Multispecific antibody

Abstract

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, a recombinant protein or immunoconjugate comprising the multispecific antibody or the antigen-binding molecule thereof, a use thereof in the preparation of a medicament for treating or preventing respiratory tract infections caused by viruses or in the preparation of a medicament for treating or preventing respiratory tract inflammation or dermatitis, and in a detection product. The multispecific antibody or the antigen-binding molecule thereof has excellent broad-spectrum neutralizing capability for coronaviruses and significant neutralizing capacity for coronavirus mutant strains, especially a variety of SARS-CoV-2 coronavirus mutant strains including Omicron subvariants BQ.1.1, XBB.1.5, XBB.1.16, EG.5, BA.2.86, and JN.1. The multispecific antibody or the antigen-binding molecule thereof can not only treat or prevent respiratory tract infection symptoms caused by viruses but also significantly relieve respiratory tract inflammation responses. The multispecific antibody or the antigen-binding molecule thereof has a good therapeutic effect on inflammatory diseases such as rhinitis, asthma, and dermatitis and has good clinical application prospects in the future.

Description

A multispecific antibody TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and 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, a recombinant protein or an immunoconjugate comprising the multispecific antibody or the antigen-binding molecule thereof, and application of the multispecific antibody or the antigen-binding molecule thereof in preparation of a drug for treating or preventing a viral respiratory infection disease or a drug for treating or preventing respiratory inflammation (such as rhinitis, asthma, etc.) or dermatitis, and application in detection products. BACKGROUND

[0002] The novel coronavirus is transmitted through the air and infects the respiratory tract, which can cause patients to have respiratory infection symptoms such as cough, fever, and sore throat. A small part of the infected people will progress to pneumonia, and severe cases can lead to systemic inflammatory storm and systemic organ failure.

[0003] The novel coronavirus continues to mutate with stronger immune escape ability, viral infection ability, and transmission capacity, leading to repeated infection of the population. At the same time, long-term infection and post-infection sequelae of the population seriously affect people's physical health.

[0004] SARS-CoV-2 belongs to the Coronaviridae family, and is a sarbecovirus in the Betacoronavirus genus, which is highly homologous to 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 S protein). The spike protein is hydrolyzed into S1 and S2 by intracellular proteases during the viral infection process, wherein S2 is a transmembrane protein, and S1 has a receptor binding domain (Receptor binding domain, abbreviated as RBD) and a N-terminal domain (N-terminal domain, abbreviated as NTD) domain. The RBD protein can recognize and bind to the human angiotensin-converting enzyme 2 (ACE-2) receptor expressed on the cell. The spike protein composed of S1 and S2 is a protein that specifically recognizes, binds to the target cell receptor, mediates the fusion of the viral membrane and the cell membrane to play a role, and mediates the viral protein of the virus-infected target cell, and is the recognition target point of the neutralizing antibody.

[0005] Many studies have reported neutralizing antibodies isolated from COVID-19 infected patients or vaccinees. These neutralizing antibodies mainly bind to the SARS-CoV-2 spike protein, on the one hand, they can inhibit the interaction between the virus particles and the receptor, or destroy the virus particles, thereby preventing the virus from entering the target cells of the infected organism; on the other hand, these neutralizing antibodies can bind to virus-infected cells and mediate the immune system to kill or phagocytose virus-infected cells, thereby eliminating virus-infected cells.

[0006] SARS-CoV-2 is an RNA virus, and when it spreads in the population, mutations are easily generated during the replication process of the viral genome, and a new mutant strain replaces the old mutant strain every half year to form an iteration of the pandemic. The Omicron mutant strain was first discovered in December 2021, and within a month, it had caused a pandemic worldwide. In the past two years, the Omicron mutant strain has evolved into BA.5, BF.7, BQ.1.1, XBB.1.5, XBB.1.16, EG.5, BA.2.86, JN.1, etc. Subtype mutant strains, especially BA.2.86 and JN.1, have 39 mutations in the spike protein sequence based on BA.2, resulting in severe immune escape, not only escaping the immune environment formed by vaccination or previous infection, but also causing many antibody or immunoglobulin drugs to be inactivated. Single antibody therapy targeting a single epitope has been difficult to cope with rapidly mutating viruses, and antibody cocktails have not significantly improved drug efficacy, so a large number of antibody drugs approved for clinical emergency use have been withdrawn, and antibody drugs in clinical trials have also been difficult to advance in the clinic.

[0007] In order to improve the neutralization breadth and neutralization ability of antibodies against COVID-19, researchers have genetically engineered existing COVID-19 neutralizing antibodies, hoping to construct antibodies that can bind to multiple epitopes of coronaviruses, inhibit viral mutation escape, and obtain antibodies with better broad-spectrum and neutralization activity, such as antibodies that can bind to multiple coronavirus immunogens, or inhibit multiple coronavirus-infected target cells, especially inhibit multiple novel coronavirus mutant strains from infecting target cells or the human body, or can eliminate virus-infected cells in the human body. SUMMARY

[0008] To solve the above technical problems, the first aspect of the present application provides a multispecific antibody or antigen binding molecule thereof, 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 (RBM) part of the receptor binding region of the coronavirus S protein;

[0010] the first antigen binding moiety binds to a part of the receptor binding motif of the receptor binding domain of the coronavirus S-protein other than the receptor binding motif.

[0011] In one embodiment of the application, the first antigen binding moiety comprises a light chain variable region VL-1 and a heavy chain variable region VH-1; the second antigen binding moiety 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 a LCDR1-1 sequence of a light chain variable region as depicted in SEQ ID NO. 1, a LCDR2-1 sequence of a light chain variable region as depicted in SEQ ID NO. 2 and a LCDR3-1 sequence of a light chain variable region as depicted in SEQ ID NO. 3;

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

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

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

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

[0017] the sequence of the heavy chain variable region VH-1 is depicted in SEQ ID NO. 14 or has more than 80% sequence homology to the sequence depicted in SEQ ID NO. 14;

[0018] the sequence of the light chain variable region VL-2 is depicted in SEQ ID NO. 15 or has more than 80% sequence homology to the sequence depicted in SEQ ID NO. 15;

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

[0020] In a specific embodiment of the present application, the above-mentioned light chain variable region VL-1 can be subjected to a small number of amino acid deletions, insertions or amino acid mutations on the basis of the above-mentioned sequence to obtain an amino acid sequence with more than 80% homology. A small number of amino acid substitutions (deletions or insertions, or amino acid mutations, or substitution of similar amino acids), especially conservative amino acid substitutions in the framework region part of the variable region, the variants obtained have high homology (more than 80% homology) with the above-mentioned sequence, and retain the original properties and functions of the light chain variable region, i.e. retain the properties and functions of the antibody specifically binding to the coronavirus, or have the function of treating, preventing the symptoms of respiratory tract infection caused by the virus, or have the function of alleviating or relieving the symptoms of respiratory inflammation, and these variants also fall within the scope of protection of the present application.

