein or a SARS-CoV-2 variant JN.1 S protein.

29. Use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the expression vector of any one of claims 6-10, the recombinant host cell of claim 11, the antibody derivative of claim 12, or the detection reagent of claim 13 in the manufacture of a detection test strip for detecting a SARS-CoV-2 S protein.

30. The use according to claim 29, characterized in that, The SARS-CoV-2 S protein is a SARS-CoV-2 variant XBB S protein or a SARS-CoV-2 variant JN.1 S protein.

31. Use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the expression vector of any one of claims 6-10, the recombinant host cell of claim 11, the antibody derivative of claim 12, or the detection reagent of claim 13 in the manufacture of a diagnostic product for diagnosing and / or aiding in the diagnosis of a SARS-CoV-2 infection disease.

32. The use according to claim 31, characterized in that, The SARS-CoV-2 is a SARS-CoV-2 variant XBB or a SARS-CoV-2 variant JN.1 S.

33. Use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the expression vector of any one of claims 6-10, the recombinant host cell of claim 11, the antibody derivative of claim 12, or the detection reagent of claim 13 for detecting a SARS-CoV-2 S protein for non-diagnostic and non-therapeutic purposes.

34. The use according to claim 33, wherein The SARS-CoV-2 S protein is a SARS-CoV-2 variant XBB S protein or a SARS-CoV-2 variant JN.1 S protein.

35. Use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the expression vector of any one of claims 6-10, or the recombinant host cell of claim 11 in the manufacture of a pharmaceutical composition for treating and / or preventing a SARS-CoV-2 infection disease.

36. The use according to claim 35, wherein The SARS-CoV-2 is a SARS-CoV-2 variant XBB or a SARS-CoV-2 variant JN.1 S.

37. Use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the expression vector of any one of claims 6-10, the recombinant host cell of claim 11, or the pharmaceutical composition of claim 17 for the manufacture of a pharmaceutical preparation for the treatment and / or prevention of a SARS-CoV-2 infection disease.

38. The use according to claim 37, wherein The SARS-CoV-2 is SARS-CoV-2 variant XBB or SARS-CoV-2 variant JN.1 S.

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