Multi-specific antibody

Abstract

The present invention relates to a multispecific antibody or an antigen-binding molecule thereof, a homodimer thereof, a nucleic acid molecule encoding the multispecific antibody or the antigen-binding molecule thereof, a vector comprising the nucleic acid molecule, a host cell comprising the vector, and a recombinant protein or immunoconjugate comprising the multispecific antibody or the antigen-binding molecule thereof, the invention also discloses application of the compounds in preparation of drugs for treating or preventing respiratory tract infection diseases caused by viruses, or preparation of drugs for treating or preventing respiratory tract inflammation or dermatitis, and application in detection of products. The multispecific antibody or the antigen binding molecule of the multispecific antibody has excellent broad-spectrum neutralizing capacity on coronavirus, and has remarkable neutralizing capacity on various SARS-CoV-2 coronavirus mutant strains including coronavirus mutant strains, especially Omicron subtype BQ.1. 1, XBB.1. 5, XBB.1. 16, EG.5, BA.2. 86 and JN.1, and the multispecific antibody or the antigen binding molecule of the multispecific antibody has the advantages that the multispecific antibody or the antigen binding molecule of the multispecific antibody or the antigen binding molecule of the multispecific antibody or the antigen binding molecule of the multispecific antibody or the antigen binding molecule of the multispecific antibody has excellent broad-spectrum neutralizing capacity on the coronavirus mutant strains, especially various SARS-CoV-2 coronavirus mutant strains; it is accidentally found that the multispecific antibody or the antigen binding molecule thereof not only can treat and prevent respiratory tract infection symptoms caused by viruses, but also can remarkably relieve respiratory tract inflammatory reaction, has a good treatment effect on rhinitis, asthma, dermatitis and other inflammatory diseases, and has a good clinical application prospect in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine; the present invention relates to a multispecific antibody or its antigen-binding molecule, and its homodimer, a nucleic acid molecule encoding the multispecific antibody or its antigen-binding molecule, a vector containing the nucleic acid molecule, a host cell containing the vector, a recombinant protein or immunoconjugate containing the multispecific antibody or its antigen-binding molecule, and their applications in the preparation of drugs for treating or preventing viral respiratory infectious diseases, or in the preparation of drugs for treating or preventing respiratory inflammations (such as rhinitis, asthma, etc.) or dermatitis, as well as their applications in detection products. Background Art

[0002] The novel coronavirus spreads through the air and infects the respiratory tract, which can cause respiratory infection symptoms such as coughing, fever, and sore throat in patients. A small number of infected individuals will progress to pneumonia, and severe cases can lead to systemic inflammatory storms and systemic organ failure in patients.

[0003] The continuous mutation of the novel coronavirus has brought stronger immune escape ability, virus infection ability, and transmissibility, resulting in repeated infections in the population. At the same time, long COVID and post-COVID sequelae after population infection seriously affect people's physical health.

[0004] SARS-CoV-2 belongs to the Coronaviridae family and is a member of the Sarbecovirus genus in the Betacoronavirus genus, the same genus as the SARS-CoV coronavirus that broke out in 2002, with an amino acid homology of up to 79%. The main envelope protein of the SARS-CoV-2 virus is the spike protein (also known as the Spike protein, abbreviated as the S protein). During the virus infection process, the spike protein is hydrolyzed by intracellular proteases into two parts, S1 and S2. Among them, S2 is a transmembrane protein, and S1 has a receptor-binding domain (Receptor binding domain, abbreviated as RBD) and an N-terminal domain (N-terminal domain, abbreviated as NTD). The RBD protein can recognize and bind to the human angiotensin-converting enzyme 2 (ACE-2) receptor expressed on the cell surface. The spike protein composed of S1 and S2 is the protein that specifically recognizes and binds to the target cell receptor of the SARS-CoV-2 virus, mediates the fusion of the virus membrane and the cell membrane, and plays a role in mediating the virus to infect the target cell. It is the recognition target of neutralizing antibodies.

[0005] Many studies at home and abroad have reported neutralizing antibodies isolated from patients infected with the novel coronavirus or from vaccinated individuals. These neutralizing antibodies mainly bind to the spike protein of SARS-CoV-2. On the one hand, they can achieve a protective effect by inhibiting the interaction between virus particles and receptors, or by disrupting virus particles to prevent the virus from entering the target cells of the infected organism. On the other hand, these neutralizing antibodies can mediate the body's immune system to kill or phagocytose virus-infected cells by binding to virus-infected cells, thereby clearing virus-infected cells.

[0006] SARS-CoV-2 belongs to an RNA virus. When it spreads in the population, the viral genome is prone to mutations during the replication process. A new mutant strain replaces the old mutant strain every six months, forming an iteration of a pandemic. The Omicron mutant strain was first discovered in December 2021 and caused a pandemic worldwide within a month. In the past two years, the Omicron mutant strain has evolved into sub-lineage mutant strains such as BA.5, BF.7, BQ.1.1, XBB.1.5, XBB.1.16, EG.5, BA.2.86, JN.1, etc. In particular, the spike protein sequences of BA.2.86 and JN.1 have 39 additional mutations on the basis of BA.2, resulting in severe immune escape. It not only escapes the immune environment formed by vaccination or previous infection in the body, but also causes the inactivation of many antibody or immunoglobulin drugs. Single antibody therapy targeting a single epitope is difficult to cope with the rapidly mutating virus, and the improvement effect of antibody cocktails on drugs is not significant. Therefore, a large number of antibody drugs approved for emergency clinical use have been withdrawn, and antibody drugs in clinical trials are also difficult to advance clinically.

[0007] In order to improve the neutralization breadth and neutralization ability of antibodies against the novel coronavirus, researchers have genetically engineered existing neutralizing antibodies against the novel coronavirus, hoping to construct antibodies that can simultaneously bind to multiple epitopes of coronaviruses, inhibit virus mutation and escape, and obtain antibodies with better broad-spectrum and neutralization activities. For example, multi- / bispecific antibodies that can bind to multiple coronavirus immunogens, or inhibit the infection of target cells by multiple coronaviruses, especially inhibit the infection of target cells or the human body by multiple novel coronavirus mutant strains, or can clear virus-infected cells in the human body. Summary of the Invention

[0008] To solve the above technical problems, in a first aspect of the present invention, there is provided a multi-specific antibody or its antigen-binding molecule, which comprises a first antigen-binding module and a second antigen-binding module;

[0009] The second antigen-binding module binds to the receptor-binding motif (Receptor binding motif, abbreviated as RBM) part of the receptor-binding region of the coronavirus S protein;

[0010] The first antigen-binding module binds to a portion outside the receptor-binding motif of the receptor-binding region of the coronavirus S protein.

[0011] In a specific embodiment of the present invention, the first antigen-binding module comprises a light chain variable region VL-1 and a heavy chain variable region VH-1; the second antigen-binding module comprises a light chain variable region VL-2 and a heavy chain variable region VH-2;

[0012] The light chain variable region VL-1 comprises the LCDR1-1 sequence of the light chain variable region shown in SEQ ID NO.1, the LCDR2-1 sequence of the light chain variable region shown in SEQ ID NO.2, and the LCDR3-1 sequence of the light chain variable region shown in SEQ ID NO.3;

[0013] The heavy chain variable region VH-1 comprises the HCDR1-1 sequence of the heavy chain variable region shown in SEQ ID NO.4, the HCDR2-1 sequence of the heavy chain variable region shown in SEQ ID NO.5, and the HCDR3-1 sequence of the heavy chain variable region shown in SEQ ID NO.6;

[0014] The light chain variable region VL-2 comprises the LCDR1-2 sequence of the light chain variable region shown in SEQ ID NO.7, the LCDR2-2 sequence of the light chain variable region shown in SEQ ID NO.8, and the LCDR3-2 sequence of the light chain variable region shown in SEQ ID NO.9;

[0015] The heavy chain variable region VH-2 comprises the HCDR1-2 sequence of the heavy chain variable region shown in SEQ ID NO.10, the HCDR2-2 sequence of the heavy chain variable region shown in SEQ ID NO.11, and the HCDR3-2 sequence of the heavy chain variable region shown in SEQ ID NO.12.

[0016] Preferably, the sequence of the light chain variable region VL-1 is as shown in SEQ ID NO.13, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.13;

[0017] The sequence of the heavy chain variable region VH-1 is as shown in SEQ ID NO.14, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.14;

[0018] The sequence of the light chain variable region VL-2 is as shown in SEQ ID NO.15, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.15;

[0019] The sequence of the heavy chain variable region VH-2 is as shown in SEQ ID NO.16, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.16.

[0020] In a specific embodiment of the present invention, the above light chain variable region VL-1 can have a small number of amino acid deletions, insertions or amino acid mutations on the basis of the above sequence to obtain an amino acid sequence with a homology of more than 80%. Variants obtained by a small number of amino acid substitutions (deletions or insertions, or amino acid mutations, or substitutions with similar amino acids), especially conservative amino acid substitutions in the framework region of the variable region, have a high homology (more than 80% homology) with the above sequence and retain the original properties and functions of the light chain variable region, that is, retain the antibody properties and functions of specifically binding to coronaviruses, or have the function of treating or preventing respiratory infection symptoms caused by viruses, or have the function of reducing or alleviating respiratory inflammation symptoms. Then, these variants also fall within the protection scope of the present invention.

[0021] Similarly, the above heavy chain variable region VH-1 can also have a small number of amino acid deletions, insertions or amino acid mutations on the basis of the above sequence. Variants obtained by conservative amino acid substitutions, especially in the framework region of the variable region, retain the original properties and functions of the heavy chain variable region, that is, retain the antibody properties and functions of specifically binding to coronaviruses, or have the function of treating or preventing respiratory infection symptoms caused by viruses, or have the function of reducing or alleviating respiratory inflammation symptoms. These variants also fall within the protection scope of the present invention.

[0022] Similarly, the above situation also applies to the light chain variable region VL-2 and the heavy chain variable region VH-2, which will not be elaborated here.

[0023] In a specific embodiment of the present invention, the first antigen-binding module is selected from any one of Fv, Fab, Fab’, dsFv or scFv; the second antigen-binding module is selected from any one of Fv, Fab, Fab’, dsFv or scFv.

[0024] In another alternative embodiment of the present invention, the first and second antigen-binding modules are Fab or Fab’ fragments. In still another alternative embodiment of the present invention, one of the first and second antigen-binding modules is a Fab or Fab’ fragment, and the other is Fv, dsFv or scFv.

[0025] Preferably, the first antigen-binding module and the second antigen-binding module are selected to have the structure of single-chain antibody fragment (scFv); specifically, the first antigen-binding module is referred to as single-chain antibody fragment scFv-1; and the second antigen-binding module is referred to as single-chain antibody fragment scFv-2.

[0026] In a specific embodiment of the present invention, scFv-1 sequentially includes the light-chain variable region VL-1, the first linker peptide, and the heavy-chain variable region VH-1 from the N-terminus to the C-terminus; alternatively, scFv-1 sequentially includes the heavy-chain variable region VH-1, the first linker peptide, and the light-chain variable region VL-1 from the N-terminus to the C-terminus;

[0027] scFv-2 sequentially includes the light-chain variable region VL-2, the second linker peptide, and the heavy-chain variable region VH-2 from the N-terminus to the C-terminus; alternatively, scFv-2 sequentially includes the heavy-chain variable region VH-2, the second linker peptide, and the light-chain variable region VL-2 from the N-terminus to the C-terminus.

[0028] Preferably, the first linker peptide or the second linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; wherein, the value of n falls within the range of 1 to 10.

[0029] In a specific embodiment of the present invention, the C-terminus of scFv-1 is connected to the N-terminus of scFv-2 through a linker peptide, and the C-terminus of scFv-2 is connected to a tag sequence; alternatively, the C-terminus of scFv-2 is connected to the N-terminus of scFv-1 through a linker peptide, and the C-terminus of scFv-1 is connected to a tag sequence.

[0030] Preferably, the linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n; wherein, the value of n falls within the range of 1 to 10.

[0031] Preferably, the tag sequence is a small molecule polypeptide tag sequence. The small molecule polypeptide tag sequence is defined as a segment of amino acid sequence with high affinity for the immobilized ligand, and is some polypeptide sequences designed for the purpose of purification and fused with the target protein for expression. The tag sequence can be selected, but is not limited to: glutathione S-transferase GST, poly-histidine ((Poly-His), streptavidin (Strep), FLAG tag, maltose binding protein (MBP).

[0032] In an alternative embodiment of the present invention, the multispecific antibody or its antigen-binding molecule of the present invention may further comprise more antigen-binding modules, which may be the same as or different from the first / second antigen-binding modules, for example, may be antigen-binding modules that bind to other antigens.

[0033] In an alternative embodiment of the present invention, the first / second antigen-binding module is selected from animal-derived antibodies, such as murine antibodies or their immunoglobulins, camel-derived antibodies or their immunoglobulins, humanized antibodies or chimeric antibodies.

[0034] In a specific embodiment of the present invention, the multispecific antibody or its antigen-binding molecule further comprises a constant region; preferably, the constant region is the heavy chain constant region and / or the light chain constant region of human immunoglobulin.

[0035] In an alternative embodiment of the present invention, the heavy chain constant region and / or the light chain constant region may be selected from human immunoglobulin A (IgA), or human immunoglobulin M (IgM), or human immunoglobulin E (IgE), or human immunoglobulin D (IgD).

[0036] In a preferred embodiment of the present invention, the heavy chain constant region is preferably the heavy chain constant region of human IgG1, 2, 3, 4; preferably, the heavy chain constant region is the Fc domain of human IgG1.

[0037] In a preferred embodiment of the present invention, the C-terminus of the scFv-1 is connected to the N-terminus of the scFv-2 through a third linker peptide, and the C-terminus of the scFv-2 is connected to the Fc domain of human IgG1 through a hinge peptide; or,

[0038] The C-terminus of the scFv-2 is connected to the N-terminus of the scFv-1 through a third linker peptide, and the C-terminus of the scFv-1 is connected to the Fc domain of human IgG1 through a hinge peptide; or,

[0039] The C-terminus of the scFv-1 is sequentially connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide, and the C-terminus of the Fc domain of human IgG1 is connected to the N-terminus of scFv-2 through a fifth linker peptide; or,

[0040] The C-terminus of the scFv-2 is sequentially connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide, and the C-terminus of the Fc domain of human IgG1 is connected to the C-terminus of scFv-1 through a fifth linker peptide.

