Monoclonal antibody aiming at human SARS-CoV-2 as well as preparation method and application thereof

Abstract

The invention provides a monoclonal antibody aiming at human SARS-CoV-2 or an antigen binding fragment of the monoclonal antibody, a related product of the monoclonal antibody, and a preparation method and application of the monoclonal antibody and the related product. The monoclonal antibody or the antigen binding fragment of the monoclonal antibody can be specifically bound with highly conservative S2 subunits in S proteins of various SARS-CoV-2 variants with relatively high affinity, and has relatively high neutralizing activity on viruses of various SARS-CoV-2 variants, so that the monoclonal antibody or the antigen binding fragment of the monoclonal antibody has extremely high potential and is developed into a broad-spectrum detection kit and a therapeutic antibody aiming at the SARS-CoV-2; the kit can be used as a treatment emergency plan standby scheme when severe infection is caused by the new SARS-CoV-2 variant, shows a remarkable application prospect in the fields of clinical detection, treatment and emergency prevention and control of SARS-CoV-2, and has an extremely high application value.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular virology, specifically to a monoclonal antibody against human SARS-CoV-2 or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] The SARS-CoV-2 virus continues to evolve, and the high frequency of mutations in its spike protein (i.e., the S protein) significantly affects the effectiveness of neutralizing antibodies. As of 2025, the dominant circulating strains globally are the XDV variant of the Omicron family and its sub-branch NB.1.8.1. These recombinant progeny carry key mutations such as L452R and F486P in the RBD region of the spike protein, leading to increased transmissibility and a significantly enhanced ability to evade existing neutralizing antibodies. For example, mutations in the RBD region of the Omicron BA.1 / BA.2 variant can reduce the neutralizing activity of some monoclonal antibodies by 10-100 times, while the latest KP.3.1.1 variant further breaches the immune barrier through the G142D mutation in the NTD region. This continuous antigenic drift has reduced the clinical protective efficacy of traditional monoclonal antibody drugs (such as Regeneron REGN-COV2 and Eli Lilly bamlanivimab) from over 80% in the early stages to below 40%, highlighting the urgency of developing broad-spectrum neutralizing antibodies.

[0003] Existing neutralizing antibody drugs have limitations. Firstly, their neutralizing activity against variant strains is insufficient: while early-approved monoclonal antibodies (such as the combination therapy of abavirin / romispirin from Brix Biotech) are effective against the original SARS-CoV-2 strain and the Delta variant, their neutralizing activity against omeprazole BA.1 / BA.2 is reduced by 5-10 times, requiring high-dose administration to maintain efficacy. Furthermore, existing antibody drugs, such as AstraZeneca's Evusheld, have only 30%-50% efficacy against omeprazole variants and require repeated injections every 6 months, resulting in poor long-term adherence. Research by Lu Bai's team at Tsinghua University shows that single antibodies are insufficient to cover all mutant epitopes, and while multi-antibody combinations ("cocktail therapy") can improve broad-spectrum efficacy, they are costly to prepare and carry the risk of cross-reactivity.

[0004] In conclusion, it is of paramount importance to develop a monoclonal antibody that can broadly and efficiently neutralize various subtypes of SARS-CoV-2 (e.g., target highly conserved and mutated epitopes in the S protein, thus enabling its use in preventing and controlling current or future SARS-CoV-2 variant infections) while also possessing high antigen-binding capacity (thus enabling its use in the efficient detection of SARS-CoV-2). Summary of the Invention

[0005] To address the needs or shortcomings of the existing technologies mentioned above, this invention provides a monoclonal antibody or its antigen-binding fragment against human SARS-CoV-2, along with related products, preparation methods, and applications. The monoclonal antibody can specifically bind to the S2 subunit of the SARS-CoV-2 S protein of various subtypes with high affinity and can broadly and efficiently neutralize various subtypes of SARS-CoV-2 virus. Therefore, it can be used for the detection, prevention, and / or treatment of current or future SARS-CoV-2 variants.

[0006] Specifically, the present invention provides the following technical solution: In a first aspect, the present invention provides a monoclonal antibody against human SARS-CoV-2 or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes: The amino acid sequences are HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; The light chain variable region includes: The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0007] In a specific implementation, the heavy chain variable region further includes four frame regions H-FR1, H-FR2, H-FR3 and H-FR4 arranged alternately with HCDR1, HCDR2 and HCDR3 in sequence, and the light chain variable region further includes four frame regions L-FR1, L-FR2, L-FR3 and L-FR4 arranged alternately with LCDR1, LCDR2 and LCDR3 in sequence; Preferably, the amino acid sequences of H-FR1 to H-FR4 are as shown in SEQ ID NO:7 to 10, respectively; and / or, the amino acid sequences of L-FR1 to L-FR4 are as shown in SEQ ID NO:11 to 14, respectively.

[0008] Preferably, the monoclonal antibody or its antigen-binding fragment comprises: The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15; and, The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16.

[0009] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment comprises: The heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO:15; and, The light chain variable region has the amino acid sequence shown in SEQ ID NO:16.

[0010] In addition, the monoclonal antibody or its antigen-binding fragment further includes a constant region; preferably, the constant region is selected from any one of the following: the constant region of IgG, IgA or IgM antibody.

[0011] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:17; and preferably, the amino acid sequence of the light chain constant region is shown in SEQ ID NO:18.

[0012] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment comprises: A heavy chain comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and, Light chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:20.

[0013] More preferably, the monoclonal antibody or its antigen-binding fragment comprises: The heavy chain, whose amino acid sequence is shown in SEQ ID NO:19; and, The light chain has the amino acid sequence shown in SEQ ID NO:20.

[0014] In some embodiments, the monoclonal antibody or its antigen-binding fragment further has a leader sequence (e.g., a signal peptide sequence) at the N-terminus of its heavy chain variable region and / or light chain variable region. In a preferred embodiment, the leader sequence at the N-terminus of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:33, its encoding gene sequence is shown in SEQ ID NO:34, and the leader sequence at the N-terminus of its light chain variable region is shown in SEQ ID NO:35, its encoding gene sequence is shown in SEQ ID NO:36.

