Humanized monoclonal antibody for neutralizing SARS-CoV-2 and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a humanized monoclonal antibody for neutralizing SARS-CoV-2 and an application thereof. Background Art

[0002] The pathogen of the novel coronavirus infection, SARS-CoV-2, belongs to the genus Betavirus of the Coronaviridae family and is a linear positive-strand RNA virus. Studies have found that SARS-CoV-2 is adjacent to SARS-CoV and SARS-CoV-like groups in the evolutionary tree. The novel coronavirus has four main 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 virus particles on receptor cells through interaction with ACE2 on the human respiratory epithelial cell membrane, while the S2 subunit mediates the fusion process of the virus envelope with the susceptible cell membrane structure. Therefore, both S1 and S2 subunits contain epitopes for neutralizing antibodies.

[0003] As a supplement to vaccines and chemotherapy, antibody-mediated measures to prevent and treat viral infections have shown good results, and their application prospects have been recognized by experts. Unlike the easy mutation of S1, 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. It has now become a recognized region in the industry for preparing protective vaccines and broad-spectrum neutralizing antibodies against SARS-CoV-2. At present, there are no therapeutic drugs for SARS-CoV-2 infection, and the treatment options for COVID-19 caused by SARS-CoV-2 infection are mainly to relieve symptoms, prevent secondary infections, reduce complications, and support organ function. Therefore, there is an urgent need in this field to develop coronavirus neutralizing antibodies, especially monoclonal antibodies, with good neutralizing effects on viruses. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a human monoclonal antibody S2 and its application for neutralizing SARS-CoV-2 in the art. The present invention uses phage display technology to construct a large-capacity human immune phage antibody library, and uses the SARS-CoV-2 virus S2 subunit as a target to screen human antibody single chain variable fragments (ScFv) to obtain a ScFv antibody molecule S2. Further studies have shown that the monoclonal antibody has a strong neutralizing effect on the SARS-CoV-2 virus.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] A first aspect of the present invention provides a human monoclonal antibody that neutralizes 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 shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively;

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

[0009] Further, the amino acid sequences of FR-H1, FR-H2, FR-H3, and 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, 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 shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, 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, SEQ ID NO: 16, respectively.

[0011] Further, 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;

[0012] Optionally, 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 to SEQ ID NO:17.

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

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

[0015] In some embodiments, the amino acid sequence corresponding to the heavy chain variable region, the amino acid sequence corresponding to the light chain variable region, the amino acid sequence corresponding to CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region, or the amino acid sequence corresponding to CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region of the monoclonal antibody provided by the present invention have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the corresponding amino acid sequence described above in the present invention. Antibody sequences are all within the scope of protection of the present invention.

[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 variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, and SARS-CoV-2 Omicron variant.

[0017] The second aspect of the present invention provides a nucleic acid molecule encoding the monoclonal antibody according to the first aspect of the present invention;

[0018] Optionally, 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;

[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 to SEQ ID NO: 18.

[0020] In the present invention, the nucleic acid molecule refers to the polymeric form of nucleotides, and includes the sense strand and antisense strand of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers thereof. In a specific embodiment, nucleotides refer to ribonucleotides, deoxyribonucleotides or modified forms of any nucleotide type, and combinations thereof. The nucleic acid molecule also includes, but is not limited to, single-stranded forms and double-stranded forms of DNA. In addition, polynucleotides, for example, cDNA or mRNA, can include any one or two 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 derived nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, replacement of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), side-joining moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified bonds (e.g., α-anomeric nucleic acids, etc.).

[0022] The third aspect of the present invention provides an expression vector, wherein the expression vector comprises the nucleic acid molecule according to the second aspect of the present invention;

[0023] Optionally, the vector is a DNA vector, an RNA vector or a vector of viral origin;

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

[0025] In the present invention, 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 and vectors that bind to the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of a nucleic acid to which it is operably linked.

[0026] In some embodiments, the expression vector includes but is not limited to: plasmid, cosmid, bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC), and vectors derived from bacteriophage or plant or animal (including human) virus. The vector may include a replication origin recognized by the host cell, and in the case of the expression vector, includes a promoter and other regulatory sequences recognized by the host cell. In a specific embodiment, the vector includes a nucleic acid molecule encoding the monoclonal antibody of the present invention as described above that is operably connected to a promoter and optional other regulatory elements. Some vectors can replicate autonomously in the host into which they are introduced (for example, a vector with a bacterial replication origin can replicate in bacteria). Other vectors can be integrated into the host's genome when introduced into the host, and thus replicated with the host genome. The vector includes but is not limited to: those suitable for the recombinant production of the monoclonal antibody of the present invention as described above, and those suitable for introducing a subject or its cell in need, so that the monoclonal antibody of the present invention as described above is provided to the subject.