[0041] Preferably, the third linker peptide, or the fourth linker peptide, or the fifth linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n 、(GGGGS) n G、(GGGGS) n GS、GS(GGGGS) n 、GS(GGGGS) n GS、GSGGSG、GSGGSGGGSGGSGGG、GGGGSGGG、(GGGGSGG) n ; wherein, the value of n falls within the range of 1 to 10.

[0042] Preferably, the hinge peptide is EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP) n 、ESKYGPPCPSCP; wherein, the value of n falls within the range of 1 to 10.

[0043] In another alternative embodiment, the hinge peptide can be selected from the heavy chains of different subtypes of human immunoglobulins, for example, it can be selected from the α1 chain, α2 chain, or the γ1 chain, γ2 chain, γ3 chain, γ4 chain, or the δ chain.

[0044] Preferably, the Fc domain of human IgG1 sequentially includes the heavy chain constant region CH2 and the heavy chain constant region CH3 from the N-terminus to the C-terminus; the sequence of the heavy chain constant region CH2 is as shown in SEQ ID NO.17; the sequence of the heavy chain constant region CH3 is as shown in SEQ ID NO.18.

[0045] The second aspect of the present invention provides a homodimer of a multispecific antibody or its antigen-binding molecule, wherein,

[0046] The homodimer of the multispecific antibody or its antigen-binding molecule is: when the above-mentioned multispecific antibody or its antigen-binding molecule is expressed in a host cell, the homodimer formed by the homologous dimerization of the domains of the heavy chain constant region. The homologous dimerization, for example, is the formation of a disulfide bond by the interaction of the amino acids in the hinge region and the domains of the heavy chain constant region.

[0047] The third aspect of the present invention provides a nucleic acid molecule encoding the multispecific antibody or its antigen-binding molecule as described above. It can be a nucleic acid molecule formed by deoxyribonucleic acid or a nucleic acid molecule formed by ribonucleic acid.

[0048] The fourth aspect of the present invention provides a vector containing the above nucleic acid molecule, that is, a vector containing the nucleic acid molecule encoding the above multispecific antibody or its antigen-binding molecule, especially an expression vector for expressing the above multispecific antibody or its antigen-binding molecule.

[0049] Preferably, the vector can be transcribed, translated, and modified to form the above multi-binding specific antibody or its antigen-binding molecule with the functional activity of inhibiting coronavirus infection, or inhibiting respiratory allergic inflammatory reaction or dermatitis inflammatory reaction.

[0050] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can transform, transduce, or transfect a host cell to enable the genetic material elements it carries to be expressed in the host cell. The vector can contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector can also contain an origin of replication. The vector may also include components to assist its entry into the cell, such as virus particles, liposomes, or protein coats, but not limited to these substances. In the embodiments of the present invention, the vector can be selected from, but not limited to: plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosome YAC, bacterial artificial chromosome BAC, or P1-derived artificial chromosome PAC), bacteriophages (such as λ phage or M13 phage), and animal viruses used as vectors, such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40).

[0051] Preferably, the vector is a viral vector; more preferably, the viral vector is an adenovirus, a lentiviral vector or an adeno-associated viral vector (such as AAV8), including recombinant forms thereof. The term "recombinant" means that the composition is manipulated (i.e., engineered) in a manner that does not generally exist in nature. In embodiments of a recombinant adeno-associated viral vector, a nucleic acid sequence encoding the antibody or antigen-binding molecule of the present invention is inserted into the viral genome.

[0052] In a specific embodiment of the present invention, the vector can be used for in vitro production of the multispecific antibody or its antigen-binding molecule of the present invention. For example, the vector containing the antibody gene of the present invention is delivered to a suitable host cell by in vitro infection, and the host cell expresses and secretes the antibody.

[0053] In another specific embodiment of the present invention, the vector can be used as a gene therapy drug. For example, a gene therapy drug delivered by a recombinant virus system (lentivirus system or adeno-associated virus system) or a non-viral vector (such as liposome or other lipid-containing complexes) stably expresses the antibody or its antigen-binding molecule of the present invention in the body (such as in a patient) in a genome-integrated or non-integrated manner for a long time.

[0054] The fifth aspect of the present invention provides a host cell comprising the above vector.

[0055] Regarding the "host cell", it can be selected, but not limited to: prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cell models such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells; or cells in an animal body (such as in a human body) that can express the protein molecule. Preferably, the host cell is HEK293 cell.

[0056] In a preferred embodiment of the present invention, the host cell can be a cell isolated from an animal body and cultured in vitro. After being modified, the cell can express the above-mentioned multi-binding specific antibody or its antigen-binding molecule in a membrane fusion form or a soluble form. Infusing it back into the patient can achieve the function of preventing or treating respiratory infectious diseases caused by viruses including coronaviruses, alleviating or reducing respiratory inflammation (such as rhinitis, asthma, etc.), and alleviating or reducing skin inflammation (such as dermatitis).

[0057] The sixth aspect of the present invention further provides a method for producing the above-mentioned multispecific antibody or its antigen-binding molecule, wherein the above-mentioned multispecific antibody or its antigen-binding molecule is produced by culturing a host cell containing a nucleic acid molecule encoding the above-mentioned multispecific antibody or its antigen-binding molecule.

[0058] The above-mentioned multispecific antibody or its antigen-binding molecule of the present invention can be produced by the above-mentioned recombinant method, or by the method of stable expression cell line, or by the method of hybridoma.

[0059] The seventh aspect of the present invention provides a method for producing a homodimer of the above-mentioned multispecific antibody or its antigen-binding molecule. Culturing the above-mentioned host cell, when the above-mentioned multispecific antibody or its antigen-binding molecule is expressed in the host cell, the domains of the heavy chain constant region undergo homodimerization to produce the homodimer of the above-mentioned multispecific antibody or its antigen-binding molecule.

[0060] Other aspects of the present invention also provide glycosylation variants of the above-mentioned multispecific antibody or its antigen-binding molecule, antibody variants engineered with cysteine or the like, antibody derivatives, immunoconjugates, and the like.

[0061] The eighth aspect of the present invention provides a recombinant protein, which comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned homodimer.

[0062] The ninth aspect of the present invention provides an immunoconjugate, which comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned homodimer.

[0063] Preferably, the conjugated part of the immunoconjugate uses one or more heterologous molecules, such as heterologous molecules with cytotoxicity that can be applied to immunoconjugates.

[0064] The tenth aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned homodimer, or comprises the above-mentioned nucleic acid molecule, or comprises the above-mentioned vector, or comprises the above-mentioned host cell, or comprises the above-mentioned recombinant protein, or comprises the above-mentioned immunoconjugate, and a pharmaceutically acceptable carrier.

[0065] In a specific embodiment of the present invention, the pharmaceutical composition can adopt known dosage forms, such as pre-filled syringes, auto-injectors, pre-filled pens; preferably, the dosage form is usually administered by injection, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion; more preferably, the pharmaceutical composition is administered by non-conventional routes, such as local, epidermal or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual or topical administration; more preferably, the pharmaceutical composition adopts nasal spray dosage form, nasal drop dosage form, nebulized inhalation dosage form, nasal lavage fluid dosage form, oral liquid dosage form, mouthwash dosage form.

[0066] In a specific embodiment of the present invention, the pharmaceutical composition is in the form of tablets, capsules or powders.

[0067] In a specific embodiment of the present invention, the pharmaceutical composition is in the form of external ointment or emulsion dosage form, external gel dosage form, external lotion or external spray.

[0068] The eleventh aspect of the present invention provides the use of the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned homodimer, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned recombinant protein, or the above-mentioned immunoconjugate in the preparation of a medicament for treating or preventing a respiratory infection disease caused by a virus.

[0069] In a specific embodiment of the present invention, the virus refers to a virus that can cause respiratory infections, such as known influenza virus, human parainfluenza virus, respiratory syncytial virus, rhinovirus, human metapneumovirus, human bocavirus, adenovirus, rubella virus, measles virus, Nipah virus, mumps virus, varicella-zoster virus, Coxsackie virus, or rotavirus, etc.

[0070] In a preferred embodiment of the present invention, the use refers to the use in the preparation of a medicament for treating or preventing a respiratory infection disease caused by a coronavirus. More preferably, the coronavirus includes SARS-CoV-2 and its mutant strains, SARS-CoV or SARS-like coronaviruses, etc. In a more preferred embodiment of the present invention, the SARS-CoV-2 mutant strain is an Alpha, Beta, Gamma, Delta or Omicron subtype mutant strain (such as BA.1, BA.2, BA.5, BF.7, BQ.1.1, XBB, XBB.1.5, XBB.1.16, EG.5, BA.2.86, JN.1, etc.).

[0071] The twelfth aspect of the present invention provides the use of the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned homodimer, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned recombinant protein, or the above-mentioned immunoconjugate in the preparation of a medicament for treating or preventing respiratory inflammation or dermatitis; preferably, the respiratory inflammation is rhinitis or asthma. Preferably, the dermatitis is atopic dermatitis.

[0072] Rhinitis refers to an inflammatory disease of the nasal mucosa, including chronic rhinitis, allergic rhinitis, acute rhinitis, etc. Its clinical manifestations include runny nose accompanied by symptoms such as olfactory loss, sneezing, nasal congestion, obstructive nasal voice, nasal dryness and itching, headache, wheezing, and dyspnea.

[0073] Asthma is a disease characterized by chronic airway inflammation, which leads to a significant increase in the airway's reactivity to various stimuli (i.e., airway hyperresponsiveness). Patients usually experience recurrent symptoms such as wheezing, shortness of breath, dyspnea, chest tightness, and coughing, especially aggravated at night or in the early morning. Chronic inflammation may also lead to airway remodeling and functional impairment, further exacerbating the condition and affecting the quality of life.

[0074] Dermatitis is a general term for skin inflammatory disorders caused by various internal and external infectious or non-infectious factors, and it is not an independent disease; the causes of dermatitis are diverse. For example, contact dermatitis caused by external factors is due to the skin contacting certain substances, such as chemical substances, plants (such as poison ivy), metals (such as nickel), etc.; for example, atopic dermatitis, which is caused by internal factors, is a chronic, recurrent inflammatory skin disease related to genetics and is often accompanied by other allergic diseases, such as allergic asthma and allergic rhinitis; for example, neurodermatitis caused by long-term scratching, etc.

[0075] The thirteenth aspect of the present invention provides a detection product, wherein the detection product comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned homodimer, or comprises the above-mentioned nucleic acid molecule, or comprises the above-mentioned vector, or comprises the above-mentioned host cell, or comprises the above-mentioned recombinant protein, or comprises the above-mentioned immunoconjugate.

[0076] The detection product is used to detect the presence or level of coronavirus in a sample.

[0077] In a specific embodiment of the present invention, the detection product includes, but is not limited to, detection reagents, detection kits, detection chips, test strips, detection instruments, etc.

[0078] The above-mentioned multispecific antibody or its antigen-binding molecule of the present invention can be labeled by chemical methods or genetic engineering methods, and the labeled antibody or its antigen-binding molecule can be used for detection; the labeled antibody or its antigen-binding molecule falls within the protection scope of the present invention.

[0079] Specific detection methods can adopt the following steps: 1) Provide a sample; 2) Contact the sample with the above-mentioned multispecific antibody or its antigen-binding molecule of the present invention; 3) Detect the immune reaction between the sample and the antibody or antigen-binding molecule.

[0080] On the other hand, the present invention also provides a method for treating or preventing respiratory infectious diseases caused by viruses, or a method for preparing a treatment or prevention of respiratory inflammation, a method for preparing a treatment or prevention of dermatitis, by administering to a patient a therapeutically effective amount of the above-mentioned multispecific antibody or its antigen-binding molecule, or its homodimer; or by administering to a patient a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned multispecific antibody or its antigen-binding molecule, or its homodimer. Description of the Drawings

[0081] Figure 1 It is the plasmid map of the pcDNA3.4-Fc expression vector used in the preparation of the antibodies in Example 1 and Example 2 of the present invention;

[0082] Figure 2 It is the plasmid map of the pcDNA3.4 expression vector used in the preparation of the antibodies in Example 3 and Example 4 of the present invention;

[0083] Figure 3 It is the SDS-PAGE diagram of Examples 1-4 of the present invention;

[0084] Figure 4 It is the operation flow chart for detecting the treatment and prevention of COVID-19 virus infection by the antibodies in Example of the present invention in mice;

[0085] Figure 5 It is the result of the treatment and prevention of COVID-19 virus XBB.1 infection by the antibody in Example 1 of the present invention in human ACE2 transgenic mice;

[0086] Figure 6 It is the result of the treatment and prevention of COVID-19 virus EG.5 infection by the antibody in Example 1 of the present invention in human ACE2 transgenic mice;

[0087] Figure 7 It is the experimental flow chart of the rhinitis mouse model establishment process and the treatment of rhinitis mice with the antibodies in Example of the present invention;

[0088] Figure 8 It is the weight change and weight change trend chart of mice during the treatment of rhinitis mice with the antibody in Example 1 of the present invention;

[0089] Figure 9 Statistical results of the number of sneezes in mice during the treatment of rhinitis mice with the antibody of Example 1 of the present invention;

[0090] Figure 10 Concentration of OVA-sIgE in serum 8 days after treating rhinitis mice with the antibody of Example 1 of the present invention;

[0091] Figure 11 Concentration of histamine in serum 8 days after treating rhinitis mice with the antibody of Example 1 of the present invention;

[0092] Figure 12 Pathological HE staining of nasal mucosa 8 days after treating rhinitis mice with the antibody of Example 1 of the present invention;

[0093] Figure 13 Experimental flowchart of asthma mouse model establishment process and treatment of asthma mice with the antibody of the present invention's Example;

[0094] Figure 14 Graph of body weight change and body weight change trend of mice during the treatment of asthma mice with the antibody of Example 1 of the present invention;

[0095] Figure 15 Statistical results of allergic asthma score of mice during the treatment of asthma mice with the antibody of Example 1 of the present invention;

[0096] Figure 16 Concentration of OVA-sIgE in bronchoalveolar lavage fluid and serum 7 days after treating asthma mice with the antibody of Example 1 of the present invention;

[0097] Figure 17 Concentration of IL-4 in bronchoalveolar lavage fluid and serum 7 days after treating asthma mice with the antibody of Example 1 of the present invention;

[0098] Figure 18 Concentration of IL-5 in bronchoalveolar lavage fluid and serum 7 days after treating asthma mice with the antibody of Example 1 of the present invention;

[0099] Figure 19 Pathological HE staining of lungs 7 days after treating asthma mice with the antibody of Example 1 of the present invention;

[0100] Figure 20 Experimental flowchart of dermatitis mouse model establishment process and treatment of asthma mice with the antibody of Example 1 of the present invention;

[0101] Figure 21 Graph of body weight change and body weight change trend of mice during the treatment of dermatitis mice with the antibody of Example 1 of the present invention;

[0102] Figure 22 The spleen mass coefficient of the mice after 20 days of treatment with the antibody of Example 1 of the present invention for dermatitis mice;

[0103] Figure 23 The trend chart of the skin lesion score of the mice during the treatment of dermatitis mice with the antibody of Example 1 of the present invention;

[0104] Figure 24 The skin photo of the mice after 20 days of treatment with the antibody of Example 1 of the present invention for dermatitis mice;

[0105] Figure 25 The concentration of TNF-α in the serum after 20 days of treatment with the antibody of Example 1 of the present invention for dermatitis mice;

[0106] Figure 26 The concentration of IL-1β in the serum after 20 days of treatment with the antibody of Example 1 of the present invention for dermatitis mice. Detailed implementation manners

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the present invention will be apparent from this specification, the drawings, and the appended claims.