[0015] In some feasible implementations, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, human antibodies, chimeric antibodies, or bispecific or multispecific antibodies.

[0016] Secondly, the present invention provides a polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above. This polynucleotide is not limited to any particular method of its production and can be obtained using genetic engineering recombination techniques or chemical synthesis methods.

[0017] In a feasible implementation, the polynucleotide is a polynucleotide group.

[0018] In some preferred embodiments, the polynucleotide group comprises: (I) A first polynucleotide encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide being a DNA molecule or an mRNA molecule transcribed therefrom comprising the nucleotide sequences shown in SEQ ID NO:21, 22, and 23 (which may encode the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, HCDR1, HCDR2, and HCDR3); and, (II) A second polynucleotide encoding the light chain variable regions LCDR1, LCDR2, and LCDR3 of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule or an mRNA molecule transcribed therefrom containing nucleotide sequences as shown in SEQ ID NO:24, 25, and 26 (which may encode amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, LCDR1, LCDR2, and LCDR3).

[0019] More preferably, the polynucleotide sequence comprises: (I) A first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide being a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:27 (which may encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:15) or an mRNA molecule transcribed therefrom; and, (II) A second polynucleotide encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule containing the nucleotide sequence shown in SEQ ID NO:28 (which may encode the light chain variable region of the amino acid sequence shown in SEQ ID NO:16) or an mRNA molecule transcribed therefrom.

[0020] In some specific embodiments, the first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention and / or the second polynucleotide encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention further includes a nucleotide sequence at the 5' end encoding a leader sequence. Preferably, the nucleotide sequence encoding the leader sequence at the N-terminus of the heavy chain variable region is shown in SEQ ID NO:34, and the nucleotide sequence encoding the leader sequence at the N-terminus of the light chain variable region is shown in SEQ ID NO:36.

[0021] Preferably, the polynucleotide sequence further includes: (III) A third polynucleotide encoding the heavy chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, said third polynucleotide is a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:29 (which may encode the heavy chain constant region of the amino acid sequence shown in SEQ ID NO:17); and, (IV) A fourth polynucleotide encoding the light chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the fourth polynucleotide is a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:30 (which may encode the light chain constant region of the amino acid sequence shown in SEQ ID NO:18).

[0022] In the most preferred embodiment, the polynucleotide group comprises: (I) A first polynucleotide encoding a heavy chain of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide being a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:31 or an mRNA molecule transcribed therefrom, which may encode a heavy chain with an amino acid sequence as shown in SEQ ID NO:19; and, (II) A second polynucleotide encoding a light chain of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:32, which may encode a light chain with an amino acid sequence as shown in SEQ ID NO:20.

[0023] Thirdly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and at least one expression regulatory element operatively linked to the polynucleotide.

[0024] Fourthly, the present invention provides an expression vector comprising a polynucleotide as described in the second aspect above, or a nucleic acid construct as described in the third aspect above.

[0025] Preferably, the expression vector is a eukaryotic expression vector.

[0026] Fifthly, the present invention provides a transformed host cell comprising the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above. The host cells include, but are not limited to: prokaryotic cells, such as Escherichia coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cells can also be cell lines, such as the 293T cell line.

[0027] Preferably, the host cell is a eukaryotic cell, and more preferably a mammalian cell.

[0028] Sixthly, the present invention provides a method for preparing a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, the method comprising: (1) Under conditions suitable for expressing the monoclonal antibody or its antigen-binding fragment, the transformed host cells as described in claim 13 are cultured to express the monoclonal antibody or its antigen-binding fragment; (2) The expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cell.

[0029] In a seventh aspect, the present invention provides a pharmaceutical conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, and an effector molecule conjugated directly or indirectly via a spacer to the monoclonal antibody or the antigen-binding fragment thereof. Preferably, the effector molecule is a detectable marker.

[0030] In an eighth aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, and / or a pharmaceutical conjugate as described in the seventh aspect above, as well as a pharmaceutically acceptable carrier and / or excipient.

[0031] In a ninth aspect, the present invention provides a kit comprising a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, a drug conjugate as described in the seventh aspect above, and / or a pharmaceutical composition as described in the eighth aspect above.

[0032] In a tenth aspect, the present invention provides the use of monoclonal antibodies or antigen-binding fragments thereof as described in the first aspect above, polynucleotides as described in the second aspect above, nucleic acid constructs as described in the third aspect above, expression vectors as described in the fourth aspect above, transformed host cells as described in the fifth aspect above, drug conjugates as described in the seventh aspect above, and / or pharmaceutical compositions as described in the eighth aspect above in any of the following aspects: (1) Use in the preparation of medicaments for the prevention and / or treatment of human SARS-CoV-2 infectious diseases; (2) Use in the preparation of products for detecting the presence or level of human SARS-CoV-2 in samples and / or for diagnosing human SARS-CoV-2 infection; (3) Application in the preparation of products for neutralizing the virulence of human SARS-CoV-2 in samples.

[0033] Optionally, the sample is a biological sample of the subject.

[0034] Eleventhly, the present invention provides a method for preventing and / or treating human SARS-CoV-2 infection in a subject, the method comprising: administering to the subject a preventive and / or therapeutically effective amount of the following substances: a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, a drug conjugate as described in the seventh aspect above, and / or a pharmaceutical composition as described in the eighth aspect above.

[0035] In a feasible implementation, the subject is a human being.

[0036] The monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition of the present invention, can be administered to a subject via any suitable route of administration, including but not limited to oral, oral, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

[0037] The substance can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., other antiviral drugs).

[0038] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, the method of administration, the patient's age and weight, and the patient's symptoms.

[0039] In a twelfth aspect, the present invention provides a method for detecting the presence or level of human SARS-CoV-2 in a sample, the method comprising using a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, a drug conjugate as described in the seventh aspect above, and / or a drug composition as described in the eighth aspect above.

[0040] In some preferred embodiments of the method, the monoclonal antibody or its antigen-binding fragment further includes a detectable marker.