[0021] Similarly, the above-mentioned heavy chain variable region VH-1 can also be subjected to a small number of amino acid deletions, insertions or amino acid mutations, especially conservative amino acid substitutions in the framework region part of the variable region, the variants obtained retain the original properties and functions of the heavy chain variable region, i.e. retain the properties and functions of the antibody specifically binding to the coronavirus, or have the function of treating, preventing the symptoms of respiratory tract infection caused by the virus, or have the function of alleviating or relieving the symptoms of respiratory inflammation, and these variants also fall within the scope of protection of the present application.

[0022] Similarly, the above-mentioned heavy chain variable region VH-2 also applies to the light chain variable region VL-2 and the heavy chain variable region VH-2, which will not be described in detail.

[0023] In a specific embodiment of the present application, the first antigen binding module is selected from any one of Fv, Fab, Fab', dsFv or scFv; and 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 application, the first and second antigen binding modules are Fab or Fab' fragments. In still another alternative embodiment of the present application, one of the first and second antigen binding modules is a Fab or Fab' fragment, and the other is a Fv, dsFv or scFv.

[0025] Preferably, the first antigen binding module and the second antigen binding module are selected from 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 one specific embodiment of the present application, the scFv-1 comprises, in order from N-terminus to C-terminus, the light chain variable region VL-1, a first linker peptide, and the heavy chain variable region VH-1; or the scFv-1 comprises, in order from N-terminus to C-terminus, the heavy chain variable region VH-1, a first linker peptide, and the light chain variable region VL-1.

[0027] The scFv-2 comprises, in order from N-terminus to C-terminus, the light chain variable region VL-2, a second linker peptide, and the heavy chain variable region VH-2; or the scFv-2 comprises, in order from N-terminus to C-terminus, the heavy chain variable region VH-2, a second linker peptide, and the light chain variable region VL-2.

[0028] Preferably, the first linker peptide or the second linker peptide is selected from the group consisting of 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 one specific embodiment of the present application, 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.

[0030] Preferably, the linker peptide is selected from the group consisting of 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 n is in the range of 1-10.

[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 to the immobilized ligand, and is a polypeptide sequence designed for the purpose of fusion expression with the target protein for the purpose of purification. 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 application, the multispecific antibody or antigen binding molecule thereof of the present application can further comprise more antigen binding modules, which can be the same as the first / second antigen binding module, or can be different, for example, can be an antigen binding module binding to other antigens.

[0033] In an alternative embodiment of the present application, the first / second antigen binding module is selected from an animal-derived antibody, for example, a murine-derived antibody or its immunoglobulin, a camel-derived antibody or its immunoglobulin, a humanized antibody or a chimeric antibody.

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

[0035] In an alternative embodiment of the present application, the heavy chain constant region and / or the light chain constant region can 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 application, the heavy chain constant region is preferably a 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 application, 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 connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide in sequence, and the C-terminus of the Fc domain of human IgG1 is connected to the N-terminus of the scFv-2 through a fifth linker peptide; or,

[0040] the C-terminus of the scFv-2 is connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide in sequence, and the C-terminus of the Fc domain of human IgG1 is connected to the C-terminus of the 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 1-10.

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

[0043] In another alternative embodiment, the hinge peptide can be selected from the heavy chains of different subtypes of human immunoglobulin, for example, can be selected from the alpha 1 chain, the alpha 2 chain, or the gamma 1 chain, the gamma 2 chain, the gamma 3 chain, the gamma 4 chain, or the delta chain.

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

[0045] The second aspect of the present application provides a homodimer of a multispecific antibody or an antigen binding molecule thereof, wherein,

[0046] The homodimer of the multispecific antibody or the antigen-binding molecule thereof is a homodimer formed by the homodimerization of the domains of the heavy chain constant region when the multispecific antibody or the antigen-binding molecule thereof is expressed in a host cell. The homodimerization is, for example, the formation of a disulfide bond by the amino acid interaction of the domains of the hinge region and the heavy chain constant region.

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

[0048] The fourth aspect of the present application provides a vector comprising the nucleic acid molecule as described above, i.e., a vector comprising the nucleic acid molecule encoding the multispecific antibody or the antigen-binding molecule thereof as described above, in particular an expression vector expressing the multispecific antibody or the antigen-binding molecule thereof as described above.

[0049] Preferably, the vector can be transcribed, translated, and modified to form the multispecific antibody or the antigen-binding molecule thereof having the functional activity of inhibiting the infection of coronavirus or inhibiting the inflammation of respiratory allergic inflammation or dermatitis inflammation as described above.

[0050] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed. The vector can be transformed, transduced, or transfected into a host cell, so that the genetic material elements carried by the vector can be expressed in the host cell. The vector can comprise various elements for controlling expression, such as a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene, etc. In addition, the vector can also contain a replication initiation site. The vector can also include components that assist its entry into the cell, such as viral particles, liposomes, or protein coats, but not only these. In the embodiments of the present application, the vector can be selected from, but not limited to: a plasmid, a phagemid, a cosmid, an artificial chromosome (such as a yeast artificial chromosome YAC, a bacterial artificial chromosome BAC, or a P1-derived artificial chromosome PAC), a bacteriophage (such as a lambda phage or a M13 phage), and animal viruses used as vectors, for example, a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus (such as a herpes simplex virus), a poxvirus, a baculovirus, a papillomavirus, a papovavirus (such as SV40).

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

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

[0053] In another specific embodiment of the application, 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 a liposome or other lipid-containing complex) to stably express the antibody or antigen-binding molecule thereof of the application in the body (e.g. in a patient) in a genome-integrated or non-integrated manner.

[0054] The fifth aspect of the application provides a host cell comprising the above-mentioned vector.

[0055] As for "host cells", the following can be selected, but are not limited to: prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 fruit fly cells or Sf9, or animal cell models such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or cells in the body (e.g. in the human body) that can express the protein molecule. Preferably, the host cell is a HEK293 cell.