[0108] To explain this specification, the following definitions will be used, and where appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0109] The term "about", when used in conjunction with a numerical value, means to cover a numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0110] As used herein, the term "comprising" or "including" means including the stated elements, integers, or steps, but not excluding any other elements, integers, or steps.

[0111] The term "antibody" is used herein in the broadest sense to cover natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antigen-binding molecules, antigen-binding fragments, antigen-binding proteins, fusion proteins, recombinant proteins, etc. that exhibit the desired antigen-binding activity.

[0112] The terms "antigen-binding molecule" and "antibody-binding fragment" are used interchangeably herein and refer to a molecule that is not a full antibody and is a specific antigen-binding portion of a full antibody. Antigen-binding molecules can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of full antibodies.

[0113] The term "bispecific" antibody refers to an antibody having at least two antigen-binding sites / antigen-binding modules, each of the at least two antigen-binding sites / antigen-binding modules binding to a different epitope of the same antigen or to different epitopes of different antigens.

[0114] The terms "antigen-binding module" and "antigen-binding site" denote the region in an antibody molecule that actually binds to an antigen and include, for example, a VH / VL pair consisting of the variable domain of the antibody light chain (VL) and the variable domain of the antibody heavy chain (VH). In some embodiments of the present invention, bispecific antibodies are provided that have two antigen-binding sites / antigen-binding modules capable of binding to two different epitopes of a coronavirus.

[0115] In some embodiments of the present invention, the antigen-binding module is selected from any one of Fv, Fab, Fab’, dsFv or scFv.

[0116] The Fab fragment is a monovalent fragment composed of VL, VH, CL, and CH1 domains. For example, the Fab fragment can be obtained by papain digestion of a whole antibody. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, edited by W.E. Paul, Raven Press, N.Y. (1993)). F(ab')2 is obtained by pepsin digestion of the whole IgG antibody, removing most of the Fc region while retaining some of the hinge region, and it has two antigen-binding F(ab) parts linked together by disulfide bonds; the F(ab')2 fragment is a dimer of Fab’ and is a bivalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. The Fv fragment consists of the VL and VH domains of a single arm of the antibody. Additionally, although the two domains VL and VH of the Fv fragment are encoded by separate genes, using recombinant methods, they can be linked by a synthetic linker that enables these two domains to be produced as a single protein chain, in which the VL region and the VH region pair to form a single-chain Fv (scFv, also known as a single-chain antibody). The dsFv, which refers to a disulfide-stabilized Fv, is a type of novel small molecule antibody developed on the basis of scFv. It is an antibody in which one amino acid residue each of VH and VL is mutated to cysteine, and the VH and VL variable regions are linked by an interchain disulfide bond, which can enhance the stability of the Fv. The above antigen-binding modules can all be obtained by chemical methods, recombinant DNA methods, or protease digestion methods.

[0117] Regarding the combination modes of the two antigen-binding modules of bispecific antibodies, according to the different structures of the antigen-binding modules, they mainly include Fab-Fab combination, Fab-Fv combination, and Fv-Fv combination.

[0118] Among them, the Fab-Fab combination bispecific antibodies mainly include: Triomab produced by the hybrid hybridomas technology of rat and mouse hybridoma cells, various bsIgGs produced by using various Fc heterodimer technologies (such as Knob-in-Hole, charge pairing, SEED, BEAT, LUZ-Y, and Duobody, etc.) and avoiding Fab mismatch technologies (such as CrossMab, shared light chain, single-chain Fab, κλ-body, Orthogonal Fab, Duetmab, and TCR-CαCβ, etc.), IgG-like molecules produced by various Fab tandem methods (such as Tandem orthogonal Fab-IgG, FIT-IgG, and BiXAb, etc.), IgG-IgG produced by using chemical cross-linking technology, and Fab-linked molecules produced by various Fab cross-linking technologies (such as F(ab')2, Dockand Lock, etc.). While combining two antigen recognition and binding domains, Fab-Fab bispecific antibodies can completely retain the high affinity of the original antibody with Fab, and the structure is closer to natural IgG, with relatively high stability. However, in order to avoid the mismatch of heavy and light chains between two different Fabs, a common light chain needs to be used or mutations need to be introduced to form a preferential pairing.

[0119] In the Fab-Fv combination bispecific antibodies, one of the binding domains that recognize antigens or epitopes is Fab, and the other is Fv. Broadly defined, Fv can include single-chain variable region antibodies (scFv), engineered polypeptides or protein domains with specific recognition functions (such as Anticalins, Bicyclic peptides, DARPins, Fynomers, etc.), ligand or receptor molecules, and engineered ligand or receptor molecules, etc. Representative structures include: scFv-Fab, scFv-IgG, DVD-IgG, Fab-scFv-Fc, etc.

[0120] In the Fv-Fv combination bispecific antibodies, both of the binding domains that recognize antigens or epitopes are Fv. Representative structures include: BiTE, Diabody, DART, TandAb, scFv-scFv-Fc, etc. This combination method is very flexible and can conveniently construct multivalent and multispecific binding molecules.

[0121] For the bispecific antibodies with the above different combination methods, the structural stability may vary, but the affinity for antigens / epitopes and the neutralization ability against viruses mainly depend on the "antigen binding module / site" of the bispecific antibodies; in other words, the bispecific antibodies with the "antigen binding module / site" determined can exist in any of the above-known combination methods.

[0122] In a specific embodiment of the present invention, a bispecific antibody solution of scFv-scFv-Fc is provided; after those skilled in the art learn the bispecific antibody solution of the present invention and the sequences of its antigen-binding modules, they can use existing bispecific antibody technologies to transform it into any of the above-known combination methods, such as Fab-Fab combination, Fab-Fv combination or other Fv-Fv combination bispecific antibodies.

[0123] In a specific embodiment of the present invention, the antigen-binding module is scFv, which comprises a heavy-chain variable region (VH region) and a light-chain variable region (VL region).

[0124] The heavy-chain variable region (VH region) and the light-chain variable region (VL region) can be further divided into complementary determining regions (CDRs) and framework regions (FRs); the CDRs are hypervariable regions, and the relatively conserved FR regions are interposed therebetween. Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In a given VH or VL amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known schemes, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London), the international ImMunoGeneTics database (IMGT) (https: / / www.imgt.org / ). The CDRs of the antibodies of the present invention can be determined according to any scheme or a combination thereof in the art and human evaluation.

[0125] The term "Fc domain" or "Fc region" is used herein to define the carboxy-terminal region of the human immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains, namely the CH2 domain, the CH3 domain, and optionally the CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from antibodies of the IgG, IgA, and IgD classes; or contains the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from antibodies of the IgM and IgE classes. Unless otherwise specified herein, the amino acid residue numbering in the Fc region or the heavy chain constant region is numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0126] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants for enhanced efficacy. For example, mutating methionine at position 428 of the Fc domain to leucine and asparagine at position 434 of the Fc domain to serine can prolong the half-life of the antibody in vivo. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fcs. Modification of the Fc can alter the half-life of the antibody in a therapeutic antibody, thus enabling less frequent dosing and hence increased convenience and reduced material use. Alteration of the Fc can also enhance the activity of the antibody in vivo, exert the effector functions of the Fc, and achieve multi-pathway multifunctional activity for more effective and rapid disease remission.

[0127] The term "linker peptide" refers to a connecting peptide segment composed of amino acids, such as glycine and / or serine residues used alone or in combination, to link the respective variable domains in an antibody. In certain embodiments, the linker peptide can be about 1 to about 100 amino acids in length, for example, about 1 to 50 amino acids in length. In one embodiment, the linker peptide is a G / S linker peptide, non-limiting examples of which are disclosed in the literature (Shen et al., Anal. Chem. 80(6): 1910 - 1917 (2008)) and the patent (WO2014 / 087010), the contents of which are incorporated herein by reference in their entirety.

[0128] As used herein, the terms "bind" or "specifically bind" mean that the binding interaction is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding module / site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0129] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between the members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant (kdis) and the association rate constant (kon). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the biolayer interferometry technique to detect the association and dissociation rates between two molecules.

[0130] The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA.

[0131] An "immunoconjugate" is a product of an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0132] The percentage of "sequence homology" with respect to an amino acid sequence is generated by determining the number of amino acid residues present in two sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence homology. The optimal alignment for determining the percentage of sequence homology can be achieved in a variety of ways known in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment within the full-length sequences being compared or within the target sequence region.

[0133] In the present invention, with respect to antibody sequences, the percentage of amino acid sequence homology is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred embodiment, by optimal alignment according to the Kabat numbering rules. In some embodiments, with respect to antibodies, sequence homology can be distributed over the entire heavy chain variable region and / or the entire light chain variable region, or the percentage of sequence homology can be limited only to the framework regions, while the sequences of the corresponding CDR regions remain 100% identical.

[0134] Similarly, with respect to antibody sequences, based on the alignment, candidate antibodies having amino acid changes in the target antibody region relative to a reference antibody can be identified.

[0135] In the present invention, "conservative substitution" refers to an amino acid change that results in the replacement of one amino acid with a chemically similar amino acid. Amino acid modifications such as substitutions can be introduced into the antibodies of the present invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0136] Tables of conservative substitutions providing functionally similar amino acids are well known in the art. In a preferred aspect, the conservative substitution residues are from the following table of conservative substitutions, preferably the preferred conservative substitution residues shown in the following table.

[0137] Table of conservative substitutions

[0138]

[0139]

[0140] The term "N-terminus" refers to the amino terminus of the protein or peptide segment, and the term "C-terminus" refers to the carboxyl terminus of the protein or peptide segment.

[0141] The antibody CDR region, "complementary determining region" or "CDR region" or "CDR" (which can be used interchangeably with "hypervariable region", "HVR" herein), is the amino acid region in the variable region of the antibody that is mainly responsible for binding to the antigen epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domain of the antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0142] A variety of schemes are known in the art for determining the CDR sequences within a given VH or VL amino acid sequence. For example, the Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs are a compromise between Kabat HVRs and Chothia structural loops and are used by the AbM antibody modeling software of Oxford Molecular. The "Contact" HVRs are based on the analysis of available complex crystal structures.

[0143] Unless otherwise specified, in the present invention, when referring to residue positions in the antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it refers to the numbered positions according to the Kabat numbering system.

[0144] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although the CDRs are different between antibodies, only a limited number of amino acid positions within the CDRs directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, a minimal overlapping region can be determined, thus providing a "minimal binding unit" for antigen binding. The minimal binding unit can be a subpart of a CDR. As will be apparent to those skilled in the art, through the structure and protein folding of the antibody, the residues of the remaining part of the CDR sequence can be determined. Accordingly, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimal binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0145] The "hinge peptide" or "hinge region" generally refers to amino acids Glu216 to Pro230 of human IgG1 (see Burton, Molec. Immunol. 22:161-206 (1986)). In certain embodiments, the hinge regions of other immunoglobulin heavy chains can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the inter-heavy chain S-S bonds in the same positions.

[0146] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. A host cell is any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0147] The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates like monkeys), rabbits, and rodents (such as mice and rats). In particular, the individual is a human.

[0148] The term "coronavirus" refers to a single-stranded RNA virus with spherical virus particles having an envelope structure. According to the classification of coronaviruses by the International Committee on Taxonomy of Viruses, it can be divided into the family Coronaviridae and the subfamily Letovirinae, and the family Coronaviridae can be further divided into four different coronavirus genera, namely alpha, beta, gamma, and delta. The novel coronavirus belongs to the beta coronavirus and can cause acute respiratory infections. The clinical manifestations of the infection are mainly fever, fatigue, and dry cough, and a small number are accompanied by symptoms such as nasal congestion, runny nose, sore throat, nausea, and diarrhea. Severe patients often have difficulty breathing, septic shock, coagulation dysfunction, multiple organ failure, etc.

[0149] The term "S protein" refers to one of the structural proteins encoded by the coronavirus genome, a spike protein protruding on the surface of the virus particle. The S protein forms a special crown structure on the virus surface in the form of a trimer. The function of the S protein is to be responsible for the virus to bind to the receptor of the host cell, mediate the fusion of the virus membrane and the receptor membrane, and can cause a targeted immune response in the body to produce neutralizing antibodies, and it is an important protein for virus infection.

[0150] Example 1

[0151] The antibody of Example 1 comprises a first antigen-binding module and a second antigen-binding module, which are the single-chain antibody fragments scFv-1 and scFv-2, respectively.