[0041] In some other preferred embodiments of the method, the method further includes using a second antibody carrying a detectable marker to detect the monoclonal antibody of the present invention or its antigen-binding fragment.

[0042] This method can be used for diagnostic purposes (e.g., the sample is from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not from a patient).

[0043] Therefore, in some specific embodiments, the present invention provides a method for diagnosing whether a subject is infected with human SARS-CoV-2, comprising: detecting the presence or absence of SARS-CoV-2 in a sample from the subject using a monoclonal antibody or antigen-binding fragment of the present invention as described in the first aspect above. In some preferred embodiments, the monoclonal antibody or antigen-binding fragment of the present invention further includes a detectable marker; in other preferred embodiments, the method further includes using a second antibody carrying a detectable marker to detect the monoclonal antibody or antigen-binding fragment of the present invention or an anti-idiotype antibody. In feasible embodiments, the subject is a human.

[0044] General methods for detecting the presence or level of a target virus or antigen in a sample using monoclonal antibodies or their antigen-binding fragments are well known to those skilled in the art. In some preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.

[0045] Beneficial effects The anti-human SARS-CoV-2 monoclonal antibody and its antigen-binding fragment provided by this invention have the following technical advantages: 1) High-efficiency binding characteristics: It can bind with high affinity to the S2 subunit of the spike protein of various SARS-CoV-2 variants, and its binding specificity and stability are significantly better than those of conventional antibodies. Furthermore, since the S2 subunit of the antigenic epitope it targets is a highly conserved epitope that is not easily mutated, this antibody can effectively avoid the problem of decreased binding efficiency caused by viral S protein mutations. 2) Broad-spectrum neutralizing activity: It exhibits high neutralizing activity against various SARS-CoV-2 variant pseudoviruses, effectively blocking the binding process of the virus to the receptors on the surface of host cells, inhibiting viral invasion and replication, and the neutralizing activity is consistent among different variants. Based on the above advantages, the present invention has clear and significant application value: In the field of detection and diagnosis, it can be used as a core reagent for the accurate screening and diagnosis of SARS-CoV-2, especially suitable for rapid detection in the context of variant strain epidemics, improving detection accuracy and applicability; In the field of therapeutic applications, it can serve as a potential therapeutic candidate for SARS-CoV-2 infection, providing a new and effective means for clinical treatment; In the field of emergency prevention and control, given its highly efficient neutralizing activity against multiple variants, it can serve as a backup plan for emergency treatment when new variants of SARS-CoV-2 cause severe infections. It can quickly respond to outbreaks of sudden variants, effectively reduce the rate of severe infection and the risk of related complications, and provide important technical support for public health security. Attached Figure Description

[0046] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0047] Figure 1This is a protein purification peak diagram of the monoclonal antibody S2MAB1 expressed in HEK293F cells as described in Example 2.

[0048] Figure 2 The SDS-PAGE identification results of the monoclonal antibody S2MAB1.

[0049] Figure 3 The binding kinetics of the monoclonal antibody S2MAB1 with the spike protein of the SARS-CoV-2 wild-type / prototype strain (PT), Omicron BA.5 / BA.5.2, BF.7, and XBB.1.5 variant strains are shown. In each figure, A represents the binding kinetics of S2MAB1 with the spike protein of the wild-type / prototype strain (PT), B represents the binding kinetics of S2MAB1 with the spike protein of the Omicron BA.5 / BA.5.2 variant strain, C represents the binding kinetics of S2MAB1 with the spike protein of the Omicron BF.7 variant strain, and D represents the binding kinetics of S2MAB1 with the spike protein of the Omicron XBB.1.5 variant strain. Furthermore, in each figure, the five curves represent five dilutions.

[0050] Figure 4 The structure of the binding complex of monoclonal antibody S2MAB1 with the spike protein monomer of wild-type / prototype SARS-CoV-2 (Protoype, PT) is shown. Figure B is obtained by rotating Figure A by 180° with the Y-axis as the rotation axis.

[0051] Figure 5 The structure of the binding complex of monoclonal antibody S2MAB1 with the spike protein monomer of SARS-CoV-2 Omicron BA.5 / BA.5.2 mutant strain is shown. Figure B is obtained by rotating Figure A by 180° with the Y-axis as the rotation axis.

[0052] Figure 6 The structure of the binding complex of monoclonal antibody S2MAB1 with the spike protein monomer of SARS-CoV-2 Omicron BF.7 mutant strain is shown. Figure B is obtained by rotating Figure A by 180° with the Y-axis as the rotation axis.

[0053] Figure 7 Results show the neutralizing efficacy of the monoclonal antibody S2MAB1 against SARS-CoV-2 wild-type / prototype (PT) pseudoviruses.

[0054] Figure 8The results show the neutralizing efficacy of the monoclonal antibody S2MAB1 against the SARS-CoV-2 Omicron BA.4 / BA.5 / BA.5.2 variants (these three strains have the same S protein, so the pseudoviruses are the same).

[0055] Figure 9 The results show the neutralizing efficacy of the monoclonal antibody S2MAB1 against the SARS-CoV-2 Omicron BF.7 variant pseudovirus.

[0056] Figure 10 The results show the neutralizing efficacy of the monoclonal antibody S2MAB1 against the SARS-CoV-2 Omicron XBB.1.5 variant pseudovirus. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0058] Unless otherwise expressly stated, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0059] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0060] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0061] The term "percentage (%) sequence identity" is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing vacancies) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity.

[0062] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies.

[0063] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0064] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Specifically, the antigen-binding fragment can be selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, bispecific antibodies, etc.

[0065] The “Fab” segment consists of a light chain, a heavy chain CH1, and a variable region.

[0066] The “Fab’” fragment contains a light chain and a heavy chain portion that includes the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab’ fragments to form the F(ab’)2 molecule.

[0067] The “F(ab')2” segment contains two light chains and two heavy chain segments containing the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0068] The “Fv” region contains variable regions from both the heavy and light chains, but lacks constant regions.

[0069] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Generally, scFv polypeptides contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.