[0056] In a preferred embodiment of the application, the host cell can be an in-vivo isolated cell cultured in vitro, which is engineered to express the above-mentioned multispecific antibody or antigen-binding molecule thereof in a membrane fusion form or a soluble form, and can achieve the functions of preventing or treating respiratory tract infection diseases caused by viruses including coronaviruses, relieving or reducing respiratory tract inflammation (such as rhinitis, asthma, etc.), and relieving or reducing skin inflammation (such as dermatitis) when reinfused into a patient.

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

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

[0059] The seventh aspect of the present application provides a method for producing a homodimer of the multispecific antibody or the antigen-binding molecule thereof, which comprises culturing the above-mentioned host cell, wherein the domains of the heavy chain constant region of the multispecific antibody or the antigen-binding molecule thereof expressed in the host cell are homodimerized to produce the homodimer of the multispecific antibody or the antigen-binding molecule thereof.

[0060] The other aspects of the present application also provide a glycosylation variant, a cysteine-engineered antibody variant, an antibody derivative, and an immunoconjugate of the multispecific antibody or the antigen-binding molecule thereof.

[0061] The eighth aspect of the present application provides a recombinant protein comprising the multispecific antibody or the antigen-binding molecule thereof, or the homodimer thereof.

[0062] The ninth aspect of the present application provides an immunoconjugate comprising the multispecific antibody or the antigen-binding molecule thereof, or the homodimer thereof.

[0063] Preferably, the conjugating moiety of the immunoconjugate comprises one or more heterologous molecules, such as a cytotoxic heterologous molecule applicable to the immunoconjugate.

[0064] The tenth aspect of the present application provides a pharmaceutical composition comprising the multispecific antibody or the antigen-binding molecule thereof, or the homodimer thereof, or the nucleic acid molecule, or the vector, or the host cell, or the recombinant protein, or the immunoconjugate, and a pharmaceutically acceptable carrier.

[0065] In one embodiment of the present application, the pharmaceutical composition can be in a known dosage form, such as a pre-filled syringe, an auto-injector, a pre-filled pen; preferably, the dosage form is generally administered by injection, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and sternal injection and infusion; more preferably, the pharmaceutical composition is administered by a non-ordinary route, such as topical, epidermal or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual or topical administration; more preferably, the pharmaceutical composition is in the form of a nasal spray, a nasal drop, an aerosol inhaler, a nasal lavage, an oral liquid, a gargle.

[0066] In one embodiment of the present application, the pharmaceutical composition is in the form of a tablet, a capsule or a powder.

[0067] In one embodiment of the present application, the pharmaceutical composition is in the form of a topical ointment or cream, a topical gel, a topical lotion or a topical spray.

[0068] In one embodiment of the present application, the pharmaceutical composition is in the form of a tablet, a capsule or a powder.

[0069] In one embodiment of the present application, the virus refers to a virus capable of causing respiratory tract infection, 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 one preferred embodiment of the present application, the use refers to the use in the preparation of a medicament for treating or preventing a respiratory tract infection disease caused by a coronavirus. More preferably, the coronavirus includes SARS-CoV-2 and its mutant strains, SARS-CoV or SARS-like coronavirus, etc. In a more preferred embodiment of the present application, the SARS-CoV-2 mutant strain is Alpha, Beta, Gamma, Delta or Omicron sub-type mutant strain (e.g. 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 application provides the use of the multispecific antibody or antigen binding molecule thereof described above, or the homodimer described above, or the nucleic acid molecule described above, or the vector described above, or the host cell described above, or the recombinant protein described above, or the immunoconjugate described above, 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., and its clinical manifestations include rhinorrhea with hyposmia, sneezing, nasal congestion, occlusive nasal tone, nasal dryness and itching, headache, wheezing, dyspnea, etc.

[0073] Asthma is a disease characterized by chronic airway inflammation, which leads to significantly increased reactivity of the airways to various stimuli (i.e., airway hyperresponsiveness). Patients typically experience recurrent episodes of wheezing, shortness of breath, breathlessness, chest tightness, and coughing, especially at night or in the morning. Chronic inflammation can also lead to remodeling and functional impairment of the airway structure, exacerbating the condition and affecting quality of life.

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

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

[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 application, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip, a detection test paper, a detection instrument, etc.

[0078] The multispecific antibody or antigen binding molecule thereof of the present application can be labeled by a chemical method or a genetic engineering method, and the labeled antibody or antigen binding molecule thereof can be used for detection; the labeled antibody or antigen binding molecule thereof falls within the protection scope of the present application.

[0079] Specific detection methods can adopt the following steps: 1) providing a sample; 2) contacting the sample with the multispecific antibody or antigen binding molecule thereof of the present application; 3) detecting the immune reaction between the sample and the antibody or antigen binding molecule thereof.

[0080] The present application also provides a method for treating or preventing a respiratory infection disease caused by a virus, or a method for preparing a treatment or prevention of respiratory inflammation, a method for preparing a treatment or prevention of dermatitis, wherein a therapeutically effective amount of the multispecific antibody or antigen binding molecule thereof of the present application or a homodimer thereof is administered to a patient; or a pharmaceutical composition comprising a therapeutically effective amount of the multispecific antibody or antigen binding molecule thereof of the present application or a homodimer thereof is administered to a patient. BRIEF DESCRIPTION OF DRAWINGS

[0081] FIG. 1 is a plasmid map of the pcDNA3.4-Fc expression vector used in the preparation of the antibodies of embodiments 1 and 2 of the present application;

[0082] FIG. 2 is a plasmid map of the pcDNA3.4 expression vector used in the preparation of the antibodies of embodiments 3 and 4 of the present application;

[0083] FIG. 3 is a SDS-PAGE diagram of embodiments 1-4 of the present application;

[0084] FIG. 4 is a flowchart of the operation of detecting the treatment and prevention of new coronavirus infection by the antibodies of embodiments of the present application in mice;

[0085] FIG. 5 is the result of the treatment and prevention of new coronavirus XBB.1 infection by the antibodies of embodiment 1 of the present application in human ACE2 transgenic mice;

[0086] FIG. 6 is the result of the treatment and prevention of new coronavirus EG.5 infection by the antibodies of embodiment 1 of the present application in human ACE2 transgenic mice;

[0087] FIG. 7 is a flowchart of the modeling process of rhinitis mice and the treatment of rhinitis mice by the antibodies of embodiments of the present application;