[0152] In a specific embodiment of the present invention, the light chain variable region VL-1 of scFv-1 comprises the LCDR1-1 sequence of the light chain variable region shown in SEQ ID NO.1, the LCDR2-1 sequence of the light chain variable region shown in SEQ ID NO.2, and the LCDR3-1 sequence of the light chain variable region shown in SEQ ID NO.3; the heavy chain variable region VH-1 of scFv-1 comprises the HCDR1-1 sequence of the heavy chain variable region shown in SEQ ID NO.4, the HCDR2-1 sequence of the heavy chain variable region shown in SEQ ID NO.5, and the HCDR3-1 sequence of the heavy chain variable region shown in SEQ ID NO.6.

[0153] In a preferred embodiment of the present invention, the sequence of the light chain variable region VL-1 of the scFv-1 is as shown in SEQ ID NO.13, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.13; the sequence of the heavy chain variable region VH-1 of the scFv-1 is as shown in SEQ ID NO.14, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.14.

[0154] Specifically in this embodiment, the sequence of the light chain variable region VL-1 of the scFv-1 is as shown in SEQ ID NO.13, and the sequence of the heavy chain variable region VH-1 of the scFv-1 is as shown in SEQ ID NO.14.

[0155] Specifically, according to the latest research progress of the inventors of the present application, the variable region of scFv-1 binds to the outside of the receptor binding motif (RBM), that is, the binding epitope of scFv-1 is not located in the RBM of the RBD of the S1 protein, but outside the motif.

[0156] In a specific embodiment of the present invention, the light chain variable region VL-2 of the scFv-2 comprises the LCDR1-2 sequence of the light chain variable region shown in SEQ ID NO.7, the LCDR2-2 sequence of the light chain variable region shown in SEQ ID NO.8, and the LCDR3-2 sequence of the light chain variable region shown in SEQ ID NO.9; the heavy chain variable region VH-2 of the scFv-2 comprises the HCDR1-2 sequence of the heavy chain variable region shown in SEQ ID NO.10, the HCDR2-2 sequence of the heavy chain variable region shown in SEQ ID NO.11, and the HCDR3-2 sequence of the heavy chain variable region shown in SEQ ID NO.12.

[0157] In a preferred embodiment of the present invention, the sequence of the light chain variable region VL-2 of scFv-2 is as shown in SEQ ID NO.15, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.15; the sequence of the heavy chain variable region VH-2 of scFv-2 is as shown in SEQ ID NO.16, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.16.

[0158] Specifically in this embodiment, the sequence of the light chain variable region VL-2 of scFv-2 is as shown in SEQ ID NO.15; the sequence of the heavy chain variable region VH-2 of scFv-2 is as shown in SEQ ID NO.16.

[0159] Specifically, the variable region of scFv-2 binds to the receptor binding motif (RBM) part of the RBD, and the binding epitope of scFv-2 is located in the RBM of the RBD of the S1 protein.

[0160] In a specific embodiment of the present invention, scFv-1 sequentially includes a light chain variable region VL-1, a first linker peptide, and a heavy chain variable region VH-1 from the N-terminus to the C-terminus; or scFv-1 sequentially includes a heavy chain variable region VH-1, a first linker peptide, and a light chain variable region VL-1 from the N-terminus to the C-terminus.

[0161] Similarly, in another specific embodiment of the present invention, scFv-2 sequentially includes the light chain variable region VL-2, a second linker peptide, and a heavy chain variable region VH-2 from the N-terminus to the C-terminus; or scFv-2 sequentially includes a heavy chain variable region VH-2, a second linker peptide, and a light chain variable region VL-2 from the N-terminus to the C-terminus.

[0162] The first linker peptide (L1) and the second linker peptide (L2), also known as the linker peptide or linker. In a specific embodiment of the present invention, the sequence of the first linker peptide or the second linker peptide is selected from linker sequences containing glycine (G) and serine (S) residues; preferably, the length of the first linker peptide or the second linker peptide is each about 1 to about 50 amino acids; preferably about 15 to about 25 amino acids; more preferably, the first linker peptide or the second linker peptide is respectively selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n; wherein, the value of n falls within the range of 1 to 10.

[0163] Specifically in this embodiment, the sequences of the first linker peptide (L1) and the second linker peptide (L2) are both (GGGGS)3.

[0164] In a specific embodiment of the present application, the C-terminus of scFv-1 is connected to the N-terminus of the scFv-2 through a third linker peptide. In another specific embodiment of the present application, it can also be that the C-terminus of scFv-2 is connected to the N-terminus of the scFv-1 through a third linker peptide.

[0165] In a specific embodiment of the present invention, the sequence of the third linker peptide (L3) is selected from linker sequences containing glycine (G) and serine (S) residues; preferably, the lengths of the third linker peptides (L3) are each about 1 to about 50 amino acids; preferably about 15 to about 25 amino acids; more preferably, the third linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; wherein, the value of n falls within the range of 1 to 10.

[0166] Specifically in this embodiment, the sequence of the third linker peptide (L3) is GS(GGGGS)4.

[0167] That is, the antigen-binding part (antigen-binding molecule) of the bispecific antibody in Example 1 is, from the N-terminus to the C-terminus: scFv-1 - L3 - scFv-2; more specifically, the antigen-binding part of the bispecific antibody in Example 1 is, from the N-terminus to the C-terminus: VL-1 - L1 - VH-1 - L3 - VL-2 - L2 - VH-2.

[0168] Of course, in other embodiments of the present application, the structure of the antigen-binding part of the bispecific antibody can also be VH-1 - L1 - VL-1 - L3 - VL-2 - L2 - VH-2, VL-1 - L1 - VH-1 - L3 - VH-2 - L2 - VL-2 or VH-1 - L1 - VL-1 - L3 - VH-2 - L2 - VL-2.

[0169] In other embodiments of the present application, the structure of the antigen-binding portion of the bispecific antibody can also be VL-2—L2—VH-2—L3—VL-1—L1—VH-1, VH-2—L2—VL-2—L3—VL-1—L1—VH-1, VL-2—L2—VH-2—L3—VH-1—L1—VL-1, or VH-2—L2—VL-2—L3—VH-1—L1—VL-1. Or constructs formed by arranging the combinations of VL1 and VH1 or VL2 and VH2 in different orders.

[0170] In a specific embodiment of the present application, the C-terminus of the antigen-binding portion / antigen-binding molecule of the present invention can be linked to a tag sequence. The tag sequence is a small peptide purification tag sequence; the small peptide tag sequence is defined as a segment of amino acid sequence that has a high affinity for the immobilized ligand and is some peptide sequences designed to be fused with the target protein for purification purposes. More preferably, the tag sequence is a polyhistidine (His) tag sequence.

[0171] In a specific embodiment of the present application, the bispecific antibody further comprises a heavy chain constant region; the heavy chain constant region is preferably the heavy chain constant region of human IgG1, 2, 3, or 4. Preferably, the heavy chain constant region is the Fc domain of human IgG1.

[0172] In a specific embodiment of the present application, the C-terminus of scFv-1 is linked to the N-terminus of scFv-2 through a third linker peptide (L3), and the C-terminus of scFv-2 is linked to the Fc domain of human IgG1 through a hinge peptide (Hinge). In another specific embodiment of the present application, it can also be that the C-terminus of scFv-2 is linked to the N-terminus of scFv-1 through a third linker peptide (L3), and the C-terminus of scFv-1 is linked to the Fc domain of human IgG1 through a hinge peptide (Hinge).

[0173] In a preferred embodiment of the present application, the hinge peptide sequence is EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP) n 、ESKYGPPCPSCP; wherein, the value of n can be 1 or more than 1.

[0174] In another preferred embodiment of the present application, the hinge peptide sequence can be selected from the heavy chains of different subtypes of human immunoglobulins, for example, it can be selected from the α1 chain, α2 chain, or the γ1 chain, γ2 chain, γ3 chain, γ4 chain, or δ chain.

[0175] In this embodiment, the sequence of the bispecific antibody is, from the N-terminus to the C-terminus: scFv-1—L3—scFv-2—Hinge—Fc.

[0176] In a specific embodiment of the present application, the Fc domain of human IgG1 contains the heavy chain constant region CH2 and the heavy chain constant region CH3 in sequence from the N-terminus to the C-terminus;

[0177] That is, the sequence of the bispecific antibody in Example 1 is, from the N-terminus to the C-terminus:

[0178] VL-1—L1—VH-1—L3—VL-2—L2—VH-2—Hinge—CH2—CH3.

[0179] In this embodiment, the sequence of the heavy chain constant region CH2 is as shown in SEQ ID NO.17; the sequence of the heavy chain constant region CH3 is as shown in SEQ ID NO.18; the sequence of the hinge peptide Hinge is as shown in SEQ ID NO.19.

[0180] Example 2

[0181] The sequence structure of the antibody in Example 2 is, from the N-terminus to the C-terminus:

[0182] scFv-2—L3—scFv-1—Hinge—CH2—CH3.

[0183] The sequences of scFv-1, scFv-2, L3, Hinge, CH2, and CH3 are the same as those in Example 1 above.

[0184] Examples 3 and 4

[0185] In a specific embodiment of the present invention, the C-terminus of scFv-1 is sequentially connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide, and the C-terminus of the Fc domain of human IgG1 is connected to the N-terminus of scFv-2 through a fifth linker peptide.

[0186] In another specific embodiment of the present invention, the C-terminus of the scFv-2 is sequentially connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide, and the C-terminus of the Fc domain of human IgG1 is connected to the C-terminus of scFv-1 through a fifth linker peptide.

[0187] In a specific embodiment of the present invention, the sequence of the fourth linker peptide (L4) or the fifth linker peptide (L5) is selected from linker sequences comprising glycine (G) and serine (S) residues; preferably, the length of the fourth linker peptide (L4) or the fifth linker peptide (L5) is each about 1 to about 50 amino acids; preferably about 15 to about 25 amino acids; more preferably, the fourth linker peptide or the fifth linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; wherein, the value of n falls within the range of 1 to 10.

[0188] Specifically in Example 3 and Example 4, the amino acid sequence of the fourth linker peptide (L4) is GS, and the amino acid sequence of the fifth linker peptide (L5) is GGS.

[0189] The sequence structure of the antibody in Example 3 is, from the N-terminus to the C-terminus in sequence:

[0190] scFv-1—L4—Hinge—CH2—CH3—L5—scFv-2.

[0191] The sequence structure of the antibody in Example 4 is, from the N-terminus to the C-terminus in sequence:

[0192] scFv-2—L4—Hinge—CH2—CH3—L5—scFv-1.

[0193] Wherein, the sequences of scFv-1, scFv-2, Hinge, CH2, and CH3 are the same as those in Example 1 above.

[0194] Preparation of the antibodies in Examples 1-4

[0195] Step 1) Construct the antibody expression vector pcDNA3.4-Fc containing the Fc gene fragment

[0196] Synthesize the gene fragments of the fully human IgG1 signal peptide gene SP, hinge peptide Hinge, heavy chain constant region CH2, and heavy chain constant region CH3 through GenScript Corporation, namely the SP-Fc gene. Connect them to the pcDNA3.4 vector by the well-known TA cloning method in the art, and finally obtain the pcDNA3.4-Fc expression vector, wherein the pcDNA3.4 vector is purchased from Thermo Fisher Scientific Inc., and its specific plasmid map is as Figure 1 shown.

[0197] Step 2) Synthesize the antibody gene sequence

[0198] As described above, the light chain variable region VL-1 of the single-chain antibody fragment scFv-1 in Example 1 is shown in SEQ ID NO.13, and the heavy chain variable region VH-1 is shown in SEQ ID NO.14; the sequence of the first linker peptide (L1) between the two is (GGGGS)3.

[0199] Correspondingly, the nucleic acid sequence encoding scFv-1 is shown in SEQ ID NO.20;

[0200] The light chain variable region VL-2 of the single-chain antibody fragment scFv-2 in Example 1 is shown in SEQ ID NO.15, and the heavy chain variable region VH-2 is shown in SEQ ID NO.16; the sequence of the second linker peptide (L2) between the two is (GGGGS)3.

[0201] Correspondingly, the nucleic acid sequence encoding scFv-2 is shown in SEQ ID NO.21.

[0202] In Example 1, the nucleotide sequence corresponding to scFv-1—L3—scFv-2 was synthesized by a conventional method.

[0203] In Example 2, the nucleotide sequence corresponding to scFv-2—L3—scFv-1 was synthesized by a conventional method.

[0204] In Example 3, the nucleotide sequences corresponding to scFv-1—L4—Hinge—Fc—L5—scFv-2 were synthesized by a conventional method respectively.

[0205] In Example 4, the nucleotide sequences corresponding to scFv-2—L4—Hinge—Fc—L5—scFv-1 were synthesized by a conventional method respectively.

[0206] Step 3) Construct an expression vector for the antibody gene

[0207] The single-chain antibody scFv-1—L3—scFv-2 of Example 1 or the single-chain antibody scFv-2—L3—scFv-1 of Example 2 synthesized in the above Step 2 was ligated into the pcDNA3.4-Fc expression vector constructed in the above Step 1 by TA cloning. The nucleotide sequences of the antibody scFv-1—L4—Hinge—Fc—L5—scFv-2 of Example 3 or the antibody scFv-2—L4—Hinge—Fc—L5—scFv-1 of Example 4 were ligated into the pcDNA3.4 expression vector containing the fully human IgG1 signal peptide gene by TA cloning ( Figure 2) and transform DH5α competent cells to construct the expression plasmid of the final bispecific antibody. After cloning, sequence information was confirmed by sequencing.

[0208] Step 4) Expression of bispecific antibody in mammalian cell 293F

[0209] The expression plasmid of the bispecific antibody was purified by a plasmid purification kit (Meiji Bio) and co-transfected into HEK293F cells for expression using EZ Trans cell transfection reagent (Li Ji Bio).

[0210] The specific transfection steps are as follows: One day before transfection, 50 ml of 293F cells were seeded in a 250 mL cell culture shake flask at a density of 1.2×10 6 cells / ml. On the day of transfection, the transfection reagent EZ-Trans was fully mixed with the expression plasmid constructed in step 3) (mass-volume ratio of DNA:EZ-Trans = 1:3) and dissolved in serum-free OPM medium to obtain an EN-Trans mixture (i.e., 60 μg DNA and 180 μL EZ-Trans dissolved in 4 mL medium). After standing for 15 minutes, the EZ-Trans-DNA mixture was uniformly added to HEK293F cells in a droplet shape. Six days after transfection, the cell culture supernatant was centrifuged to obtain the supernatant for subsequent extraction and purification steps of the bispecific antibody. (Note: When the antibody is expressed in host cells, it is secreted as a protein, and the Fc domain of human IgG1 will undergo homodimerization to form a homodimer, which is secreted in the cell culture supernatant; after subsequent extraction and purification, it is identified.)