[0027] In some embodiments, the selection of the specific vector depends on the recombination procedure followed and the host used. The vector is introduced into the host cell and can be realized by calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, liposome transfection or electroporation in particular. The vector can replicate autonomously or can replicate with the chromosome into which they have been integrated. In some embodiments, the vector comprises one or more selective markers. The selection of the marker can depend on the host cell selected. These include but are not limited to: kanamycin, neomycin, puromycin, hygromycin, bleomycin, thymidine kinase gene from herpes simplex virus and dihydrofolate reductase gene from mouse.

[0028] The fourth aspect of the present invention provides a recombinant host cell, wherein the host cell comprises the expression vector described in the third aspect of the present invention.

[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, etc. Specific eukaryotic host cells are selected based on their ability to perform the desired post-translational modification on the heavy chain and / or light chain of the monoclonal antibody of the present invention as described above.

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

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

[0032] (1) An antibody derivative, wherein the antibody derivative is a complex formed by directly or indirectly coupling the monoclonal antibody according to the first aspect of the present invention to a detectable marker;

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

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

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

[0036] (5) A therapeutic conjugate, comprising the monoclonal antibody according to the first aspect of the present invention;

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

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

[0039] In some embodiments, the monoclonal antibodies, recombinant host cells, therapeutic conjugates, pharmaceutical compositions or pharmaceutical formulations described above of the present invention can be administered in vivo by various routes, including but not limited to: intravenous, subcutaneous, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal and intrathecal, or in other ways, such as by implantation.

[0040] In some embodiments, the pharmaceutical composition or pharmaceutical preparation can be formulated into a preparation in solid, semisolid, 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 may also include a variety of pharmaceutically acceptable carriers. Various pharmaceutically acceptable carriers may be used, including but not limited to: solvents, adjuvants and diluents. In addition, various pharmaceutically acceptable auxiliary substances may also be used, such as pH regulators and buffers, tension regulators, stabilizers, wetting agents, etc. Non-limiting exemplary carriers include saline, buffered saline, glucose, water, glycerol, ethanol and combinations thereof.

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

[0043] In some embodiments, the pharmaceutical compositions or pharmaceutical formulations of the present invention as described above can be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0044] In some embodiments, pharmaceutical compositions or pharmaceutical preparations as described above can also be formulated into sustained-release microcapsules together with biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable preparations include polylactic-glycolic acid (PLGA) polymers. Non-limiting exemplary non-biodegradable preparations include polyglycerol fatty acid esters. Certain methods of preparing such preparations known to those skilled in the art can be used to prepare the microcapsules obtained by sustained release.

[0045] A sixth aspect of the present invention provides any of the following methods:

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

[0047] (2) A method for preparing the recombinant host cell according to the fourth aspect of the present invention, the method comprising: introducing the nucleic acid molecule according to the second aspect of the present invention or the expression vector according to the third aspect of the present invention 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 described in the first aspect of the present invention, the antibody derivative described in the fifth aspect of the present invention, or the detection reagent with a sample to be tested, and detecting the formation of an immune complex between the monoclonal antibody and the 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 described in the first aspect of the present invention, the antibody derivative described in the fifth aspect of the present invention or the detection reagent with a system in need;

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

[0051] In the present invention, 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, a test sample 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 cellular system, a subcellular system, a tissue system, an organoid system, or an organ system.

[0053] A seventh aspect of the present invention provides any of the following applications:

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

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

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

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

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

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

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

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

[0062] The present invention also provides a method for diagnosing and / or assisting in the diagnosis of SARS-CoV-2 infectious diseases, the method comprising: contacting the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, the antibody derivative described in the fifth aspect of the present invention, the detection reagent, the detection kit or the detection test strip with a test sample derived from a subject, and detecting the formation of an immune complex between the monoclonal antibody and the SARS-CoV-2S protein, so as to diagnose and / or assist in the diagnosis of whether the subject suffers from a SARS-CoV-2 infectious disease or the risk of the subject suffering from a SARS-CoV-2 infectious disease.