[0088] FIG. 8 is a graph of the body weight change and body weight change trend of mice during the treatment of rhinitis mice by the antibodies of embodiment 1 of the present application;

[0089] FIG. 9 is a statistical result of the number of sneezing of mice during the treatment of rhinitis mice by the antibodies of embodiment 1 of the present application;

[0090] Figure 10 is the concentration of OVA-sIgE in serum of mice treated with the antibody of Example 1 of the present application for 8 days;

[0091] Figure 11 is the concentration of histamine in serum of mice treated with the antibody of Example 1 of the present application for 8 days;

[0092] Figure 12 is the pathological HE staining of nasal mucosa of mice treated with the antibody of Example 1 of the present application for 8 days;

[0093] Figure 13 is the experimental flow chart of the process of modeling asthma mice and treating asthma mice with the antibody of Example of the present application;

[0094] Figure 14 is the weight change and weight change trend of mice treated with the antibody of Example 1 of the present application for 7 days;

[0095] Figure 15 is the statistical result of allergic asthma score of mice treated with the antibody of Example 1 of the present application for 7 days;

[0096] Figure 16 is the concentration of OVA-sIgE in bronchoalveolar lavage fluid and serum of mice treated with the antibody of Example 1 of the present application for 7 days;

[0097] Figure 17 is the concentration of IL-4 in bronchoalveolar lavage fluid and serum of mice treated with the antibody of Example 1 of the present application for 7 days;

[0098] Figure 18 is the concentration of IL-5 in bronchoalveolar lavage fluid and serum of mice treated with the antibody of Example 1 of the present application for 7 days;

[0099] Figure 19 is the pathological HE staining of lung of mice treated with the antibody of Example 1 of the present application for 7 days;

[0100] Figure 20 is the experimental flow chart of the process of modeling dermatitis mice and treating dermatitis mice with the antibody of Example 1 of the present application;

[0101] Figure 21 is the weight change and weight change trend of mice treated with the antibody of Example 1 of the present application for 20 days;

[0102] Figure 22 is the spleen mass coefficient of mice treated with the antibody of Example 1 of the present application for 20 days;

[0103] Figure 23 is the skin lesion score trend of mice treated with the antibody of Example 1 of the present application for 20 days;

[0104] Figure 24 is the skin photo of mice treated with the antibody of Example 1 of the present application for 20 days;

[0105] Figure 25 is the concentration of TNF-α in serum of mice treated with the antibody of Example 1 of the present application for 20 days;

[0106] Figure 26 is the concentration of IL-1 β in serum after 20 days of treatment of mice with dermatitis with the antibody of Example 1 of the present application. DETAILED DESCRIPTION

[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 application 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 application will be apparent from the description and drawings, and from the claims.

[0108] To interpret the specification, the following definitions will be employed, and whenever appropriate and feasible, the singular forms also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0109] The term "about" when used in connection with a numerical value means encompassing numerical values 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," or "having" means including but not limited to.

[0111] The term "antibody" is used herein in the broadest sense to include various structural forms of natural and artificial antibodies, 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, and the like that exhibit the desired antigen-binding activity.

[0112] The terms "antigen-binding molecule" and "antibody-binding fragment" are used interchangeably herein to refer to molecules that are specific portions of intact antibodies that bind to antigens. Antigen-binding molecules can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.

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

[0114] The terms "antigen binding module", "antigen binding site" refer to the region of an antibody molecule that actually binds to an antigen, including, for example, a VH / VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). In some embodiments of the present application, bispecific antibodies are provided, which 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 application, the antigen binding module is selected from any one of Fv, Fab, Fab', dsFv or scFv.

[0116] The Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, for example, Fab fragments can be obtained by papain digestion of whole antibodies. The Fab' monomer is essentially a Fab fragment with the hinge region (for more detailed description of other antibody fragments see: Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993)). F(ab')2 is bivalent fragment of the antibody of the IgG type, which is produced by the proteolytic digestion of whole IgG antibodies with the enzyme pepsin, which removes part of the Fc region leaving some hinge region; F(ab')2 fragments are Fab' dimers, which are bivalent antibody fragments. F(ab')2 can be reduced under neutral conditions to form Fab' monomers, which are monovalent antibody fragments. The Fv fragment consists of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent antibody fragment known as single chain Fv (scFv, also known as single chain antibody). The dsFv, refers to disulfide-stabilized Fv (dsFv), is a new type of small molecule antibody developed on the basis of scFv, which is an antibody in which each of the amino acid residues of VH and VL is mutated to cysteine, and the VH and VL variable regions are connected by inter-chain disulfide bond, which can enhance the stability of Fv. The above antigen binding modules can be obtained by chemical methods, recombinant DNA methods or protease digestion.

[0117] As for the combination of the two antigen binding modules of the bispecific antibody, according to the structure of the antigen binding module, it mainly includes Fab-Fab combination, Fab-Fv combination and Fv-Fv combination.

[0118] Among them, the Fab-Fab combination bispecific antibody mainly includes: Triomab produced by hybrid hybridomas through rat and mouse hybridoma cell re-fusion technology, various bsIgG produced by Fc heterodimer technology (such as Knob-in-Hole, charge pairing, SEED, BEAT, LUZ-Y and Duobody, etc.) and avoiding Fab mismatch technology (such as CrossMab, common light chain, single-chain Fab, Kappa Lambda body, Orthogonal Fab, Duetmab and TCR-CaCp, 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 chemical cross-linking technology, and Fab connection molecules produced by various Fab cross-linking technologies (such as F(ab')2, Dock and Lock, etc.). The Fab-Fab bispecific antibody uses Fab to completely retain the high affinity of the original antibody while combining two antigen recognition binding domains, and has higher stability due to its structure closer to natural IgG. However, in order to avoid the mismatch of heavy and light chains between two different Fabs, common light chains or mutations are needed to form a preferential pairing.

[0119] In the Fab-Fv combination bispecific antibody, one of the binding domains that recognize antigens or antigen epitopes is Fab, and the other is Fv. Broadly speaking, 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.), ligands or receptor molecules, and engineered ligands or receptor molecules, etc. Representative structures include: scFv-Fab, scFv-IgG, DVD-IgG, Fab-scFv-Fc, etc.

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

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

[0122] In one embodiment of the application, a bispecific antibody format of scFv-scFv-Fc is provided; one skilled in the art, upon knowing the bispecific format of the application and the sequences of its antigen binding modules, can engineer it into any of the above known combinations using existing bispecific technology, for example, a Fab-Fab combination, a Fab-Fv combination, or other Fv-Fv combination bispecific antibody.