[0211] Step 5) Extraction and purification of the antibody

[0212] The cell supernatant collected in the above step 4) was filtered through a 0.45 μM filter membrane, and the supernatant was diluted with binding buffer 1×PBS. The bispecific antibody containing IgG1 Fc in the supernatant was purified using a protein-G column (Tian Di Ren He Biotechnology Co., Ltd., Changzhou), and the purification method was referred to the instruction manual of the protein-G column. The purified bispecific antibody.

[0213] The absorbance at 280 nm was measured using a Nanodrop 2000 (ThermoFisher), and the antibody concentration was calculated. After affinity purification, the purity of the antibody was analyzed and identified by SDS-PAGE. 5 μl of the purified sample was mixed with 20 μl of 5× loading buffer, heated in a metal water bath at 100 °C for 10 minutes, and 10 μl of the heated sample mixture was loaded onto PAGE gels (Nanjing Genscript Biotech Co., Ltd.). The samples were separated by electrophoresis according to their molecular weights. After the gel with separated samples was stained with Coomassie Brilliant Blue R250 for 3 hours, it was decolorized with a decolorizing solution, and the SDS-PAGE detection results of the expressed and purified bispecific antibody were obtained by photographing with a GelDoc Go Gel Imaging System (BIO-RAD), as Figure 3 shown. The SDS-PAGE of the antibodies in Examples 2-4 below is all shown in Figure 3 .

[0214] Effect data

[0215] I. Production of SARS-CoV-2 and its mutant strains, SARS-CoV, bat SARS-like coronavirus (BtSL-CoV) WIV-1, and Rs3367 pseudovirus

[0216] SARS-CoV-2 and its mutant strains, SARS-CoV, BtSL-CoV WIV-1, Rs3367, and pseudovirus are non-replication-defective retroviral particles with their respective spike membrane proteins (Spike, S) on the surface and carrying a luciferase reporter gene. They can mimic the infection process of SARS-CoV-2 and its mutant strains, BtSL-CoV WIV-1, Rs3367, and SARS-CoV viruses on host cells (such as human hepatoma cell line Huh-7 and 293T cell line 293T-ACE2 stably expressing the human ACE2 receptor), and express the luciferase reporter gene in the infected cells. Since the infection with pseudovirus does not produce infectious virus particles, relevant operations can be safely carried out in a biosafety level 2 laboratory.

[0217] SARS-CoV-2, BtSL-CoV WIV-1, Rs3367, and SARS-CoV pseudoviruses were obtained by co-transfecting 293T cells with their respective S protein expression plasmids and an HIV Env-deficient backbone plasmid (pNL4-3.Luc.R-E-) carrying a luciferase reporter gene.

[0218] The S gene sequences of SARS-CoV-2, SARS-CoV, BtSL-CoV WIV-1, and Rs3367 were designed based on the NCBI GenBank sequences NC_045512, ABD72979.1, KC881007.1, and KC881006.1. After codon optimization, the gene sequences were synthesized by Nanjing Genscript and ligated into the pcDNA3.1 eukaryotic expression vector to construct the S protein expression plasmids of SARS-CoV-2, SARS-CoV, BtSL-CoV WIV-1, and Rs3367. Among them, for the SARS-CoV-2 mutant pseudoviruses Alpha, Beta, Gamma, Delta, Lambda, and Omicron, corresponding point mutations and deletion mutations were required for the S protein expression plasmids. The pNL4-3.Luc.R-E- backbone plasmid was derived from the NIH AIDS Reagent Program of the United States. All plasmids were amplified by transforming DH5α competent cells and purified using a plasmid purification kit produced by GeneCreate Biological Engineering Co., Ltd. The purification operation process was referred to the instruction manual of the kit.

[0219] 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco) and seeded into 10 cm cell culture dishes before transfection. After 24 hours of culture, the backbone plasmid (pNL4-3.Luc.R-E-) and the expression plasmids of SARS-CoV, BtSL-CoV WIV-1, Rs3367, SARS-CoV-2, and its mutant strains were co-transfected into 293T cells at a ratio of 3:1 using the EZTrans cell transfection reagent (LiJi Biological). For the detailed transfection method, please refer to the instruction manual of the EZ Trans cell transfection reagent. After 48 hours of transfection, the supernatant containing pseudovirus was collected, centrifuged at 2500 rpm for 10 minutes to remove cell debris, and then aliquoted and stored at -80 °C in the refrigerator for the detection of neutralizing antibodies.

[0220] II. Detection of the neutralizing activities of the antibodies in Examples 1-4 of the present application against the pseudoviruses of SARS-CoV-2 and its mutant strains (Alpha, Beta, Gamma, Delta, Lambda, and Omicron), as well as various coronaviruses including SARS-CoV, the bat coronavirus BtSL-CoV WIV-1, and Rs3367

[0221] The neutralizing abilities of different concentrations of bispecific antibodies against the infection of pseudoviruses to Huh-7 cells were tested on 96-well cell culture plates to detect their neutralizing activities against SARS-CoV-2 and its mutant strains, SARS-CoV, BtSL-CoV WIV-1, and Rs3367 viruses.

[0222] The detection method is generally as follows: 1) Huh-7 cells are seeded in a 96-well cell plate at a density of 1×10 4 per well and cultured in a cell incubator at 37°C with 5% CO2 for 24 hours; 2) The antibodies of the examples and comparative examples are diluted to different concentrations with cell culture medium, mixed with an equal volume of pseudovirus dilution containing 100 TCID50, and incubated at 37°C for 1 hour; 3) The cell culture medium is discarded, 50 μl of virus-antibody complex is added to each well, duplicate wells are set, and at the same time, an antibody-free group, a virus-free group, and a positive serum control group are set; 4) After culturing for 12 hours, 150 μl of maintenance medium is added to each well and the culture is continued at 37°C for 48 hours; 5) The cells are lysed using a luciferase detection kit (Luciferase Assay System, Promega Cat.# E1500) and the luciferase activity of each well is detected. The specific detection method refers to the kit instruction manual; the chemiluminescence RLU value of each well is detected using a multimode microplate reader (Perkin Elmer); 6) The neutralization inhibition percentage of the pseudovirus by antibodies at different concentrations is calculated based on the ratio of the RLU values of the antibody and the virus control, and the half-maximal inhibitory concentration IC50 (unit: μg / ml) of the antibody against the virus is calculated using PRISM 7 software (GraphPad).

[0223] Experimental groups 1-4: Use the antibodies of Examples 1-4 of the present application;

[0224] Control group 1: Use the IgG1 monoclonal antibody constructed with the above-mentioned VL1 and VH1 (Comparative Example 1);

[0225] Control group 2: Use the IgG1 monoclonal antibody constructed with the above-mentioned VL2 and VH2 (Comparative Example 2);

[0226] Control group 3: An equal-proportion mixture of Comparative Example 1 and Comparative Example 2;

[0227] The detection results are shown in Table 1 below:

[0228] Table 1

[0229]

[0230]

[0231] Table 1 shows the neutralization IC50 results of the antibodies of Examples 1-4 and the control group antibodies against multiple SARS-CoV-2 mutant strains and other multiple coronaviruses.

[0232] As can be seen from Table 1:

[0233] 1) The bispecific antibodies of Examples 1-4 have potent neutralizing ability against pseudoviruses of major SARS-CoV-2 variants including Alpha, Beta, Delta, Omicron, etc., as well as SARS-CoV, bat coronavirus BtSL-CoVWIV-1, and Rs3367, demonstrating the broad-spectrum nature of the antibodies of Examples 1-4 in neutralizing coronaviruses.

[0234] 2) A surprising finding is that for the subtype mutants of Omicron, Comparative Example 1 has relatively weak neutralization, and the IC50 values of Comparative Example 2 for 12 Omicron variants are greater than 50 μg / mL. However, the geometric median of the IC50 of the combined bispecific antibody (Example 1) is 0.03 μg / mL, unexpectedly showing significant potent and broad-spectrum neutralizing ability. Although the bispecific antibodies of Examples 2-4 are less effective than the bispecific antibody of Example 1, they are also significantly superior to Comparative Examples 1-3, indicating that the bispecific antibody formed by combining two antigen-binding units has a synergistic effect.

[0235] 3) The bispecific antibody of Example 1 can still maintain potent neutralizing activity against multiple SARS-CoV mutants including Omicron mutant subtypes BQ.1.1, XBB.1.5, XBB.1.16, EG.5, BA.2.86, and JN.1. Even for the BA.2.86 and JN.1 mutants (with 34 additional amino acid mutations in the S protein compared to the BA.2 mutant), the bispecific antibody of Example 1 in this application unexpectedly shows significant neutralizing ability, has excellent broad-spectrum nature, and is significantly superior to the bebtelovimab antibody approved by the FDA for COVID-19 clinical treatment.

[0236] III. Evaluation of the preventive and therapeutic effects of the bispecific antibody of Example 1 in this application against SARS-CoV mutants XBB.1 and EG.5-infected mice

[0237] Animal studies have been approved by the Animal Experiment Committee of the First Affiliated Hospital of Guangzhou Medical University (approval number: 20230615). Six-week-old human ACE2-K18 transgenic mice (n = 5 per group) were used to evaluate the preventive and therapeutic effects of the bispecific antibody of Example 1 in animal studies. The mice were infected intranasally with a dose of 10 5 FFU of the SARS-CoV mutants XBB.1 or EG.5 virus. Twenty-four hours before or 24 hours after infection, the bispecific antibody of Example 1 in this application was administered by intraperitoneal injection (200 μg / mouse) (i.p.) or intranasal drip (20 μg / mouse) (i.n.). Two days after virus infection, lung samples of the mice were collected for virus titer determination. The amount of virus in each gram of lung tissue was determined 48 hours after virus infection; the operation procedure is shown in Figure 4 .

[0238] Mice treated with PBS were used as the control group, and the results are shown in Figure 5 and Figure 6 . Whether before the mice were infected with the XBB.1 or EG.5 virus (prevention group) or after the virus infection (treatment group), the bispecific antibody in Example 1 was administered to the mice by subcutaneous injection, and the viral load in the lung tissue of the mice could be reduced to the lower limit of detection (undetectable), which was significantly better than that of the control group mice. For the infection of the COVID-19 variant XBB.1, prophylactic and therapeutic administration by intranasal drip could reduce the viral load in the lung tissue of the mice to an undetectable level. In addition, compared with the control group mice, the symptoms of respiratory tract infection in the mice treated with the bispecific antibody in Example 1, such as sneezing, runny nose, and aggravated wheezing, were significantly alleviated.

[0239] These research findings also surprised and delighted the inventors, who also hope to promptly disclose the technical materials and conduct clinical promotion to contribute to the prevention and control of the new round of epidemic caused by the currently prevalent Omicron mutant subtypes.

[0240] In summary, based on the efficacy data of the bispecific antibodies in Examples 1, 2, 3, and 4 of the present application, it is proved that the bispecific antibody composed of the combination of the antibody specifically binding to the RBM motif and the antibody specifically binding to the outside of the RBM has excellent broad-spectrum neutralization ability against coronaviruses. In particular, it not only has a strong and broad-spectrum neutralization ability against multiple Omicron mutant subtypes, but also has a strong and broad-spectrum neutralization ability against the Sarbecovirus related to SARS-CoV.

[0241] IV. Rhinitis treatment effect of the antibody in Example 1 of the present application

[0242] The method for constructing a mouse rhinitis model is as follows:

[0243] BALB / c mice, age: 6 - W, 30 females;

[0244] Randomly divided into five groups: normal group (blank group), rhinitis group, treatment group of Example 1, hormone (budesonide) treatment group, IgG treatment group; 6 mice in each group;

[0245] Sensitization: Each modeling mouse was intraperitoneally injected with OVA (1 mg / ml) aluminum hydroxide (20 mg / ml) solution, once every other day for 1 - 14 days, a total of 7 times, to complete the sensitization process;

[0246] Challenge: On days 21 - 27, each modeling mouse was intranasally dripped with 50 μg OVA / saline solution, 10 μL on each side, once a day for 7 times, to complete the challenge process.

[0247] Drug administration method:

[0248] In the administration group (including the treatment group of Example 1, the hormone (budesonide) treatment group, and the IgG treatment group), drug treatment (drug concentration 1 mg / ml) was performed 30 minutes after each challenge, 20 μl per mouse, 10 μl in each nostril; the drug treatment was given twice a day, with an interval of eight hours.

[0249] Observation: Body weight: 2 times / week; Observation score: 1 time every 2 days, during the challenge phase, within 15 minutes after giving OVA.

[0250] Endpoint plan: On the 8th day after administration, all mice were euthanized with CO2, and samples (nasal mucosa, serum) were collected. The entire operation process is shown in Figure 7 .

[0251] Result evaluation:

[0252] (1) Observation indicators: ① Body weight measurement 2 times / week; ② Observation score: Count the number of sneezes of mice within 10 minutes after challenge on the 23rd, 25th, and 27th days.

[0253] (2) Detection indicators:

[0254] ① Pathology: On the 8th day after administration, the nasal mucosa of all mice was fixed with paraformaldehyde for pathological HE staining: inflammatory cells, eosinophils, mast cells, goblet cells, and pathological abnormalities were described.

[0255] ② Cytokines & immunoglobulins: On the 8th day after administration, the levels of OVA-sIgE and histamine in the sera of all mice were detected by ELISA.

[0256] The test results are as follows Figures 8 - 11 shown:

[0257] Figure 8 The upper figure above shows the relationship between the body weight (weight) of mice in each group and time, Figure 8 and the lower figure is the relationship between the percentage change in mouse body weight and time. Compared with the other four groups, after treating rhinitis mice with the hormone (budesonide) treatment group for 7 days, the body weight of the mice decreased significantly (p < 0.001), and the weight reduction exceeded 13.0%, indicating that budesonide had an obvious inhibitory effect on the growth and development of mice. In contrast, the antibody treatment of Example 1 did not have an obvious impact on the body weight and growth of mice during the same period, showing better safety and clinical feasibility.