[0070] A "bispecific antibody" is a small antigen-binding fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker so short as to prevent pairing between the two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0071] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction between the monoclonal antibody of this invention and the 2019-nCoV RBD protein, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0072] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The term "non-binding" protein or cell refers to a protein or cell that does not bind to itself, or does not bind to it with a high affinity, i.e., the KD of the binding protein or cell is 1.0 × 10⁻⁶. -6 M or higher, more preferably 1.0 × 10 -5 M or higher, more preferably 1.0 × 10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0 × 10 -2 M or higher.

[0073] For IgG antibodies, the term "high affinity" refers to a KD of 1.0 × 10⁻⁶ for the antigen. -6 M or lower, preferably 5.0 × 10 -8 M or lower, more preferably 1.0 × 10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype refers to a KD of 10. -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0074] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see U.S. Patent No. 8,278,036, belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0075] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following embodiments are given only for illustrative purposes and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0076] The sequence information of the monoclonal antibody S2MAB1 described in the following examples is shown in Table 1 below.

[0077] Table 1. Sequence information of monoclonal antibody S2MAB1 Sequence description SEQ ID NO: Sequence information S2MAB1 - HCDR1 1 GFTFSDS S2MAB1 - HCDR2 2 SDSGDT S2MAB1 - HCDR3 3 CARDRSSLAEATSNWFDSW S2MAB1 - LCDR1 4 KSSQNILYSSNNNNYLA S2MAB1 - LCDR2 5 WASIRES S2MAB1 - LCDR3 6 CQQHYSIPLTF S2MAB1 - H - FR1 7 QVQLVESGGGLVKPGRSLRLSCAAS S2MAB1 - H - FR2 8 YMTWIRQAPGKGLEWVSYI S2MAB1 - H - FR3 9 RYYADSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYY S2MAB1 - H - FR4 10 GQGTLVTVSS S2MAB1 - L - FR1 11 DIVLTQSPDSLAVSLGERATINC S2MAB1 - L - FR2 12 WYQQKPGQPPKLLIH S2MAB1 - L - FR3 13 GVPDRFSGSGSGTDFTLTISSLQAEDVAVYY S2MAB1 - L - FR4 14 GGGTKVEIK S2MAB1 - VH 15 QVQLVESGGGLVKPGRSLRLSCAASGFTFSDSYMTWIRQAPGKGLEWVSYISDSGDTRYYADSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCARDRSSLAEATSNWFDSWGQGTLVTVSS S2MAB1 - VL 16 DIVLTQSPDSLAVSLGERATINCKSSQNILYSSNNNNYLAWYQQKPGQPPKLLIHWASIRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSIPLTFGGGTKVEIK S2MAB1 - CH 17 KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK S2MAB1 -CL 18 SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS S2MAB1 -Heavy chain 19 QVQLVESGGGLVKPGRSLRLSCAASGFTFSDSYMTWIRQAPGKGLEWVSYISDSGDTRYYADSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCARDRSSLAEATSNWFDSWGQGTLVTVSSTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK S2MAB1 -Light chain 20 DIVLTQSPDSLAVSLGERATINCKSSQNILYSSNNKNYLAWYQQKPGQPPKLLIHWASIRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSIPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS S2MAB1 - HCDR1 coding sequence 21 GGATTCACCTTCAGTGACTCC S2MAB1 - HCDR2 coding sequence 22 AGTGATAGTGGTGATACC S2MAB1 - HCDR3 coding sequence 23 TGTGCGAGAGATCGCTCGAGTCTAGCAGAGGCTACTTCTAACTGGTTCGACTCCTGG S2MAB1 - LCDR1 coding sequence 24 AAGTCCAGCCAGAATATTTTATATAGCTCCAACAATAACAACTACTTAGCT S2MAB1 - LCDR2 coding sequence 25 TGGGCATCTATCCGGGAATCC S2MAB1 - LCDR3 coding sequence 26 TGTCAGCAACATTACAGTATTCCGCTCACTTTC S2MAB1 - VH gene 27 CAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTCAAGCCTGGCCGGAGCCTGCGGCTGAGCTGCGCCGCTAGCGGCTTTACATTCAGCGATAGCTACATGACCTGGATCCGGCAGGCCCCTGGAAAAGGCCTGGAATGGGTGTCCTACATCAGCGACTCCGGCGACACCAGATACTACGCCGATAGCGTGAAGGGCAGATTCACCATCTCTAGAGACAACGCCAAGAATAGCCTGTACCTGCAGATGAACAGCCTCCGCCCCGAGGACACAGCCGTGTACTATTGTGCTAGAGATAGAAGCAGCCTGGCCGAGGCCACAAGCAACTGGTTCGACTCCTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCT S2MAB1 - VL gene 28 GACATCGTGCTGACACAGAGCCCCGACAGCCTGGCTGTGTCCCTGGGCGAGCGGGCCACCATTAACTGCAAGTCCAGCCAGAATATCCTGTACAGCAGCAACAACAAGAACTACCTGGCCTGGTATCAGCAGAAACCTGGCCAGCCTCCAAAGCTGCTGATCCACTGGGCCAGCATCAGAGAGAGCGGAGTGCCTGATAGATTCAGCGGCAGCGGCTCTGGCACAGACTTCACCCTGACCATCAGCTCTCTCCAGGCCGAAGATGTGGCCGTCTACTACTGTCAGCAACACTACTCTATCCCCCTGACCTTTGGCGGCGGAACCAAGGTGGAAATCAAG S2MAB1 - CH gene 29 AAAGGCCCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCAAAGTGGATAAACGCGTGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACTATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA S2MAB1 - CL gene 30 AGCGTGTTTATCTTCCCTCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTCTGTCTCCTGAACAACTTCTATCCCAGGGAGGCCAAGGTCCAGTGGAAAGTGGACAACGCCCTGCAAAGCGGCAATAGCCAGGAGTCCGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTGTCCAGCACCCTGACCCTCAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTGACCCATCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACAGGGGCGAATGCAGC S2MAB1 - Heavy chain gene 31 CAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTCAAGCCTGGCCGGAGCCTGCGGCTGAGCTGCGCCGCTAGCGGCTTTACATTCAGCGATAGCTACATGACCTGGATCCGGCAGGCCCCTGGAAAAGGCCTGGAATGGGTGTCCTACATCAGCGACTCCGGCGACACCAGATACTACGCCGATAGCGTGAAGGGCAGATTCACCATCTCTAGAGACAACGCCAAGAATAGCCTGTACCTGCAGATGAACAGCCTCCGCCCCGAGGACACAGCCGTGTACTATTGTGCTAGAGATAGAAGCAGCCTGGCCGAGGCCACAAGCAACTGGTTCGACTCCTGGGGCCAGGGCACCCTGGTGACCGTGTCTTCTACCGTCTCCTCAGCCAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCAAAGTGGATAAACGCGTGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACTATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA S2MAB1 - Light chain gene 32 GACATCGTGCTGACACAGAGCCCCGACAGCCTGGCTGTGTCCCTGGGCGAGCGGGCCACCATTAACTGCAAGTCCAGCCAGAATATCCTGTACAGCAGCAACAACAAGAACTACCTGGCCTGGTATCAGCAGAAACCTGGCCAGCCTCCAAAGCTGCTGATCCACTGGGCCAGCATCAGAGAGAGCGGAGTGCCTGATAGATTCAGCGGCAGCGGCTCTGGCACAGACTTCACCCTGACCATCAGCTCTCTCCAGGCCGAAGATGTGGCCGTCTACTACTGTCAGCAACACTACTCTATCCCCCTGACCTTTGGCGGCGGAACCAAGGTGGAAATCAAGCGAACTGTGGCTGCACCAAGCGTGTTTATCTTCCCTCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTCTGTCTCCTGAACAACTTCTATCCCAGGGAGGCCAAGGTCCAGTGGAAAGTGGACAACGCCCTGCAAAGCGGCAATAGCCAGGAGTCCGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTGTCCAGCACCCTGACCCTCAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTGACCCATCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACAGGGGCGAATGCAGC S2MAB1 -H chain leader sequence 33 METDTLLLWVLLLWVPGSTGD The S2MAB1 -H chain leader sequence encodes a gene 34 ATGGAGACGGATACGCTGCTCCTGTGGGTTTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC S2MAB1-L chain leader sequence 35 MSCGFCCSGFQVPLV The S2MAB1-L chain leader sequence encodes a gene. 36 ATGTCCTGTGGGTTTTGCTGCTCTGGGTTCCAGGTTCCACTGGTG