[0123] In one embodiment of the application, the antigen binding module is a scFv, comprising a heavy chain variable region (VH region) and a light chain variable region (VL region).

[0124] As to the heavy chain variable region (VH region) and the light chain variable region (VL region), they can be further subdivided into complementary determining regions (CDRs) and framework regions (FRs); the CDRs are hypervariable regions, which are interspersed with FR regions that are more conserved. Each of VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The precise amino acid sequence boundaries of each CDR in a given VHor VLamino acid sequence can be determined using any one of a number of well-known schemes or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., 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 application can be determined with boundaries according to any of the schemes or combinations thereof in the art and human evaluation.

[0125] The term "Fc domain" or "Fc region" is used herein to define a carboxy-terminal region of human immunoglobulin heavy chains that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. Native immunoglobulin "Fc domains" comprise two or three constant domains, i.e., CH2 domains, CH3 domains, and optionally CH4 domains. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of a heavy chain derived from an IgG, IgA, and IgD class antibody; or the second, third and fourth constant domains (CH2, CH3 and CH4 domains) of a heavy chain derived from an IgM and IgE class antibody. Unless otherwise specified herein, amino acid residue positions in an Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as EU index) as set forth 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 an antibody provided herein to thereby generate Fc region variants with enhanced efficacy. For example, mutation of the methionine at position 428 of the Fc domain to a leucine, together with mutation of the asparagine at position 434 of the Fc domain to a serine, can increase 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 Fc. Modifications to the Fc can alter the half-life of the antibody in a therapeutic antibody, allowing for less frequent dosing and thus increased convenience and reduced material use. Changes to the Fc can also enhance the activity of the antibody in vivo, exerting effector functions of the Fc, allowing for multifunctional activity through multiple pathways for more effective and rapid relief of disease.

[0127] The term "linker peptide" refers to a linking peptide segment composed of amino acids, such as glycine and / or serine residues, used alone or in combination, to link individual variable domains in an antibody. In certain embodiments, the linker peptide can be from about 1 to about 100 amino acids long, for example, from about 1 to 50 amino acids long. In one embodiment, the linking peptide is a G / S linking peptide, non-limiting examples of which are disclosed in the literature (Shen et al., Anal. Chem. 80(6): 1910-1917 (2008)) and patents (WO 2014 / 087010), the contents of which are incorporated herein in their entirety.

[0128] As used herein, the term "binds" or "binds specifically" means that the binding 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 particular antigen can be determined by enzyme linked immunosorbent assay (ELISA) or routine binding assays known in the art.

[0129] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally 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 particular method for measuring affinity is the Biacore® Bio-layer Interferometry technique to measure the dissociation rate of association between two molecules.

[0130] The term "antigen" refers to a molecule that elicits an immune response. Such an immune response can involve antibody production or activation of specific immune cells, or both. The skilled artisan will understand that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA.

[0131] An "immunoconjugate" is a conjugate of an antibody and one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0132] The percent "sequence identity" with respect to a specified amino acid sequence is determined by ascertaining the number of positions at which a candidate amino acid sequence and the specified sequence have an amino acid residue in common, dividing the number of positions having an amino acid residue in common by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Optimal alignment of sequences for determining percent sequence identity can be achieved by using a number of alignment programs known in the art, for example, by using the publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length or a specified region of a sequence being compared.

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

[0134] Similarly, in terms of antibody sequences, based on alignment, candidate antibodies having amino acid changes in a region of the antibody relative to a reference antibody can be determined.

[0135] In the present application, "conservative substitutions" refer to amino acid changes that result in the replacement of one amino acid with a chemically similar amino acid. Amino acid modifications such as substitutions can be introduced into an antibody of the application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

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

[0137] Conservative substitution groups

[0138] The term "N-terminal" refers to the amino terminus of a protein or peptide segment, and the term "C-terminal" refers to the carboxyl terminus of a protein or peptide segment.

[0139] An antibody CDR region, "complementarity determining region" or "CDR region" or "CDR" (used interchangeably herein with "hypervariable region," "HVR"), is a region of an antibody variable region that is primarily responsible for binding with the antigen epitope. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domain of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0140] A number of schemes for determining the CDR sequences within a given VH or VL amino acid sequence are well known in the art. For example, the Kabat Complementarity Determining Regions (CDRs) are determined 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 location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVR is a compromise between the Kabat HVR and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures (Deinum et al., J. Mol. Biol. 303: 467-478 (2000)).

[0141] Unless otherwise indicated, in the present application, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), the numbering is according to the Kabat numbering system.

[0142] Antibodies having different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although the CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, a minimal overlap region can be determined, providing a "minimal binding unit" for antigen binding. The minimal binding unit can be a sub-portion of a CDR. As will be apparent to one of skill in the art, by the structure and protein folding of the antibody, the residues of the remainder of the CDR sequence can be determined. Thus, variants of any of the CDRs given herein are also contemplated by the present application. For example, in a variant of one CDR, the amino acid residues of the minimal binding unit can remain the same, while the remaining CDR residues according to the Kabat or Chothia definition can be replaced by conservative amino acid residues.

[0143] A "hinge peptide" or "hinge region" generally refers to amino acids Glu216 to Pro230 of human IgGl (see Burton, Molec. Immunol. 22:161-206 (1986)). In certain embodiments, the hinge region of other immunoglobulin heavy chains can be aligned with the IgGl sequence by placing the first and last cysteine residues that form an inter-heavy chain S-S bond in the same position.

[0144] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, and includes the progeny of the cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the application, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as E. coli cells. Host cells include cells in culture as well as cells within a transgenic animal, transgenic plant, or cultured plant tissue or animal tissue.

[0145] The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0146] The term "coronavirus" refers to a single-stranded RNA virus, the virus particle is spherical particles with envelope structure. According to the International Committee on the Taxonomy of Viruses classification of coronaviruses, it can be divided into the family of orthocoronaviruses and the subfamily of letoviruses, and the family of orthocoronaviruses can be divided into four different coronavirus genera of alpha, beta, gamma and delta. The novel coronavirus belongs to the beta coronavirus and can cause acute respiratory infection. The clinical manifestations of infection are mainly fever, fatigue, and dry cough, and a few have symptoms such as nasal congestion, runny nose, sore throat, nausea, diarrhea, etc. Severe patients often have dyspnea, septic shock, coagulation dysfunction, multiple organ failure, etc.