[0258] Figure 9Shows the scoring results of sneezing in rhinitis mice after drug treatment. The average number of sneezes in the rhinitis group mice within 10 minutes was 5.1 times, indicating successful modeling. In the hormone (budesonide) treatment group, after 7 days of budesonide treatment, the average number of sneezes in mice on the 5th, 6th, and 7th days decreased to 2.8 times (p = 0.0031); while after treatment with the antibody of Example 1, the average number of sneezes in the corresponding three days was 2.7 times (p = 0.0013), suggesting that the antibody can effectively relieve rhinitis symptoms and has an efficacy comparable to that of hormone drugs. In contrast, the IgG treatment group failed to significantly improve the sneezing symptoms.

[0259] IgE is considered the "key to start" the onset of allergic rhinitis, and its elevated level is closely related to the occurrence, development, and severity of the disease. When an allergic individual first comes into contact with allergens such as pollen and dust mites, the immune system recognizes them as a threat and activates B cells to produce specific IgE antibodies. IgE can bind to the high-affinity receptors (FcεRI) on the surface of mast cells and basophils, forming a sensitized state (asymptomatic period). When exposed to the same allergen again, IgE binds to it, triggering mast cell degranulation and releasing inflammatory mediators such as histamine and leukotrienes, resulting in nasal mucosal vasodilation, glandular secretion increase, and nerve ending sensitization, and then triggering typical symptoms such as nasal itching, sneezing, runny nose, and nasal congestion.

[0260] Figure 10 Shows the changes in OVA-sIgE in the serum of rhinitis mice after drug intervention. After 8 days of treatment with the antibody of Example 1, the OVA-sIgE concentration decreased significantly from 39.3 ng / mL in the rhinitis group to 19.5 ng / mL (p < 0.0001), with a decrease of 50%, approaching the normal group level (11.3 ng / mL). The OVA-sIgE level in the hormone (budesonide) treatment group also decreased, to 15.5 ng / mL (p < 0.0001). The IgG treatment group failed to significantly reduce the OVA-sIgE concentration in the serum.

[0261] As the "initiator" and "amplifier" of allergic rhinitis symptoms, histamine plays a key role in acute attacks and chronic inflammation progression. It is mainly synthesized by mast cells and basophils and stored in cell granules in a bound state. Once the nasal cavity comes into contact with an allergen, IgE binds to mast cells and triggers a degranulation reaction, releasing a large amount of histamine. Histamine not only stimulates the trigeminal nerve endings to cause nasal itching and sneezing, but also activates goblet cells and submucosal glands, increasing mucin secretion; at the same time, it induces vasodilation and plasma exudation, causing clear nasal discharge and mucosal edema, thus aggravating nasal obstruction symptoms.

[0262] Figure 11Shows the changes in the level of histamine in the serum of rhinitis mice after drug treatment. After 8 days of antibody treatment in Example 1, the histamine concentration in the mouse serum decreased from 7.2 ng / mL to 3.5 ng / mL (p<0.0001), a decrease of 51%, which was similar to the concentration in the normal group (3.8 ng / mL). The histamine concentration in the hormone (budesonide) treatment group was 4.3 ng / mL (p<0.0001). The IgG treatment group failed to significantly reduce the histamine concentration in the serum. It can be seen that the antibody in Example 1 is more effective than budesonide in inhibiting histamine release and can more effectively relieve rhinitis-related symptoms, especially the frequency of sneezing.

[0263] Figure 12 The nasal mucosa of rhinitis group mice after drug treatment was subjected to pathological HE staining. Compared with the normal group, the comprehensive pathological manifestations of rhinitis group mice were epithelial layer damage (cilia shedding, cell necrosis), interstitial edema, vascular dilation, glandular hyperplasia with secretory retention. A large number of inflammatory cells infiltrated in the lamina propria and submucosa of the nasal mucosa. The inflammatory cells were distributed in sheets or foci around blood vessels and glands, indicating a chronic inflammatory response. The number of goblet cells increased significantly, showing multifocal hyperplasia, and squamous metaplasia occurred in some areas. The hyperplasia of goblet cells led to hyperfunction of mucus secretion, forming mucopurulent secretions, which were related to the symptoms of nasal congestion and runny nose. After treatment with hormone (budesonide) or the antibody described in Example 1, the pathological changes were significantly improved. The nasal mucosa structure of the treated group mice tended to be complete, and only a small amount of inflammatory cell infiltration was visible in the lamina propria and submucosa. The hyperplasia of goblet cells was significantly reduced, and no obvious mucus retention or epithelial metaplasia was observed. The above results indicate that both budesonide hormone and the antibody in Example 1 have obvious anti-inflammatory effects and can effectively relieve the chronic inflammatory response in the rhinitis model.

[0264] In summary, the antibody in Example 1 has a similar mechanism of action to the hormone (budesonide) treatment group by reducing the OVA-sIgE level and inhibiting the release of histamine from mast cells, and both can effectively control the symptoms of allergic rhinitis. However, the hormone budesonide has an adverse effect on the growth and development of mice, while the antibody does not show similar side effects. Therefore, the antibody in Example 1 is more safe and has potential for clinical application in the treatment of allergic rhinitis, especially in pediatric patients.

[0265] V. Asthma treatment effect of the antibody in Example 1 of this application

[0266] The method for constructing a mouse rhinitis model is as follows:

[0267] BALB / c mice, age: 6 - W, 35 female mice;

[0268] Randomly divided into five groups: normal group (blank group), asthma group, treatment group of Example 1, hormone (budesonide) treatment group, IgG treatment group; 7 mice in each group;

[0269] Sensitization: OVA-V (20 μg / mouse); 1 mg of aluminum hydroxide was dissolved in normal saline (100 μl / mouse). The solution was mixed using a three-way valve and continued to be mixed for 30 min to allow aluminum hydroxide to effectively adsorb the antigen. Mice were intraperitoneally injected for sensitization on days 0 / 7 / 14 of the experiment; the sensitization process was completed; mice in the blank control group were treated with normal saline in the same way.

[0270] Challenge: Prepare 3% (w / v) OVA-II (30 g / L) dissolved in normal saline, atomize it and introduce it into a closed atomization chamber for mice to inhale, once a day for 30 min each time. Mice were atomized and challenged from day 21 to 27 of the experiment; mice in the blank control group were treated with normal saline in the same way.

[0271] Drug administration method: In the drug administration group, 30 min after each challenge, drug treatment was carried out (drug concentration 1 mg / ml), 20 μl per mouse, 10 μl in each nostril; drug treatment was carried out twice a day, with an interval of eight hours. Animals in the normal group (blank group) and the asthma group were dripped with normal saline in the same way. The total treatment lasted for 7 days. The entire operation process is shown in Figure 13 .

[0272] Detection indexes:

[0273] 1. Body weight: During the sensitization stage, the body weight of animals was measured and recorded twice a week; during the challenge stage, the body weight was measured and recorded on days 20, 22, 24, 26, and 28.

[0274] 2. Symptom observation: 30 min after drug administration during the challenge stage, clinical symptoms were recorded: hunched back, curled up, increased and deepened breathing.

[0275] 3. Serum retention: On the 8th day after drug administration, all mice were euthanized with CO2, blood was collected, allowed to stand at room temperature for 0.5 hour and then centrifuged in a centrifuge. Centrifugation conditions: 4°C / 3000 rpm / 15 min; after centrifugation, the serum was separated and stored in aliquots at -80°C for subsequent ELISA detection (OVA-sIgE, IL-4, IL-5, IL-13, histamine).

[0276] 4. Collection of bronchoalveolar lavage fluid: Pre-cool PBS for later use. Make a small incision in the trachea of the mouse using a syringe needle or micro-scissors. Insert the lavage needle into the trachea and fix it with a ligature thread. Connect a 1 ml syringe to lavage the left lung of the mouse. Each time, draw 0.3 ml of pre-cooled PBS with the syringe and slowly instill it into the left lung through the lavage needle. Slowly aspirate back and forth with the syringe 3 times for sufficient lavage, then recover the fluid in the lung and place it in a sterile 1.5 ml centrifuge tube, and keep it on ice. Each mouse is lavaged 3 times in total (0.3 + 0.3 + 0.4 = 1 ml PBS), and the fluid recovery rate is about 70%. Centrifuge at 4°C / 2000 rpm for 10 minutes, take the supernatant, aliquot it, and store it at -80°C for subsequent ELISA detection (OVA-sIgE, IL-4, IL-5, IL-13, histamine).

[0277] 5. Detection of inflammatory factors: Enzyme-linked immunosorbent assay was used to detect the concentrations of inflammatory factors (IL-4, IL-5, IL-13, histamine) in the sera and bronchoalveolar lavage fluids of all mice, and the concentrations of OVA-sIgE and histamine in the sera. The experiment was carried out according to the manufacturer's instructions

[0278] 6. Pathological indicators: The lower right lobe of the lung was washed with PBS, fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned for subsequent staining experiments (H&E staining was used to observe the pathological changes of lung tissue and evaluate the degree of lung tissue inflammation; PAS staining was used to evaluate the proliferation degree of airway goblet cells).

[0279] The detection results are as follows Figures 14 - 19 shown as

[0280] Figure 14 shows the change trend of the body weight of the mice. Compared with the other four groups, after treating asthmatic mice with the hormone drug budesonide in the hormone (budesonide) treatment group for 7 days, the body weight of the mice decreased significantly (p < 0.0001), and the reduction amplitude of the body weight exceeded 10.6%, indicating that the hormone budesonide has an obvious inhibitory effect on the growth and development of mice. In contrast, after treating asthmatic mice with the bispecific antibody in Example 1 for 7 days, the body weight of the mice did not change significantly, indicating that this treatment has no adverse effects on the growth of mice, and has better safety and clinical feasibility.

[0281] Figure 15The symptom scores of asthmatic mice after drug intervention were shown, including manifestations such as hunchback, curling up and rapid breathing, with each symptom scored 1 point. The score of the asthma model group was 2.6, indicating successful establishment of the asthma model. In the hormone (budesonide) treatment group, after 7 days of treatment with budesonide, the asthma symptom score decreased to 1.1 points (p = 0.0064). After treatment with the antibody of Example 1, the symptom score also decreased to 1.1 points (p = 0.0042), indicating that the antibody could significantly relieve asthma-related symptoms. The IgG monoclonal antibody in the IgG treatment group did not show obvious therapeutic effect, indicating that it had no significant improvement on asthma symptoms.

[0282] In this study, the effects of drug intervention on the levels of OVA-specific IgE (OVA-sIgE) in bronchoalveolar lavage fluid (BALF) and serum of asthmatic mice were first detected. As Figure 16 shown in the upper and lower figures, drug intervention mainly affected the content of OVA-sIgE in BALF and had no significant effect on the level in serum. After 8 days of treatment with the antibody of Example 1, the concentration of OVA-sIgE in BALF decreased significantly from 29.2 ng / mL to 0.7 ng / mL (p < 0.0006), with a decrease amplitude as high as 97%, approaching the level of 0 ng / mL in the normal group. In the hormone (budesonide) treatment group, after treatment with budesonide, it only decreased to 4.9 ng / mL (p < 0.006), and the IgG monoclonal antibody in the IgG treatment group did not show obvious therapeutic effect. The above results indicate that the antibody of Example 1 is more effective than hormone drugs in reducing OVA-sIgE.

[0283] IL-4 (Interleukin-4) plays a key role in Th2 cell differentiation and the pathogenesis of asthma. In asthma patients, after inhaled allergens (such as dust mites, pollen) are captured by antigen-presenting cells (such as dendritic cells), it induces the differentiation of naive CD4+ T cells into the Th2 phenotype, and this process is mainly mediated by IL-4. IL-4 promotes the differentiation and maturation of Th2 cells by activating the JAK / STAT signaling pathway. Mature Th2 cells further secrete cytokines such as IL-4, IL-5 and IL-13, induce type II immune responses, and promote class switch recombination of B cells to generate IgE antibodies. IgE binds to the FcεRI receptor on the surface of mast cells and basophils, triggering a degranulation reaction when encountering allergens again, releasing mediators such as histamine and leukotrienes, resulting in acute asthma symptoms such as bronchoconstriction and mucus secretion. Then, we evaluated the regulatory effect of the antibody of Example 1 on the level of IL-4 (see Figure 17(upper and lower figures). After 8 days of treatment, the antibody of Example 1 could significantly reduce the IL-4 concentrations in BALF and serum: the IL-4 in serum decreased from 50.0 pg / mL to 13.1 pg / mL (p < 0.0002), with a decrease of 73.8%, even lower than 27.7 pg / mL in the normal group; the IL-4 concentration in BALF also significantly decreased from 16.7 pg / mL to 6.8 pg / mL (p < 0.0023). In contrast, although budesonide treatment could significantly reduce the IL-4 concentration in BALF (decreased to 3.1 pg / mL, p < 0.0001), it had no obvious effect on serum IL-4, indicating that its effect was more locally targeted. The antibody of Example 1 achieved a more extensive inflammation control by simultaneously regulating the systemic (serum) and local (lung) IL-4 levels.

[0284] In addition, IL-5 is an important cytokine secreted by Th2 cells, which can promote the differentiation and proliferation of eosinophils in the bone marrow. During rhinitis and asthma, IL-5 induces a large number of eosinophils to aggregate in the nasal mucosa and respiratory tract, releasing inflammatory mediators (such as ECP, MBP, etc.), causing tissue damage, increased vascular permeability and mucus secretion, resulting in typical symptoms such as nasal congestion and runny nose.

[0285] We further detected the effect of the antibody of Example 1 on the IL-5 level (see Figure 18 the upper and lower figures), and the results showed that the serum IL-5 concentration decreased from 16 pg / mL to 10.3 pg / mL (p < 0.0043) after antibody treatment, approaching 11.6 pg / mL in the normal group. Budesonide could significantly reduce the IL-5 concentrations in both BALF and serum (both p < 0.0001), indicating that it had a more comprehensive inhibitory effect on the IL-5 pathway.