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

[0064] In the present invention, the effective amount refers to the amount of the monoclonal antibody, recombinant host cell, pharmaceutical composition or pharmaceutical preparation (i.e., active ingredient) as described above of the present invention 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 may or may not include the amount of each ingredient that may be effective when administered alone. The exact amount required will vary from subject to subject, depending on the species, age and overall condition of the subject, the severity of the condition being treated, the specific drug type used, the mode of administration, etc.

[0065] In the present invention, treatment refers to relieving or alleviating at least one symptom associated with the condition, or slowing down or reversing the progression of the condition. In addition, the treatment also means stopping, delaying the onset (i.e., the period before the clinical manifestation of the disease) and / or reducing the risk of developing or worsening the disease. In addition, the treatment may also include: (1) preventing or delaying the appearance of at least one clinical or subclinical symptom of the state, disease or condition in a subject who may suffer from or be susceptible to the state, disease or condition but has not yet experienced or displayed clinical or subclinical symptoms of the state, disease or condition; or (2) inhibiting the state, disease or condition, i.e., preventing, reducing or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing the disappearance of at least one of the state, disease or condition or its clinical or subclinical symptoms. In the present invention, the prevention refers to preventing the spread of infection in a subject exposed to the virus, such as preventing the virus from entering the subject's cells.

[0066] In some embodiments, non-limiting examples of 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 pulmonary syndrome, loss of smell, loss of taste, sore throat, nasal discharge, gastrointestinal symptoms (e.g., diarrhea), organ failure (e.g., renal failure and renal dysfunction), septic shock, and death.

[0067] In the present invention, the subject refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), veterinary birds, and experimental animal models of diseases (e.g., mice, rats, ferrets, monkeys, etc.). In a preferred embodiment, the subject of the present invention is a human.

[0068] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0069] The present invention discloses for the first time a new human monoclonal antibody S2 for neutralizing SARS-CoV-2, and a derivative comprising the human monoclonal antibody S2, wherein the derivative comprises a nucleic acid molecule encoding the antibody, an expression vector comprising the nucleic acid molecule, a host cell comprising the expression vector, a pharmaceutical composition comprising the antibody or the host cell, a detection reagent, a detection kit, etc. The human monoclonal antibody S2 provided by the present invention has a strong neutralizing activity against SARS-CoV-2, lays a foundation for the diagnosis and treatment of diseases related to SARS-CoV-2 infection and the development of related drugs, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a graph showing the results of a micro-neutralization experiment. DETAILED DESCRIPTION

[0071] The present invention is further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and cannot be construed as limiting the present invention. It can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The reagents and raw materials used in the present invention are easily available to those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention 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 invention, and should not and will not limit the present invention described in detail in the claims.

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

[0073] 1. Virus culture (all operations are performed in a BSL-3 laboratory): SARS-CoV-2 Widetype 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 infected persons in Jiangsu in 2020, 2023 and 2024. After the virus was inoculated into Vero-E6 cells, it was cultured at 37°C and 5% CO2 for 5 days, the supernatant was collected and the 50% tissue infection dose (TCID50) was determined.

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

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

[0076] 3.1 Materials

[0077] Primers: Specific light chain (Vκ and Vλ), IgG heavy chain (VH) and overlap-PCR primers were designed (Table 1), including 12 pairs for Vκ, 24 pairs for Vλ, 6 pairs for VH and 1 pair for overlap-PCR.

[0078] Table 1 Primers for constructing humanized 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] Peripheral blood from 10 COVID-19 patients in the convalescent period was mixed with an equal amount of normal saline and mononuclear cells were aspirated according to the instructions of lymphocyte separation medium. After washing three times with normal saline, RNA was extracted according to the instructions of total RNA extraction kit.

[0084] 3.2.2 PCR amplification of antibody variable region genes

[0085] The two extracted total RNAs were mixed and reverse transcribed to obtain the first strand of cDNA. The reverse transcription conditions were as follows: 55°C for 30 min, 85°C for 5 min, and 4°C for 30 min. The cDNA was then used as a template for PCR amplification of human antibody Vκ, Vλ, and VH genes. The PCR reaction conditions were: 94°C pre-denaturation for 10 min, then 94°C for 20 s, 57°C for 45 s, and 72°C for 1 min, 25 cycles, and finally 72°C extension for 20 min, followed by gel electrophoresis and gel cutting for purification and recovery.