[0147] The term "S protein" refers to one of the structural proteins encoded by the coronavirus genome, the spike protein protruding on the surface of the virus particle, and the S protein forms a special crown structure on the surface of the virus in the form of a trimer. The role of the S protein is to be responsible for the binding of the virus to the receptor of the host cell, to mediate the fusion of the virus membrane with the receptor membrane, and to cause a specific immune response in the body, to produce neutralizing antibodies, and to be an important protein for viral infection.

[0148] Example 1

[0149] The antibody of Example 1 comprises a first antigen binding moiety and a second antigen binding moiety, which are the single-chain antibody fragments scFv-1 and scFv-2, respectively.

[0150] In a specific embodiment of the present application, the light chain variable region VL-1 of scFv-1 comprises a LCDR1-1 sequence of light chain variable region as shown in SEQ ID NO. 1, a LCDR2-1 sequence of light chain variable region as shown in SEQ ID NO. 2, and a LCDR3-1 sequence of light chain variable region as shown in SEQ ID NO. 3; the heavy chain variable region VH-1 of scFv-1 comprises a HCDR1-1 sequence of heavy chain variable region as shown in SEQ ID NO. 4, a HCDR2-1 sequence of heavy chain variable region as shown in SEQ ID NO. 5, and a HCDR3-1 sequence of heavy chain variable region as shown in SEQ ID NO. 6.

[0151] In a preferred embodiment of the present application, the sequence of the light chain variable region VL-1 of scFv-1 is as shown in SEQ ID NO. 13, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO. 13; the sequence of the heavy chain variable region VH-1 of scFv-1 is as shown in SEQ ID NO. 14, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO. 14.

[0152] In particular in the present embodiment, the sequence of the light chain variable region VL-1 of scFv-1 is as shown in SEQ ID NO. 13, and the sequence of the heavy chain variable region VH-1 of scFv-1 is as shown in SEQ ID NO. 14.

[0153] Specifically, according to the latest research progress of the present inventor, the variable region of scFv-1 is combined outside the receptor binding motif (RBM), i.e. the binding epitope of scFv-1 is not located in the RBM of RBD of S1 protein, but outside the motif.

[0154] In a specific embodiment of the present application, the light chain variable region VL-2 of scFv-2 comprises a LCDR1-2 sequence of light chain variable region as shown in SEQ ID NO. 7, a LCDR2-2 sequence of light chain variable region as shown in SEQ ID NO. 8, and a LCDR3-2 sequence of light chain variable region as shown in SEQ ID NO. 9; the heavy chain variable region VH-2 of scFv-2 comprises a HCDR1-2 sequence of heavy chain variable region as shown in SEQ ID NO. 10, a HCDR2-2 sequence of heavy chain variable region as shown in SEQ ID NO. 11, and a HCDR3-2 sequence of heavy chain variable region as shown in SEQ ID NO. 12.

[0155] In a preferred embodiment of the present application, the sequence of the light chain variable region VL-2 of the scFv-2 is as set forth in SEQ ID NO. 15, or has more than 80% sequence homology with the sequence set forth in SEQ ID NO. 15; the sequence of the heavy chain variable region VH-2 of the scFv-2 is as set forth in SEQ ID NO. 16, or has more than 80% sequence homology with the sequence set forth in SEQ ID NO. 16.

[0156] In particular in the present embodiment, the sequence of the light chain variable region VL-2 of the scFv-2 is as set forth in SEQ ID NO. 15; the sequence of the heavy chain variable region VH-2 of the scFv-2 is as set forth in SEQ ID NO. 16.

[0157] In particular, the variable region of scFv-2 binds to the receptor binding motif (RBM) portion of RBD, and the binding epitope of scFv-2 is located in the RBM of RBD of S1 protein.

[0158] In a particular embodiment of the present application, scFv-1 comprises, in order from N-terminus to C-terminus, the light chain variable region VL-1, a first linker peptide, and the heavy chain variable region VH-1; or, scFv-1 comprises, in order from N-terminus to C-terminus, the heavy chain variable region VH-1, a first linker peptide, and the light chain variable region VL-1.

[0159] Likewise, in another particular embodiment of the present application, scFv-2 comprises, in order from N-terminus to C-terminus, the light chain variable region VL-2, a second linker peptide, and the heavy chain variable region VH-2; or, scFv-2 comprises, in order from N-terminus to C-terminus, the heavy chain variable region VH-2, a second linker peptide, and the light chain variable region VL-2.

[0160] The first linker peptide (L1) and the second linker peptide (L2) are also known as connecting peptide or linker. In a particular embodiment of the present application, the sequence of the first linker peptide or the second linker peptide is selected from the linker sequences comprising glycine (G) and serine (S) residues; preferably, the length of the first linker peptide or the second linker peptide is about 1 to about 50 amino acids, respectively; preferably, about 15 to about 25 amino acids; more preferably, the first linker peptide or the second linker peptide is selected from the group consisting of 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 n has a value falling within a range of 1 to 10.

[0161] In particular in the present embodiment, the sequence of the first linker peptide (L1) and the second linker peptide (L2) is (GGGGS)3.

[0162] In one particular embodiment of the present application, the C-terminus of scFv-1 is connected to the N-terminus of scFv-2 via a third linker peptide. In another particular embodiment of the present application, the C-terminus of scFv-2 can also be connected to the N-terminus of scFv-1 via a third linker peptide.

[0163] In one particular embodiment of the present application, the sequence of the third linker peptide (L3) is selected from the group consisting of linker sequences comprising glycine (G) and serine (S) residues; preferably, the length of the third linker peptide (L3) is from about 1 to about 50 amino acids; preferably, from about 15 to about 25 amino acids; more preferably, the third linker peptide is selected from the group consisting of 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 n has a value falling within a range of 1 to 10.

[0164] In particular in the present embodiment, the sequence of the third linker peptide (L3) is GS(GGGGS)4.

[0165] That is, the antigen-binding portion (antigen-binding molecule) of the bispecific antibody of Example 1, from N-terminus to C-terminus, is: scFv-1—L3—scFv-2; more particularly, the antigen-binding portion of the bispecific antibody of Example 1, from N-terminus to C-terminus, is: VL-1—L1—VH-1—L3—VL-2—L2—VH-2.

[0166] Of course, in other embodiments of the present application, the structure of the antigen-binding portion 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.