[0286] Figure 19 Pathological HE staining was performed on the nasal mucosa of asthmatic mice after drug treatment. Compared with the normal group, a large number of inflammatory cells were infiltrated in the lung interstitium and around the airways of asthmatic mice, and the inflammatory cells were distributed in sheets or foci around bronchi, blood vessels and alveolar septa, indicating a chronic inflammatory reaction. The number of goblet cells increased significantly, showing multifocal hyperplasia, and squamous metaplasia occurred in some areas. The hyperplasia of goblet cells led to hyperfunction of mucus secretion, forming mucopurulent secretions, which were related to airway obstruction and cough symptoms. Alveolar inflammation, exudation in the alveolar cavity, and inflammatory cells filling the alveolar cavity, and "hemophagocytosis" (erythrocyte extravasation) formed in some areas. Interstitial edema, vascular dilation, and thickening of the alveolar septum. The pathological HE staining of asthmatic mice treated with control 2 IgG was similar to that of the asthmatic group. After treatment of asthmatic mice with the hormone-positive drug and the antibody in Example 1, the pathological HE staining was relatively similar, with only a small amount of inflammatory cells aggregating around the bronchi, a small amount of exudation in the alveolar cavity, and no obvious hyperplasia of goblet cells.

[0287] In summary, the hormone budesonide mainly inhibits eosinophil-mediated inflammatory responses at both the pulmonary and systemic levels by regulating the level of IL-5, thereby alleviating asthma symptoms. The antibody of Example 1, on the other hand, more significantly regulates the level of IL-4, inhibits the activity of Th2 cells and the immune responses triggered by them, blocks the class switch of B cells to IgE, and thus reduces the asthma response, with an immune regulatory mechanism different from that of budesonide. The differences between the two in the anti-inflammatory pathway provide an important reference for clinical combination or alternative treatment strategies. Although the overall efficacy of both is comparable, compared with the possible adverse effects on body development caused by hormonal drugs, the treatment with the antibody of Example 1 shows better safety in mice, indicating good clinical application prospects.

[0288] VI. Therapeutic effect of the antibody of Example 1 of the present application on dermatitis

[0289] Construction of a mouse dermatitis model:

[0290] BALB / c mice, age: 6 - W, 40 female;

[0291] Randomly divided into five groups: normal group (blank group), dermatitis group, treatment group of Example 1, hormone (dexamethasone) treatment group, IgG treatment group; 8 mice in each group;

[0292] All mice were adaptively fed for one week. After anesthetizing the mice with tribromoethanol, the back hair was removed with depilatory cream and razor, with an area of about 2 cm * 3 cm. Then the mice were randomly grouped. Except for the normal group (blank group), on the 1st - 3rd days of the experiment, 200 μL of 0.5% DNCB (dissolved in a mixture of acetone: olive oil (3:1)) was evenly applied to the depilated area to sensitize the mice. Next, on the 11th, 14th, 17th, 20th, 23rd, 26th, 29th, and 30th days of the experiment, 20 μL and 100 μL of 1% DNCB were respectively applied to each ear and dorsal skin. At the same time, from the 11th to 30th days of the experiment, the mice in the dosing groups were treated with drugs daily.

[0293] Drug administration method: One hour after excitation on the 11th - 30th days, the administration volume for topical application was 50 μL, administered 2 times a day for a total of 20 times. The entire operation process is shown in Figure 20 .

[0294] Detection indicators:

[0295] 1) Body weight measurement: Monitor body weight 3 times a week

[0296] Measurement of ear and dorsal skin thickness: Take images with a camera every week to record the clinical symptoms of the ears and dorsal skin. Measure the thickness of the ears and dorsal skin of each mouse every week and record it.

[0297] 2) Lesion score:

[0298] During the experiment, the severity of skin lesions in mice was scored, including four aspects: erythema / bleeding, edema / exudation, dryness / desquamation, and epidermal exfoliation. The scores represent the severity level, divided into four grades: none (0), mild (1), moderate (2), and severe (3). The sum of the four symptoms is the final score, with the score ranging from 0 to 12 points. (Note: The scoring time points are set as the 1st, 11th, 13th, 16th, 19th, 22nd, 25th, 28th, and 30th days).

[0299] Endpoint sampling on the 30th day:

[0300] (1) Peripheral blood (Blood): One hour after administration, the animals were anesthetized and peripheral blood was collected. After standing at room temperature for 4 - 5 hours, the serum was obtained by centrifugation and stored at -80°C;

[0301] (2) Spleen: The spleen was weighed and the spleen mass coefficient was calculated;

[0302] (3) Ear and back skin: After the animals were euthanized, the back skin tissues and ear skin tissues of each group of mice were fixed with paraformaldehyde solution. (The ear skin was retained after fixation)

[0303] Detection indicators:

[0304] (1) ELISA: ELISA kits were used to detect the contents of total TNF-α, IL-1β, IL-13, and IgE in the sera of each group of mice;

[0305] (2) Histopathological analysis: The fixed back skin tissues were dehydrated, fully infiltrated with wax, then paraffin-embedded, sectioned, baked, stained with HE, stained with toluidine blue, and sealed with neutral gum. Finally, they were observed under a microscope, and the slides were analyzed and photographed (one slide at a magnification of 100X and one at 200X).

[0306] Data analysis

[0307] In this experiment, the scientific research statistics and graphing software GraphPad Prism 8.4.3 were used to analyze the data. One-way analysis of variance (One-way, ANOVA) was used for comparison among multiple groups, and Student's t-test was used for comparison between two groups. All statistical analyses were performed with two-tailed analysis, and the statistical significance level was set at p ≤ 0.05.

[0308] The detection results are as follows Figures 21 - 26 shown as follows:

[0309] Figure 21Shows the change trend of body weight during the treatment of mice with dermatitis. In the hormone (dexamethasone) treatment group, after continuous treatment with the hormone drug dexamethasone for 20 days, the body weight of the mice decreased significantly (p<0.0001), and the average weight loss exceeded 10%, indicating that dexamethasone has an obvious inhibitory effect on the growth and development of mice. In contrast, after 20 days of antibody treatment in Example 1, there was no obvious change in the body weight of the mice, suggesting that this treatment has no adverse effect on growth and has better safety and clinical feasibility.

[0310] In a mouse dermatitis model, the spleen mass coefficient (spleen weight / body weight) is often used as an important indicator to evaluate the degree of activation of the immune system. In allergic contact dermatitis (ACD) and atopic dermatitis (AD) models, an increase in the spleen mass coefficient is usually closely related to the enhancement of the inflammatory response. Figure 22 The results showed that the mean spleen mass coefficient of the mice in the dermatitis group was 0.46, which was significantly higher than that of the normal group (blank group) mice at 0.36 (p<0.0019), verifying the effectiveness of the model construction. In terms of treatment effect, in the IgG treatment group, the spleen mass coefficient of the mice receiving IgG treatment was 0.44, which was also significantly higher than that of the normal group (p<0.0033), suggesting that this treatment failed to effectively inhibit the inflammatory response. In contrast, after 20 days of dexamethasone treatment, the spleen mass coefficient of the mice in the hormone (dexamethasone) treatment group decreased significantly from 0.46 to 0.154 (p<0.0001). Such a drastic decrease is often related to damage to the spleen function or structure, suggesting that while dexamethasone strongly inhibits the immune response, it may be accompanied by spleen damage and other adverse reactions. The spleen mass coefficient of the mice in the antibody treatment group of Example 1 was 0.42. Although it was still higher than that of the normal group (p = 0.045), compared with the hormone (dexamethasone) treatment group and the IgG treatment group, its value was closer to the normal level, indicating that this antibody can effectively inhibit the inflammatory response while having less impact on the immune system. In addition, antibody treatment did not cause obvious body weight changes or spleen function damage within the same period, showing good safety and potential clinical application value.

[0311] Figure 23The dermatitis symptom scores of mice in each group were shown, and the scoring items included erythema / bleeding, edema / exudation, dryness / desquamation, and epidermal exfoliation, etc. Each item was scored from 1 to 3 points according to the severity. The total dermatitis score of the model group mice could reach up to 7 points and still remained at 5.6 points at the end of the experiment, further verifying the stability and effectiveness of the construction of the dermatitis model. After 20 days of treatment in the hormone (dexamethasone) treatment group, the dermatitis score decreased significantly from the highest 4.3 points to 1.1 points (p = 0.0064), showing its good anti-inflammatory effect. After 20 days of antibody treatment in Example 1, the score decreased from the highest 4.8 to 2.4 points (p = 0.0042), also showing a significant symptom relief effect. In contrast, there was no obvious score improvement in the IgG treatment group, suggesting that this IgG antibody had no effective intervention on dermatitis symptoms. In summary, the antibody treatment in Example 1 had a good effect in relieving dermatitis symptoms, showing its potential therapeutic value.

[0312] Figure 24 It was a comparison graph of the back skin states of mice in each group after 20 days of treatment. The degree of skin improvement from excellent to poor was as follows: normal group > hormone (dexamethasone) treatment group > treatment group of Example 1 > IgG group, which was consistent with the skin score results.

[0313] In acute irritant dermatitis, TNF-α and IL-1β can activate mast cells and macrophages, induce the release of histamine and proteases, and then trigger erythema, edema, and exudation reactions; among them, TNF-α can also promote the abnormal proliferation and apoptosis of keratinocytes, leading to epidermal thickening, and stimulate fibroblasts and endothelial cells to secrete matrix metalloproteinases (MMPs), exacerbating tissue damage.

[0314] This study first evaluated the regulatory effects of various treatment methods on the level of TNF-α in serum. As Figure 25 shown, after 20 days of antibody treatment in Example 1, the concentration of TNF-α decreased significantly from 728 pg / mL to 499.3 pg / mL (p = 0.0001), even lower than 586 pg / mL in the normal group. The IgG treatment group failed to reduce the concentration of TNF-α. The concentration of TNF-α in the hormone (dexamethasone) treatment group decreased to 410.3 pg / mL (p < 0.0001), showing that the hormone (dexamethasone) had the same effect as the antibody treatment in Example 1, indicating that this antibody could effectively inhibit the expression of inflammatory factors, thereby relieving dermatitis symptoms.

[0315] In addition, IL-1β can enhance the degranulation reaction of mast cells by activating the NLRP3 inflammasome, release inflammatory mediators such as histamine and chymotrypsin, and induce fibroblasts to secrete MMP-1 and MMP-9, resulting in epidermal barrier damage and collagen degradation. Therefore, we further detected the changes in the level of IL-1β in serum. As Figure 26As shown, the concentration of IL-1β in the dermatitis group of mice was significantly higher than that in the blank group (p = 0.0032). After 20 days of antibody treatment in Example 1, the level of IL-1β decreased significantly from 217.6 pg / mL to 128.5 pg / mL (p = 0.0028), slightly lower than 143.2 pg / mL in the normal group; the IgG treatment group failed to reduce the concentration of IL-1β; after dexamethasone treatment, the level of IL-1β was 193.9 pg / mL, and no significant decrease was observed, indicating that the antibody in Example 1 was more effective than dexamethasone in inhibiting IL-1β.

[0316] In summary, this study first confirmed that the antibody in Example 1 achieved precise intervention in the inflammatory cascade reaction by simultaneously neutralizing TNF-α and IL-1β, effectively blocking the activation of mast cells and macrophages and the release of their inflammatory mediators, thereby alleviating dermatitis symptoms such as erythema, edema, and exudation. In contrast, dexamethasone mainly relies on inhibiting TNF-α to exert its anti-inflammatory effect, and the mechanism is relatively single. Although the overall curative effects of the two are comparable, the antibody in Example 1 has a more precise mechanism of action and is expected to become an important basis for a new generation of immunomodulatory treatment strategies. More importantly, compared with the potential kidney damage and adverse effects on the body development caused by hormonal drugs, this antibody treatment showed better safety in mice, indicating good clinical application prospects.

[0317] Based on the public content and spirit of this application obtained by those skilled in the art, some simple adjustments can be made on the basis of the bispecific antibody sequence in Example 1 of this application. For example, the front and back orders of the light chain and heavy chain can be swapped on the basis of the bispecific antibody sequence in Example 1 to obtain antibodies such as VH-1—L1—VL-1—L3—VL-2—L2—VH-2—Hinge—CH2—CH3, VL-1—L1—VH-1—L3—VH-2—L2—VL-2—Hinge—CH2—CH3, or VH-1—L1—VL-1—L3—VH-2—L2—VL-2—Hinge—CH2—CH3.

[0318] It is also possible to swap the front and back orders of the light chain and heavy chain on the basis of the bispecific antibody sequence in Example 2 to obtain antibodies such as VH-2—L2—VL-2—L3—VL-1—L1—VH-1—Hinge—CH2—CH3, VL-2—L2—VH-2—L3—VH-1—L1—VL-1—Hinge—CH2—CH3, or VH-2—L2—VL-2—L3—VH-1—Linker-b—VL-1—Hinge—CH2—CH3;

[0319] It is also possible to swap the front-back order of the light chain and the heavy chain based on the bispecific antibody sequence of Example 3 to obtain, such as VH-1—L1—VL-1—L4—Hinge—CH2—CH3—L5—VL-2—L2—VH-2, VL-1—L1—VH-1—L4—Hinge—CH2—CH3—L5

[0320] —VH-2—L2—VL-2 or an antibody of VH-1—L1—VL-1—L4—Hinge—CH2—CH3—L5—VH-2—L2—VL-2;

[0321] It is also possible to swap the front-back order of the light chain and the heavy chain based on the bispecific antibody sequence of Example 4 to obtain, such as VH2—L2—VL2—L4—Hinge—CH2—CH3—L5—VL1—L1—VH1, VL2—L2—VH2—L4—Hinge—CH2—CH3—L5—VH1—L1—VL1 or an antibody of VH2—L2—VL2—L4—Hinge—CH2—CH3—L5—VH1—L1—VL1.

[0322] It can be reasonably speculated that they all have effects similar to those of the bispecific antibodies of Examples 1, 2, 3, and 4; these equivalent replacement schemes all fall within the protection scope of this application.

[0323] Those skilled in the art can also perform conventional equivalent replacements on the linker peptide sequence, Fc domain sequence, hinge peptide sequence, etc. based on the bispecific antibody sequences of Examples 1, 2, 3, and 4 of this application; those skilled in the art can also perform amino acid insertion, replacement, or deletion treatments that do not affect the overall effect of the antibody based on the bispecific antibody sequence of the present invention; these equivalent replacement schemes all fall within the protection scope of this application.