[0086] 3.2.3 ScFv gene splicing

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

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

[0089] The purified scFv gene and pComb3XSS plasmid were digested with sfiI, and the target fragments recovered after gel purification were connected and transferred into competent E. coli XL1-Blue. They were added to 20 mL of 2YT culture medium and cultured at 37°C for 45 min. After centrifugation, the precipitate was spread on a 2YT plate and cultured at 30°C overnight. The next day, all the bacterial mosses grown on the plate were collected in 2YT medium and cultured at 37°C until OD600 was 0.8. A final concentration of 1×10 9 PFU / mL of helper phage VCSM13 was cultured at 37℃ for 1h. Kanamycin was added at a final concentration of 50μg / mL, and cultured for another 8h at 37℃. The precipitate was discarded after centrifugation at 900g for 20min, and 5×PEG / NaCl was added to the supernatant. After mixing, the mixture was placed on ice for 6h, and centrifuged at 900g for 45min. The precipitate was resuspended in 3mL of PBS and filtered through a 0.22μm filter membrane. The filtrate was the humanized phage single-chain antibody library.

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

[0091] 200 μL of the amplified phage library was incubated with the solid-phase coated SARS-CoV-2-S protein for 4 rounds of “adsorption-elution-amplification” affinity screening. The 4th round of eluate was used to infect Escherichia coli XL1-Blue in the logarithmic growth phase, and then coated on a 2×YT culture plate and cultured at 37°C overnight. 200 single colonies were randomly picked and inoculated into 96-well deep-well plates (containing 100 μg / mL ampicillin, 12.5 μg / mL tetracycline, and 1 g / mL glucose), and cultured at 37°C overnight with shaking. The next day, they were inoculated into new 96-well deep-well plates (containing 100 μg / mL ampicillin and 30 μg / mL tetracycline) at a ratio of 1:10 and cultured at 37°C for 6 h with shaking. Helper phage VCSM13 (final concentration of 1×10 9 PFU / mL), incubated at 37°C for 1h, added kanamycin (final concentration of 50μg / mL) and shaken at 30°C overnight to prepare phage single-chain antibody, and coated the ELISA plate with 0.1μg / well SARS-CoV-2S2 protein. The secondary antibody was HRP-labeled anti-M13 antibody diluted 1:2000 with PBS buffer (containing 5g / mL skim milk powder). Phage-ELISA was performed and OD450 value was determined. When Positive / Negative ≥2.1, it was defined as positive, and the bacterial solution of the positive clone was sent to a commercial company for sequencing.

[0092] 3.3 Results

[0093] Screening of single-chain antibodies against SARS-CoV-2S2 protein: Four rounds of affinity screening were performed on the human single-chain antibody library using SARS-CoV-2-S protein as the antigen. Single-chain antibodies specific to the SARS-CoV-2S2 protein were selectively enriched, and the output / input ratio was increased by nearly 50 times. 200 phage monoclones were randomly selected for Phage-ELISA test and OD450 values ​​were measured. The results showed that one single-chain antibody could specifically bind to the SARS-CoV-2S2 protein and had a high affinity, named S2. The sequence information of S2 is shown in Table 2.

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

[0095]

[0096]

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

[0098] 4.1 Antibody Preparation

[0099] The antibody clone fragment scFv screened through the phage library was handed over to a commercial company for full-molecule expression after sequencing. 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 seeded in 96-well plates and cultured at 37°C and 5% CO2 until the monolayer cell density was ≥80%.

[0102] (2) The antibody to be tested was serially diluted two-fold to eight concentrations, namely: 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 1.56 μg / mL.

[0103] (3) Mix 100 TCID50 of virus with an equal volume of sterile antibody and incubate at 37° for 1 h.

[0104] (4) Add 100 μL of the incubated antigen-antibody complex to the Vero-E6 cell culture wells, set up duplicate wells, and set up a positive control group (antibodies with known neutralizing titers), a negative control group (normal human serum), a virus control group, and a normal cell control group.

[0105] (5) The cell culture was placed in an incubator at 37°C and 5% CO2 for 3-7 days, and the cytopathic effect (CPE) was observed under an inverted microscope. When the CPE level in the virus control cell wells used in the neutralization test reached 76-100%, the experimental results were observed and recorded. Monoclonal antibodies that could inhibit the appearance of CPE by more than 50% were considered to have neutralizing inhibitory effects.

[0106] 4.3 Results

[0107] The results of micro-neutralization experiments showed that IgG-S2 had a neutralizing inhibitory effect on the three variants of SARS-CoV-2 WT, XBB, and JN.1 (e.g. Figure 1 shown).