[0167] In other embodiments of the application, the structure of the antigen binding portion of the bispecific antibody can also be VL-2— L2— VH-2— L3— VL-1— LI— VH-1, VH-2— L2— VL-2— L3— VL-1— LI— VH-1, VL-2— L2— VH-2— L3— VH-1— LI— VL-1, or VH-2— L2— VL-2— L3— VH-1— LI— VL-1, or a construct in which the combination of VL1 and VH1 or VL2 and VH2 is arranged in a different order.

[0168] In a specific embodiment of the application, the C-terminus of the antigen binding portion / antigen binding molecule of the application can be linked to a tag sequence. The tag sequence is a small polypeptide purification tag sequence; a small polypeptide tag sequence is defined as a stretch of amino acids with high affinity to an immobilized ligand, and is a polypeptide sequence designed for fusion expression with a protein of interest for the purpose of purification. More preferably, the tag sequence is a poly-histidine (His) tag sequence.

[0169] In a specific embodiment of the application, the bispecific antibody further comprises a heavy chain constant region; the heavy chain constant region is preferably a heavy chain constant region of human IgGl, 2, 3, 4. Preferably, the heavy chain constant region is the Fc domain of human IgGl.

[0170] In a specific embodiment of the application, the C-terminus of scFv-1 is linked to the N-terminus of scFv-2 via a third linker peptide (L3), and the C-terminus of scFv-2 is linked to the Fc domain of human IgGl via a hinge peptide (Hinge). In another specific embodiment of the application, the C-terminus of scFv-2 can be linked to the N-terminus of scFv-1 via a third linker peptide (L3), and the C-terminus of scFv-1 is linked to the Fc domain of human IgGl via a hinge peptide (Hinge).

[0171] In a preferred embodiment of the application, the hinge peptide sequence is EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP) n , ESKYGPPCPSCP; wherein n can be 1 or more than 1.

[0172] In another preferred embodiment of the application, the hinge peptide sequence can be selected from the heavy chain of different subtypes of human immunoglobulin, for example, can be selected from the a1 chain, a2 chain, or the g1 chain, g2 chain, g3 chain, g4 chain, or d chain.

[0173] In this embodiment, the sequence of the bispecific antibody is, from N-terminus to C-terminus, scFv-1—L3—scFv-2—Hinge—Fc.

[0174] In one specific embodiment of the present application, the Fc domain of human IgG1 comprises, from N-terminus to C-terminus, heavy chain constant region CH2 and heavy chain constant region CH3;

[0175] That is, the sequence of the bispecific antibody of Example 1 is, from N-terminus to C-terminus:

[0176] VL-1—L1—VH-1—L3—VL-2—L2—VH-2—Hinge—CH2—CH3.

[0177] 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; and the sequence of the hinge peptide Hinge is as shown in SEQ ID NO. 19.

[0178] Example 2

[0179] The sequence structure of the antibody of Example 2 is, from N-terminus to C-terminus:

[0180] scFv-2—L3—scFv-1—Hinge—CH2—CH3.

[0181] The sequences of scFv-1, scFv-2, L3, Hinge, CH2 and CH3 are the same as those of Example 1 described above.

[0182] Examples 3 and 4

[0183] In one specific embodiment of the present application, the C-terminus of scFv-1 is connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide in sequence, 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.

[0184] In another specific embodiment of the present application, the C-terminus of scFv-2 is connected to the N-terminus of the Fc domain of human IgG1 through a fourth linker peptide and a hinge peptide in sequence, 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.

[0185] In one specific embodiment of the present application, the sequence of the fourth linker peptide (L4) or the fifth linker peptide (L5) is selected from a linker sequence comprising glycine (G) and serine (S) residues; preferably, the fourth linker peptide (L4) or the fifth linker peptide (L5) is each about 1 to about 50 amino acids in length; preferably, about 15 to about 25 amino acids in length; more preferably, the fourth linker peptide or the fifth linker peptide is selected from the group consisting of 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 n is in the range of 1 to 10.

[0186] In particular 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.

[0187] The sequence structure of the antibody of Example 3 is, in order from N-terminus to C-terminus:

[0188] scFv-1—L4—Hinge—CH2—CH3—L5—scFv-2.

[0189] The sequence structure of the antibody of Example 4 is, in order from N-terminus to C-terminus:

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

[0191] Wherein the sequences of scFv-1, scFv-2, Hinge, CH2, and CH3 are the same as described above in Example 1.

[0192] Preparation of the antibodies of Examples 1-4

[0193] Step 1) Construction of antibody expression vector pcDNA3.4-Fc containing Fc gene fragment

[0194] The gene fragments of the full human IgG1 signal peptide gene SP, the hinge peptide Hinge, the heavy chain constant region CH2, and the heavy chain constant region CH3, i.e. the SP-Fc gene, were synthesized by Kingsway Biotech. The SP-Fc gene was ligated to the pcDNA3.4 vector by TA cloning, which is well known in the art, to obtain the pcDNA3.4-Fc expression vector. The pcDNA3.4 vector was purchased from Thermo Fisher Scientific, and the specific plasmid map is shown in Figure 1.

[0195] Step 2) Synthesis of antibody gene sequence

[0196] As described above, the light chain variable region VL-1 of the single-chain antibody fragment scFv-1 of Example 1 is as shown in SEQ ID NO. 13, and the heavy chain variable region VH-1 is as shown in SEQ ID NO. 14; the sequence of the first linker peptide (L1) between the two is (GGGGS)3.

[0197] Correspondingly, the nucleic acid sequence encoding scFv-1 is as shown in SEQ ID NO. 20;

[0198] The light chain variable region VL-2 of the single-chain antibody fragment scFv-2 of Example 1 is as shown in SEQ ID NO. 15, and the heavy chain variable region VH-2 is as shown in SEQ ID NO. 16; the sequence of the second linker peptide (L2) between the two is (GGGGS)3.

[0199] Correspondingly, the nucleic acid sequence encoding scFv-2 is as shown in SEQ ID NO. 21.

[0200] Example 1, the corresponding nucleotide sequence of scFv-1—L3—scFv-2 is synthesized by conventional method.

[0201] Example 2, the corresponding nucleotide sequence of scFv-2—L3—scFv-1 is synthesized by conventional method.

[0202] Example 3, the corresponding nucleotide sequence of scFv-1—L4—Hinge—Fc—L5—scFv-2 is synthesized by conventional method respectively.

[0203] Example 4, the corresponding nucleotide sequence of scFv-2—L4—Hinge—Fc—L5—scFv-1 is synthesized by conventional method respectively.