[0324] Based on the bispecific antibody scheme of scFv-scFv and the sequence of its antigen-binding module obtained by those skilled in the art, existing bispecific antibody technologies can be used to transform it into a bispecific / multispecific antibody of Fab-Fab combination, Fab-Fv combination, or other Fv-Fv combinations.

[0325] As can be seen from the above content, the multispecific antibody or its antigen-binding molecule that neutralizes coronaviruses of the present invention has excellent broad-spectrum and potent neutralizing ability against coronaviruses and can effectively block the infection of multiple coronaviruses. Therefore, based on learning this technical content, those skilled in the art can further develop corresponding recombinant proteins, fusion proteins, and immunoconjugates, as well as drugs for treating or preventing diseases caused by coronaviruses, and detection products for detecting coronaviruses, and drugs for relieving and treating inflammatory diseases of the respiratory tract (such as rhinitis, asthma, etc.) or dermatitis and other inflammatory diseases.

[0326] Application Example

[0327] This application example describes a method for using the bispecific antibodies of Embodiments 1-4 of the present application to prevent or treat respiratory infectious diseases, respiratory inflammations, or dermatitides caused by viruses (including coronaviruses).

[0328] Although specific administration methods, dosages, and regimens are provided, those skilled in the art will understand that changes can be made without substantially affecting the treatment. Based on the guidance disclosed herein, respiratory infectious diseases, respiratory inflammations, or dermatitides caused by viruses (including coronaviruses) can be treated or prevented by administering a therapeutically effective amount of the antibodies described herein.

[0329] The specific administration method is as follows:

[0330] 1) Pre-treatment of the subject: In specific embodiments, the subject is treated prior to administering a therapeutic agent that includes a pharmaceutical therapy for respiratory infections or respiratory inflammations caused by one or more viruses known to those skilled in the art. However, such pre-treatment is not always required and can be determined by a skilled clinician.

[0331] 2) Administration of the therapeutic composition

[0332] After screening the subject, the above-mentioned therapeutically effective dose of the antibodies of Embodiments 1-4 of the present application is administered to the patient. Additional drugs such as antiviral agents can be administered to the subject simultaneously with, before, or after administering the disclosed agent. Administration is achieved by any method known in the art such as injection (including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-orbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion), or by non-conventional routes of administration, such as local, epidermal, or mucosal routes of administration, for example, intranasal, oral, vaginal, rectal, sublingual, or topical administration. The amount of the composition administered to prevent, reduce, inhibit, and / or treat the condition of the subject depends on the subject being treated, the severity of the condition, and the mode of administration to the subject. Ideally, the therapeutically effective amount of the agent is an amount sufficient to prevent, reduce, and / or inhibit, and / or treat the condition of the subject without causing substantial cytotoxic effects in the subject. The effective amount can be readily determined by those skilled in the art, for example, by conventional tests establishing a dose-response curve. Similarly, these compositions can be formulated with an inert diluent or a pharmaceutically acceptable carrier. In one specific example, the antibody is administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks, depending on the specific stage of the viral infection. In one example, the antibody is administered continuously. In another example, the antibody is administered at 50 μg per kg twice a week for 2-3 weeks. The therapeutic composition can be administered for a long period of time (such as for several months or years).

[0333] 3) Evaluation

[0334] After administering one or more therapies, monitor the reduction of the viral level in the patient, or the reduction of one or more related clinical symptoms, or the relief of respiratory discomfort symptoms. In certain instances, starting 2 days after treatment, perform one or more analyses on the subject. Monitor the subject using any method known in the art. For example, a biological sample including a throat swab can be obtained from the subject and the change in the viral level can be evaluated.

[0335] 4) Additional treatment

[0336] In a specific embodiment, if the subject is stable or has a minor, mixed, or partial response to the treatment, additional treatment can be performed after re-evaluation with the same regimen and pharmaceutical formulation for the desired time they previously received.

[0337] It should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0338] The series of detailed descriptions listed above are only specific descriptions of the feasible embodiments of the present invention, and they are not intended to limit the protection scope of the present invention. Any equivalent embodiments or changes made without departing from the technical spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A multispecific antibody or its antigen-binding molecule, characterized in that: The multispecific antibody or its antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module; The second antigen-binding module binds to the receptor-binding motif portion of the receptor-binding region of the coronavirus S protein; The first antigen-binding module binds to a portion other than the receptor-binding motif of the receptor-binding region of the coronavirus S protein.

2. The multispecific antibody or its antigen-binding molecule according to claim 1, characterized in that: The first antigen-binding module comprises a light chain variable region VL-1 and a heavy chain variable region VH-1; The second antigen-binding module comprises a light chain variable region VL-2 and a heavy chain variable region VH-2; The light chain variable region VL-1 comprises the LCDR1-1 sequence of the light chain variable region shown in SEQ ID NO.1, the LCDR2-1 sequence of the light chain variable region shown in SEQ ID NO.2, and the LCDR3-1 sequence of the light chain variable region shown in SEQ ID NO.3; The heavy chain variable region VH-1 comprises the HCDR1-1 sequence of the heavy chain variable region shown in SEQ ID NO.4, the HCDR2-1 sequence of the heavy chain variable region shown in SEQ ID NO.5, and the HCDR3-1 sequence of the heavy chain variable region shown in SEQ ID NO.6; The light chain variable region VL-2 comprises the LCDR1-2 sequence of the light chain variable region shown in SEQ ID NO.7, the LCDR2-2 sequence of the light chain variable region shown in SEQ ID NO.8, and the LCDR3-2 sequence of the light chain variable region shown in SEQ ID NO.9; The heavy chain variable region VH-2 comprises the HCDR1-2 sequence of the heavy chain variable region shown in SEQ ID NO.10, the HCDR2-2 sequence of the heavy chain variable region shown in SEQ ID NO.11, and the HCDR3-2 sequence of the heavy chain variable region shown in SEQ ID NO.

12.

3. The multispecific antibody or its antigen-binding molecule according to claim 2, characterized in that: The sequence of the light chain variable region VL-1 is as shown in SEQ ID NO.13, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.13; The sequence of the heavy chain variable region VH-1 is as shown in SEQ ID NO.14, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.14; The sequence of the light chain variable region VL-2 is as shown in SEQ ID NO.15, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.15; The sequence of the heavy chain variable region VH-2 is as shown in SEQ ID NO.16, or it has a sequence homology of more than 80% with the sequence shown in SEQ ID NO.

16.

4. The multispecific antibody or its antigen-binding molecule according to claims 2-3, characterized in that: The first antigen-binding module is selected from any one of Fv, Fab, Fab’, dsFv or scFv; The second antigen-binding module is selected from any one of Fv, Fab, Fab’, dsFv or scFv.

5. The multispecific antibody or its antigen-binding molecule according to claim 4, characterized in that: The first antigen-binding module is a single-chain antibody fragment scFv-1; the second antigen-binding module is a single-chain antibody fragment scFv-2; The scFv-1 sequentially comprises the light-chain variable region VL-1, the first linker peptide and the heavy-chain variable region VH-1 from the N-terminus to the C-terminus; alternatively, the scFv-1 sequentially comprises the heavy-chain variable region VH-1, the first linker peptide and the light-chain variable region VL-1 from the N-terminus to the C-terminus; The scFv-2 sequentially comprises the light-chain variable region VL-2, the second linker peptide and the heavy-chain variable region VH-2 from the N-terminus to the C-terminus; alternatively, the scFv-2 sequentially comprises the heavy-chain variable region VH-2, the second linker peptide and the light-chain variable region VL-2 from the N-terminus to the C-terminus; Preferably, the first linker peptide or the second linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; wherein, the value of n falls within the range of 1 to 10.

6. The multispecific antibody or its antigen-binding molecule according to any one of claims 2 to 5, characterized in that: The C-terminus of the scFv-1 is connected to the N-terminus of the scFv-2 through a linker peptide, and the C-terminus of the scFv-2 is connected to a tag sequence; or, The C-terminus of the scFv-2 is connected to the N-terminus of the scFv-1 through a linker peptide, and the C-terminus of the scFv-1 is connected to a tag sequence; Preferably, the linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; wherein, the value of n falls within the range of 1 to 10; Preferably, the tag sequence is a small molecule polypeptide tag sequence; more preferably, the tag sequence is glutathione S-transferase GST, polyhistidine (Poly-His), streptavidin (Strep), FLAG tag, maltose-binding protein (MBP).

7. The multispecific antibody or its antigen-binding molecule according to any one of claims 2 to 5, characterized in that: The multispecific antibody or its antigen-binding molecule comprises a constant region; preferably, the constant region is the heavy-chain constant region and / or the light-chain constant region of human immunoglobulin; The heavy-chain constant region is preferably the heavy-chain constant region of human IgG1, 2, 3, 4; Preferably, the heavy-chain constant region is the Fc domain of human IgG1.

8. The multispecific antibody or its antigen-binding molecule according to claim 7, characterized in that: The C-terminus of the scFv-1 is connected to the N-terminus of the scFv-2 through a third linker peptide, and the C-terminus of the scFv-2 is connected to the Fc domain of human IgG1 through a hinge peptide; or, The C-terminus of the scFv-2 is connected to the N-terminus of the scFv-1 through a third linker peptide, and the C-terminus of the scFv-1 is connected to the Fc domain of human IgG1 through a hinge peptide; or, The C-terminus of the scFv-1 is sequentially connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide, and the C-terminus of the Fc domain of human IgG1 is connected to the N-terminus of scFv-2 through a fifth linker peptide; or, The C-terminus of the scFv-2 is sequentially linked to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide, and the C-terminus of the Fc domain of human IgG1 is linked to the C-terminus of scFv-1 through a fifth linker peptide; Preferably, the third linker peptide, fourth linker peptide or fifth linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n , (GGGGS) n G, (GGGGS) n GS, GS(GGGGS) n , GS(GGGGS) n GS, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n , wherein the value of n falls within the range of 1 to 10; Preferably, the hinge peptide is selected from EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP) n , ESKYGPPCPSCP, wherein the value of n falls within the range of 1 to 10; alternatively, the hinge peptide may be selected from the α1 chain, α2 chain, γ1 chain, γ2 chain, γ3 chain, γ4 chain, or δ chain of human immunoglobulin.

9. The multispecific antibody or its antigen-binding molecule according to claim 8, wherein: The Fc domain of human IgG1 sequentially contains the heavy chain constant region CH2 and the heavy chain constant region CH3 from the N-terminus to the C-terminus; Preferably, the sequence of the heavy chain constant region CH2 is as shown in SEQ ID NO.17; Preferably, the sequence of the heavy chain constant region CH3 is as shown in SEQ ID NO.

18.

10. A homodimer of a multispecific antibody or its antigen-binding molecule, wherein: The homodimer of the multispecific antibody or its antigen-binding molecule is: when the multispecific antibody or its antigen-binding molecule described in any one of claims 7 to 9 is expressed in a host cell, the homodimer formed by the homologous dimerization of the domains of the heavy chain constant region.

11. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the multispecific antibody or its antigen-binding molecule described in any one of claims 1 to 9.

12. A vector containing the nucleic acid molecule described in claim 11; preferably, the vector is an expression vector; preferably, the vector is a viral vector; preferably, the vector is a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

13. A host cell containing the vector described in claim 12; preferably, the host cell is a mammalian cell.

14. A method for producing a multispecific antibody or an antigen-binding molecule thereof as described in any one of claims 1 to 9, or for producing a homodimer as described in claim 10, characterized in that: It is obtained by transfection of a host cell with the vector described in claim 12 or its vector system; preferably, a lentiviral vector containing the nucleic acid molecule described in claim 11 or its vector system is used; preferably, an adeno-associated viral vector containing the nucleic acid molecule described in claim 11 or its vector system is used.

15. A recombinant protein, characterized in that: The recombinant protein contains the multispecific antibody or its antigen-binding molecule described in any one of claims 1 to 9, or contains the homodimer described in claim 10.

16. An immunoconjugate, which contains the multispecific antibody or its antigen-binding molecule described in any one of claims 1 to 9, or contains the homodimer described in claim 10; preferably, one or more heterologous molecules are conjugated to the multispecific antibody or its antigen-binding molecule described in any one of claims 1 to 9 or the homodimer described in claim 10; more preferably, the heterologous molecule is a cytotoxin.

17. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the multispecific antibody or its antigen-binding molecule as described in any one of claims 1 to 9, or comprises the homodimer as described in claim 10, or comprises the nucleic acid molecule as described in claim 11, or comprises the vector as described in claim 12, or comprises the host cell as described in claim 13, or comprises the recombinant protein as described in claim 15, or comprises the immunoconjugate as described in claim 16, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is in the form of an injection or a dosage form for mucosal administration; more preferably, the pharmaceutical composition is in the form of an intraperitoneal injection, an intravenous injection or an intramuscular injection; more preferably, the pharmaceutical composition is in the form of a nasal spray, a nasal drop, an aerosol inhalation, a nasal lavage solution, an oral liquid, a mouthwash; more preferably, the pharmaceutical composition is in the form of a tablet, a capsule or a powder; more preferably, the pharmaceutical composition is in the form of an external ointment or cream, an external gel, an external lotion or an external spray.

18. A detection product, characterized in that: The detection product comprises the multispecific antibody or its antigen-binding molecule as described in any one of claims 1 to 9, or comprises the homodimer as described in claim 10, or comprises the nucleic acid molecule as described in claim 11, or comprises the vector as described in claim 12, or comprises the host cell as described in claim 13, or comprises the recombinant protein as described in claim 15, or comprises the immunoconjugate as described in claim 16.

19. Use of the multispecific antibody or its antigen-binding molecule as described in any one of claims 1 to 9, or the homodimer as described in claim 10, or the nucleic acid molecule as described in claim 11, or the vector as described in claim 12, or the host cell as described in claim 13, or the recombinant protein as described in claim 15, or the immunoconjugate as described in claim 16 in the preparation of a medicament for treating or preventing a respiratory tract infectious disease caused by a virus, in the preparation of a medicament for treating or preventing a respiratory tract inflammation, and in the preparation of a medicament for treating or preventing dermatitis; preferably, the virus is a coronavirus; preferably, the respiratory tract inflammation is rhinitis or asthma, and preferably, the dermatitis is atopic dermatitis.

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