[0078] Example 1: Screening of cells secreting human SARS-CoV-2 antibodies Biological samples from volunteers used in the following experiments were obtained with their informed consent.

[0079] The volunteer received two doses of the adenovirus type 5 (Ad5) vector COVID-19 vaccine (Kivexa, CanSino Biologics Inc.) and underwent a BA.5.2 / BF.7 breakthrough infection (ethics committee approval number: 2023-30-PJ01), and presented with mild clinical symptoms. Anticoagulated whole blood was collected approximately four weeks post-infection.

[0080] 1. Preparation of single-cell suspensions of peripheral blood mononuclear cells (PBMCs) Single-cell RNA sequencing and preliminary analysis were performed by LC-BioTechnologies, with single-cell suspension preparation completed on the day of blood collection. PBMCs were resuspended in 1 mL of RPMI 1640 medium (Corning, catalog number 10-040-CVR) containing 0.04% bovine serum albumin (Roche, catalog number 10711454001) until cell viability exceeded 85%, at which point a single-cell suspension was prepared. For samples taken one month after blood transfusion (BTI), LC-BioTechnologies technicians isolated PBMCs from whole blood and used them directly for single-cell library construction.

[0081] 2. Single-cell library construction and sequencing Single-cell libraries were prepared using the Chromium Next GEM Single-Cell 5' Kit Version 2 (Dual-Exponential Labeling) (10xGenomics, USA, Catalog No. CG000331). Cell suspension, 10x barcode gel beads, and reaction reagents were dispensed into the channels of the Chromium Next GEM Chip G using microfluidic technology, ultimately forming water-in-oil emulsion beads (GEMs). This bead system facilitated efficient integration of gel beads into the cell, including mRNA release, cDNA generation via reverse transcription, and sequencing barcodes. After disrupting the liquid phase oil layer, cDNA amplification was performed, and cDNA quality was assessed after purification. The library construction process included enzyme digestion, end repair, A base addition, and adapter ligation. Target length fragments were screened and recovered, followed by library quality evaluation and quantification. The final sequencing library structure, from 5' to 3', consists of: Illumina P5 adapter, P5 sequencing primer, 16 bp barcode, 12 bp molecular tag (UMI), poly(dT)VN sequence, inserted cDNA fragment, P7 sequencing primer, sample index, and Illumina P7 adapter.

[0082] 3. Single-cell expression and immune repertoire data generation Raw data obtained from high-throughput sequencing underwent quality control filtering and sequence alignment with the human reference genome (GRCh38). Quantitative analysis of the cellular transcriptome was achieved using the 10x Genomics CellRanger analysis pipeline, combining cell barcoding and molecular tags. The final single-cell expression matrix was constructed based on quality control standards (including control cell count, median gene value, and sequencing saturation). Using the CellRanger V(D)J analysis module with default parameters, immune repertoire sequence data was generated. By aligning high-quality reads with known V(D)J gene reference sequences, single-cell V(D)J gene expression profiles and clonogenic information were obtained.

[0083] 4. Single-cell data analysis The Seurat R package (v4.3.0) was used for cross-sample single-cell data integration and cluster analysis. The quality control criteria were: cells with a mitochondrial gene proportion >20% were filtered out, cells with UMI counts between 600 and 3,000 were retained, and cells with a ribosomal gene proportion >50% were excluded. This triple quality control strategy effectively eliminated dying cells, RNA-degraded cells, and potential cell duplexes. The DoubletFinder R package (v2.0.3) was used for duplex identification and secondary removal. Data standardization was performed using Seurat, and the top 2000 genes with the highest coefficient of variation were selected for principal component analysis and cluster calculations. The Harmony algorithm R package (v0.1.1) was used to correct for batch effects between samples and groups during cell clustering. The top 30 principal components were selected for cell clustering using principal component significance assessment and inflection point analysis, and differential resolution parameters were used to determine cell cluster boundaries. The Unified Manifold Approximation and Projection (UMAP) algorithm was applied for dimensionality reduction and single-cell visualization. For detailed implementation instructions, please refer to the online tutorial (https: / / satijalab.org / seurat / v4.0 / pbmc3k_tutorial.html).