Claims

1. A human 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, and CDR-H3 in the heavy chain variable region are shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively; The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region are shown in SEQ ID NO: 11, GKN and SEQ ID NO: 15, respectively.

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

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 to SEQ ID NO:8; Optionally, 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 to SEQ ID NO:

17.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody according to any one of claims 1 to 3; Optionally, 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; 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 to SEQ ID NO:

18.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 4; Optionally, the vector is a DNA vector, an RNA vector or a vector of viral origin; Optionally, the virus-derived vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, an adenoviral vector, a poxvirus vector or a herpesvirus vector.

6. A recombinant host cell, characterized in that The host cell comprises the expression vector according to claim 5.

7. Any of the following products: (1) 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 to 3 to a detectable marker; (2) A detection reagent, characterized in that the detection reagent comprises the monoclonal antibody according to any one of claims 1 to 3, the recombinant host cell according to claim 6 and / or the antibody derivative; (3) A detection kit, characterized in that the detection kit comprises the monoclonal antibody, the antibody derivative and / or the detection reagent according to any one of claims 1 to 3; (4) A test strip, characterized in that the test strip comprises the monoclonal antibody, the antibody derivative and / or the detection reagent according to any one of claims 1 to 3; (5) A therapeutic conjugate, characterized in that the therapeutic conjugate comprises the monoclonal antibody according to any one of claims 1 to 3; (6) A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the monoclonal antibody and / or the therapeutic conjugate according to any one of claims 1 to 3; (7) A pharmaceutical preparation, characterized in that the pharmaceutical preparation comprises the monoclonal antibody, the therapeutic conjugate and / or the pharmaceutical composition according to any one of claims 1 to 3.

8. Any of the following methods: (1) A method for producing the monoclonal antibody according to any one of claims 1 to 3, characterized in that: The method comprises: culturing the recombinant host cell of claim 6, and isolating the monoclonal antibody of any one of claims 1 to 3 from the culture product of the recombinant host cell; (2) A method for preparing the recombinant host cell according to claim 6, characterized in that the method comprises: introducing the nucleic acid molecule according to claim 4 or the expression vector according to claim 5 into a host cell to obtain the recombinant host cell; (3) A method for detecting SARS-CoV-2 S protein for non-diagnostic and non-therapeutic purposes, characterized in that the method comprises: contacting the monoclonal antibody described in any one of claims 1 to 3, the antibody derivative described in claim 7, or the detection reagent with a sample to be tested, and detecting the formation of an immune complex between the monoclonal antibody and the SARS-CoV-2 S protein; (4) A method for inhibiting the activity of SARS-CoV-2 S protein in vitro, characterized in that the method comprises: contacting the monoclonal antibody described in any one of claims 1 to 3, the antibody derivative described in claim 7 or the detection reagent with a system in need; Optionally, the SARS-CoV-2S protein is SARS-CoV-2S protein, SARS-CoV-2 variant XBB S protein or SARS-CoV-2 variant JN.1S protein.

9. Any of the following applications: (1) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 or the recombinant host cell according to claim 6 in the preparation of a detection reagent for detecting SARS-CoV-2 S protein; (2) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the recombinant host cell according to claim 6, the antibody derivative or the detection reagent according to claim 7 in the preparation of a detection kit or test strip for detecting SARS-CoV-2 S protein; (3) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the recombinant host cell according to claim 6, the antibody derivative or the detection reagent according to claim 7 in the preparation of a diagnostic product for diagnosing and / or assisting in the diagnosis of SARS-CoV-2 infection diseases; (4) Use of the monoclonal antibody of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the recombinant host cell of claim 6, the antibody derivative or the detection reagent of claim 7 for non-diagnostic and non-therapeutic purposes for detecting SARS-CoV-2 S protein; (5) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 or the recombinant host cell according to claim 6 in the preparation of a pharmaceutical composition for treating and / or preventing SARS-CoV-2 infection; (6) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the recombinant host cell according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a pharmaceutical preparation for treating and / or preventing SARS-CoV-2 infection.

10. The use according to claim 9, characterized in that: The SARS-CoV-2S protein is SARS-CoV-2S protein, SARS-CoV-2 variant XBB S protein or SARS-CoV-2 variant JN.1S protein; Optionally, the SARS-CoV-2 is SARS-CoV-2, SARS-CoV-2 variant XBB or SARS-CoV-2 variant JN.1S.

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