[0084] 5. Cell type annotation After UMAP nonlinear dimensionality reduction, all cells were classified and integrated based on shared features. Cell identification was performed based on the stable expression characteristics of specific molecules in different cell subpopulations. The Seurat built-in FindAllMarker() function was used to calculate the top 50 highly expressed genes for each cell cluster, and a comprehensive cell type determination was completed by combining existing research literature. Cell clusters that simultaneously expressed two or more classic cell type markers were identified as residual duplexes and removed. In the T / B lymphocyte subset annotation, a small number of chimeric duplexes were further removed based on the co-expression characteristics of platelet, megakaryocyte, and lymphocyte markers.

[0085] 6. Single-cell gene set activity analysis The `AddModuleScore()` function built into the `Seurat` package is used to calculate the activity score of each gene set within a single cell. The algorithm proceeds as follows: First, the average value of all genes in the gene set is calculated. Based on this average, the gene expression matrix is ​​divided into different intervals. Control genes (default 100) that are not members of the gene set are randomly selected from each interval as background reference values. Finally, the average values ​​of the target gene and the background genes are calculated separately, and the gene set enrichment score is obtained through difference calculation. The feature gene set data is sourced from the `msigdb` R package (v7.5.1).

[0086] 7. Statistical Analysis Single-cell data analysis was performed using R software (v4.3.2), and visualization was completed using Cytoscape (v3.8.0). The Wilcoxon rank-sum test (Seurat's default setting) was used to identify differentially expressed genes (DEGs) between groups, and p-values ​​were corrected using the Bonferroni method. Statistical significance was set at p < 0.05. Differences in single-cell gene set scores were analyzed using either the Wilcoxon rank-sum test or the Kruskal-Wallis test.

[0087] The results showed that preliminary cell type annotation indicated four subsets of T cells (naive T cells, activated T cells, γδ T cells, and mucosa-associated inertial T cells (MAIT)); and five types of innate immune cells (NK cells, CD14 monocytes, CD16 monocytes, dendritic cells (DCs), and plasmacytic dendritic cells (pDCs)). B cells, megakaryocytes, platelets, erythrocytes, and a small number of hematopoietic stem cells were also identified.

[0088] Cluster analysis identified 12 B cell subsets, including classic naive B cells, naive B cells highly expressing IGHV3-20 (Naïve_B_IGHV3-20), IGHV4-31 (Naïve_B_IGHV4-31), and IGHV3-53 (Naïve_B_IGHV3-53), and naive B cells expressing the interferon-stimulated gene (ISG) (Naïve_B-ISG). Memory B cells were further subdivided into CD27+ memory B cells, FCRL5+ memory B cells, and memory B cells highly expressing ISG (Memory_B_ISG). Germinal center B cells (GC_B), plasmablasts, plasma cells, and intermediate transitional memory B cell subsets (ITMB, marked as IGHD+ CD27+) were also detected. Assessment of BCR signaling pathway activity in different subsets showed that the overall BCR signaling activity in the BTI group was significantly higher than that in the control group, suggesting that B cells were in an activated or proliferating state.

[0089] Example 2: Human SARS-CoV-2 antibody was obtained by integrating bioinformatics analysis and experimental verification. 1. BCR VDJ gene alignment and analysis

[0090] BCR analysis followed a standard procedure, retaining only cells with both pairable functional heavy chains (IGH) and light chains (IGK / IGL). In cases where multiple IGH or IGK / IGL chains were detected in the same cell, only the chain with the highest expression level was retained. Each unique IGH-IGK / IGL pair was defined as a clonal type. Single-cell immune repertoire data were integrated and analyzed using the scRepertoire R software package (v1.7.1) to calculate clonal diversity and VDJ gene usage frequency. Clonal types carrying TCR or BCR information were mapped to single-cell transcriptomes for subsequent analysis. For B cells, subtype classification (IgA, IgD, IgG, IgM, and IgE) was determined based on the BCR constant region.

[0091] The results showed that B cells possessed both transcriptomic expression characteristics and paired BCR heavy and light chain sequencing data, with a detection rate >94%. The top five BCRs with the highest cloning frequency (IgM / IgD types) may be candidate antibody sources targeting BA.5 and BF.7 subvariants. The proportion of BCRs with medium-to-high cloning amplification was extremely low. A total of 83 amplified BCRs were identified (corresponding to 180 B cells). Subsequent analysis indicated that amplified B cells were mainly enriched in plasmablasts, infiltrating rejection-peripheral B cells (ITMB), memory B cells, and plasma cells. High-frequency amplified BCRs may be a potential antibody repositories for the host against BA.5 / BF.7 subvariants. BCR class switching analysis revealed multiple class switching events within the same B cell subset, suggesting that antibody type switching is still active. IgM-type BCRs were dominant, with a significantly higher proportion of IgM-type BCRs in naive B cells than in other subsets. IgA and IgG-type BCRs were mainly distributed in memory B cells and plasma cells. Classification analysis of amplified BCRs showed that most amplified B cells were still IgM type, while IgG and IgA types also accounted for a certain proportion.

[0092] 2. Acquisition of Monoclonal Antibodies Select a heavy chain-light chain pairing sequence that matches the amplified IgG2 type BCR with a high number of clones (multiple groups can be selected for testing), entrust Genscript Biotech (Nanjing, China) to synthesize it, and clone it into the pCAGGS expression plasmid.

[0093] The amplified heavy and light chains were co-transfected into 293F cells in an equimolar ratio for protein expression, followed by purification using an AKTA system (Cytiva, USA) and a Protein A HP chromatography column. Elution was performed with 0.1 M glycine at pH 2.7, and PBS was used as buffer for displacement and concentration steps to obtain the purified monoclonal antibody, which is the monoclonal antibody of this invention and is named "S2MAB1".

[0094] The protein purification peak diagram and SDS-PAGE identification results of the expressed monoclonal antibody S2MAB1 are shown below. Figure 1 and Figure 2 As shown. By Figure 1 and Figure 2 It can be seen that the above method successfully obtained high-purity monoclonal antibody S2MAB1.

[0095] Example 3: Expression and purification of SARS-CoV-2 spike protein Expression constructs (pCAGGS) of the extracellular domain of the S protein from SARS-CoV-2 PT, BA.5, BF.7, and XBB.1.5 viral strains were transfected into 293F cells and expressed for 5 days. Proteins were collected by centrifugation at 6,500 rpm for 60 minutes, and the supernatant was filtered through a 0.22 µm filter. Protein purification was performed using an AKTA Start system (Cytiva, USA) with a HisTrap HP column. The running buffer system consisted of binding buffer (20 mM Tris, 500 mM NaCl, pH 7.5) and elution buffer (20 mM Tris, 500 mM NaCl, 1 M imidazole, pH 7.5). Real-time UV monitoring guided the elution of the target protein, and a 30% imidazole concentration was determined to be optimal. The purified protein was then collected by fractionation, concentrated using a 100 kDa ultrafiltration tube (Millipore, USA, catalog number UFC9100), and further purified and quality controlled using Superose 6 Increase 10 / 300GL (Cytiva, USA).

[0096] The purified expressed proteins were identified by SDS-PAGE analysis. The results showed that the extracellular domains of the S protein of SARS-CoV-2 PT, BA.5, BF.7 and XBB.1.5 viral strains were successfully obtained in high purity through the above procedure.

[0097] Example 4: Determination of the broad-spectrum binding ability of SARS-CoV-2 spike protein monoclonal antibody to spike protein and the basis of binding structure.

[0098] To elucidate the characteristics of B cell receptors (BCRs) corresponding to antigen-specific antibodies, the cross-relationships of different subvariant spike protein (SP)-related antibody subsets, and the amplification characteristics of antibody-corresponding BCRs, we purified total IgG from the serum of convalescent patients containing amplified BCRs. Simultaneously, spike proteins from variants such as PT, BA.5.2, and BF.7 were expressed in HEK293 cells for pFab / IgG binding capacity assays. In humoral immune responses, B cells undergo a cascade reaction triggered by antigen stimulation, leading to their directed differentiation into antibody-secreting plasma cells.

[0099] IgG and IgA antibodies constitute the main targets for antibody drug development. This study successfully isolated a functional IgG2 antibody from an amplified BCR sequence library. After cloning, expression, and purification, its biological characteristics were evaluated through functional validation experiments. The binding properties of the antibody to the antigen S protein were detected using an Octet 96 molecular interaction analyzer.

[0100] The antigen concentration was set to 40 µg / mL, and the antibody concentration was set in a gradient mode for antigen-antibody binding activity testing. The results showed that the antibody exhibited broad-spectrum SARS-CoV-2 spike protein binding activity, and could bind to the S protein of subtypes such as PT, BA.5.2, and BF.7.

[0101] Specifically, the binding kinetics curves of the monoclonal antibody S2MAB1 with the spike proteins of SARS-CoV-2 PT, BA.5, BF.7, and XBB.1.5 are as follows: Figure 3 As shown in Figures A, B, C, and D, the IC50 values ​​are 232 nM, 204 nM, 207 nM, and 281 nM, respectively. These results suggest that the antibody possesses strong broad-spectrum binding affinity.

[0102] AlphaFold3 was used to predict the complex structures of the monoclonal antibody S2MAB1 Fab (containing VH and VL) with PT, BA.5, and BF.7 spike protein monomers. The results are as follows: Figures 4-6 As shown, the Fab of the monoclonal antibody S2MAB1 binds to the S2 subunit along with the monomers of the PT, BA.5, and BF.7 spike proteins. It is known in the art that the S2 subunit of the SARS-CoV-2 spike protein is highly conserved and resistant to mutation; therefore, these results also help explain the molecular mechanism by which this antibody broadly binds to various subtypes of the SARS-CoV-2 spike protein.

[0103] Example 5: Validation of the broad-spectrum neutralizing efficacy of a human SARS-CoV-2 spike protein-specific monoclonal antibody

[0104] In this embodiment, the SARS-CoV-2 pseudovirus system was used, and the broad-spectrum neutralizing ability of the monoclonal antibody S2MAB1 against different variants of SARS-CoV-2 was evaluated through pseudovirus neutralization experiments.

[0105] By constructing a pseudovirus system containing multiple variants such as PT, XBB.1.5, BA.4 / 5, and BF.7, the titer of this broad-spectrum neutralizing antibody was detected.

[0106] Specifically, using pCAGGS as the backbone vector, truncated S protein expression plasmids expressing the spike protein of each SARS-CoV-2 strain with an 18-amino acid deletion at the C-terminus were constructed. These plasmids were co-transfected with the pseudovirus packaging backbone virus G*VSV-delG (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) into the HEK-293T cell line to generate pseudoviruses of each SARS-CoV-2 strain. Antibodies were serially diluted fourfold (initial concentration 50 μg / mL, final concentration 0.19 ng / mL, 10 consecutive fourfold dilutions). 1000 infection units of pseudovirus were pre-incubated with antibody dilution at 37°C for 1 hour and then seeded into 96-well plates pre-coated with Vero cells (ATCC CCL81). After 15 hours of culture, the viral infection units were quantified using a CQ1 automated imaging cytometer (Yokogawa, Japan). All samples were analyzed in duplicate. The half-maximal pseudovirus neutralizing titer (pVNT50) was calculated using a GraphPad Prism (v8.4.3) nonlinear regression model. The results are presented as geometric mean titers (95% confidence intervals). The following nonlinear regression equation was used for calculation: Y = 100 / [1 + 10(LogpVNT50 - X) × Hill slope], where the Hill slope characterizes the steepness of the family of curves.

[0107] The results are as follows Figure 7-10 As shown, the IC50 values ​​of the monoclonal antibody S2MAB1 for neutralizing PT, BA.5, BF.7, and XBB.1.5 pseudoviruses were 2.141 μg / mL, 1.553 μg / mL, 0.8047 μg / mL, and 2.325 μg / mL, respectively. This indicates that the monoclonal antibody exhibits excellent neutralizing activity against PT, BA.5.2, BF.7, and even XBB.1.5 pseudoviruses. This finding confirms that amplified BCR libraries obtained through mixed immune backgrounds can be screened for broad-spectrum neutralizing antibodies.

[0108] The above results suggest that the monoclonal antibody of the present invention has a strong broad-spectrum neutralizing ability against different variants of SARS-CoV-2.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against human SARS-CoV-2 or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes: The amino acid sequences are HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; The light chain variable region includes: The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

2. The monoclonal antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region also includes four frame regions H-FR1, H-FR2, H-FR3 and H-FR4 arranged alternately with HCDR1, HCDR2 and HCDR3 in sequence; the light chain variable region also includes four frame regions L-FR1, L-FR2, L-FR3 and L-FR4 arranged alternately with LCDR1, LCDR2 and LCDR3 in sequence. Preferably, the amino acid sequences of H-FR1 to H-FR4 are as shown in SEQ ID NO:7 to 10, respectively; and / or, the amino acid sequences of L-FR1 to L-FR4 are as shown in SEQ ID NO:11 to 14, respectively.

3. The monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

15. And / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

16. Preferably, the monoclonal antibody or its antigen-binding fragment comprises: The heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO:15; and, The light chain variable region has the amino acid sequence shown in SEQ ID NO:

16.

4. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein, The monoclonal antibody or its antigen-binding fragment further includes a constant region; Preferably, the constant region comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:17 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:

18.

5. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, wherein, The monoclonal antibody or its antigen-binding fragment comprises: A heavy chain comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and, Light chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:20; Preferably, the monoclonal antibody or its antigen-binding fragment comprises: The heavy chain, whose amino acid sequence is shown in SEQ ID NO:19; and, The light chain has the amino acid sequence shown in SEQ ID NO:

20.

6. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-5, characterized in that, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, human antibody, chimeric antibody, or bispecific or multispecific antibody.

7. A polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6.

8. The polynucleotide of claim 7, wherein, The polynucleotide is a polynucleotide group, which includes: (I) A DNA molecule containing the nucleotide sequence shown in SEQ ID NO:21, 22, 23, or an mRNA molecule transcribed therefrom; and, (II) A DNA molecule or an mRNA molecule transcribed therefrom containing the nucleotide sequences shown in SEQ ID NO:24, 25, 26.

9. The polynucleotide of claim 7 or 8, wherein, The polynucleotide sequence includes: (I) A DNA molecule containing the nucleotide sequence shown in SEQ ID NO:27 or an mRNA molecule transcribed therefrom; and, (II) A DNA molecule containing the nucleotide sequence shown in SEQ ID NO:28 or an mRNA molecule transcribed therefrom; Preferably, the polynucleotide sequence further includes: (III) A DNA molecule containing the nucleotide sequence shown in SEQ ID NO:29 or an mRNA molecule transcribed therefrom; and, (IV) A DNA molecule containing the nucleotide sequence shown in SEQ ID NO:30 or an mRNA molecule transcribed therefrom.

10. The polynucleotide according to any one of claims 7-9, wherein, The polynucleotide sequence includes: (I) A DNA molecule or its corresponding mRNA molecule with the nucleotide sequence shown in SEQ ID NO:31; and, (II) DNA molecules or their corresponding mRNA molecules with nucleotide sequences as shown in SEQ ID NO:

32.

11. A nucleic acid construct comprising a polynucleotide as described in any one of claims 7-10, and at least one expression regulatory element operatively linked to said polynucleotide.

12. An expression vector comprising a polynucleotide as described in any one of claims 7-10, or a nucleic acid construct as described in claim 11.

13. A transformed host cell, wherein the transformation comprises a polynucleotide as described in any one of claims 7-10, a nucleic acid construct as described in claim 11, or an expression vector as described in claim 12.

14. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6, the method comprising: (1) Under conditions suitable for expressing the monoclonal antibody or its antigen-binding fragment, the transformed host cells as described in claim 13 are cultured to express the monoclonal antibody or its antigen-binding fragment; (2) The expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cell.

15. A drug conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6, and an effector molecule conjugated directly or indirectly via a spacer to the monoclonal antibody or the antigen-binding fragment thereof; Preferably, the effector molecule is a detectable marker.

16. A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6, a polynucleotide as described in any one of claims 7-10, a nucleic acid construct as described in claim 11, an expression vector as described in claim 12, a transformed host cell as described in claim 13, and / or a pharmaceutical conjugate as described in claim 15, and a pharmaceutically acceptable carrier and / or excipient.

17. A kit comprising a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, a polynucleotide as described in any one of claims 7-10, a nucleic acid construct as described in claim 11, an expression vector as described in claim 12, a transformed host cell as described in claim 13, a pharmaceutical conjugate as described in claim 15, and / or a pharmaceutical composition as described in claim 16.

18. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, the polynucleotide as described in any one of claims 7-10, the nucleic acid construct as described in claim 11, the expression vector as described in claim 12, the transformed host cell as described in claim 13, the drug conjugate as described in claim 15, and / or the pharmaceutical composition as described in claim 16 in any of the following aspects: (1) Use in the preparation of medicaments for the prevention and / or treatment of human SARS-CoV-2 infectious diseases; (2) Use in the preparation of products for detecting the presence or level of human SARS-CoV-2 in samples and / or for diagnosing human SARS-CoV-2 infection; (3) Application in the preparation of products for neutralizing the virulence of human SARS-CoV-2 in samples.

19. The application as described in claim 18, characterized in that, The sample is a biological sample from the subject.

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