[0204] Step 3) Construction of expression vector of antibody gene

[0205] 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-mentioned step 2) was connected to the pcDNA3.4-Fc expression vector constructed in the above-mentioned step 1) by TA cloning. The nucleotide sequence 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 was connected to the pcDNA3.4 expression vector containing the full human IgG1 signal peptide gene (Figure 2) by TA cloning. And the DH5α competent cells were transformed to construct the expression plasmid of the final bispecific antibody, and the sequence information was confirmed by sequencing after cloning.

[0206] Step 4) Expression of bispecific antibody in mammalian cells 293F

[0207] The expression plasmid of the bispecific antibody was purified by plasmid purification kit (Meibio), and co-transfected into HEK293F cells for expression by using EZ Trans cell transfection reagent (Li Ji Biological).

[0208] The specific transfection steps are as follows: 50 ml of 293F cells were plated in a 250 mL cell culture flask at a density of 1.2 x 10 6 cells / ml the day before transfection, and on the transfection day, the transfection reagent EZ-Trans was mixed with the expression plasmid constructed in step 3) (mass volume ratio DNA: EZ-Trans = 1:3) to dissolve 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), and the EZ-Trans-DNA mixture was added to the HEK293F cells in a raindrop-like manner after standing for 15 minutes. Six days after transfection, the cell culture supernatant was obtained by centrifugation for subsequent extraction and purification 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, identification is performed.)

[0209] Step 5) Extraction and purification of antibody

[0210] The cell supernatant collected in the above-mentioned step 4) was filtered with a 0.45 μM filter, and the supernatant was diluted with 1 x PBS binding buffer. The bispecific antibody containing IgG1 Fc in the supernatant was purified by using a protein-G column (Tiandihuan Biotech Co., Ltd., Changzhou), and the purification method was referred to the instructions for use of the protein-G column. The bispecific antibody obtained by purification.

[0211] The 280 nm absorbance was measured using a Nanodrop 2000 (ThermoFisher) and the antibody concentration was calculated. After affinity purification, the antibody was analyzed for purity and identified by SDS-PAGE. 5 μl of the purified sample was mixed with 20 μl of 5x loading buffer and placed in a 100°C metal water bath for 10 minutes. 10 μl of the heated sample mixture was loaded onto a PAGE gel (Nanjing KingsRiver Biological Technology Co., Ltd.). The sample was separated by electrophoresis according to molecular weight. After staining the gel with Coomassie Brilliant Blue R250 for 3 hours, the gel was destained with destaining solution. The SDS-PAGE detection results of the expressed and purified bispecific antibody were obtained by photographing on a GelDoc Go Gel Imaging System (BIO-RAD), as shown in Figure 3. The SDS-PAGE of the antibodies of Examples 2-4 described below are shown in Figure 3.

[0212] Effect data

[0213] I. Production of SARS-CoV-2 and its mutant strains, SARS-CoV, bat SARS coronavirus (BtSL-CoV) WIV-1, Rs3367 pseudovirus

[0214] SARS-CoV-2 and its mutant strains, SARS-CoV, BtSL-CoV WIV-1, Rs3367, pseudovirus are surface respectively with their corresponding Spike membrane protein (S), non-replicating defective retrovirus particles carrying luciferase reporter gene, which can simulate the infection process of SARS-CoV-2 and its mutant strains, BtSL-CoV WIV-1, Rs3367, SARS-CoV virus to host cells (such as human hepatoma cell line Huh-7, 293T cell line 293T-ACE2 stably expressing human ACE2 receptor), and express luciferase reporter gene in infected cells. Since pseudovirus infection does not produce virus particles with infectivity, it can be safely operated in a biosafety level 2 laboratory.

[0215] SARS-CoV-2, BtSL-CoV WIV-1, Rs3367 and SARS-CoV pseudovirus were obtained by co-transfecting 293T cells with their respective S protein expression plasmids and HIV Env-deficient backbone plasmids (pNL4-3.Luc.R-E-) carrying luciferase reporter genes.

[0216] The S gene sequences of SARS-CoV-2, SARS-CoV, BtSL-CoV WIV-1 and Rs3367 were designed according to NCBI GenBank sequences NC_045512, ABD72979.1, KC881007.1 and KC881006.1, and synthesized by Nanjing Kingsriver Biotech Co., Ltd. after codon optimization, and then connected to the pcDNA3.1 eukaryotic expression vector to construct SARS-CoV-2, SARS-CoV, BtSL-CoV WIV-1 and Rs3367 S protein expression plasmids. Among them, SARS-CoV-2 mutant pseudovirus Alpha, Beta, Gamma, Delta, Lambda and Omicron need to be subjected to corresponding point mutations and deletion mutations on the S protein expression plasmid. The pNL4-3.Luc.R-E- backbone plasmid is derived from the U.S. NIH AIDS Reagent Program. All plasmids are amplified by transforming DH5a competent cells, and purified using a plasmid purification kit produced by Meibio, and the purification process is referred to the kit instructions.

[0217] 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco), and inoculated into 10 cm cell dishes before transfection. After 24 hours of culture, the backbone plasmid (pNL4-3.Luc.R-E-) was co-transfected into 293T cells with the expression plasmids of SARS-CoV, BtSL-CoV WIV-1, Rs3367, SARS-CoV-2 and its mutant strains at a ratio of 3:1 using EZ Trans cell transfection reagent (Li Ji Biotech). The detailed transfection method is described in the instructions of EZ Trans cell transfection reagent. After 48 hours of transfection, the supernatant containing the pseudovirus was collected, centrifuged at 2500 rpm for 10 minutes to remove cell debris, and then aliquoted and stored at -80°C for the detection of neutralizing antibodies.

[0218] Secondly, the neutralization activity of the antibodies of embodiments 1-4 to SARS-CoV-2 and its mutants (Alpha, Beta, Gamma, Delta, Lambda and Omicron), as well as the pseudovirus of various coronaviruses including SARS-CoV, bat coronavirus BtSL-CoV WIV-1 and Rs3367 was detected.

[0219] Different concentrations of bispecific antibodies were tested on 96-well cell plates to inhibit the infection of Huh-7 cells by pseudovirus to detect their neutralization ability to SARS-CoV-2 and its mutants, SARS-CoV, BtSL-CoV WIV-1 and Rs3367 virus.

[0220] The detection method is as follows: 1) Huh-7 cells are inoculated in a 96-well cell plate at 1 × 10 4 cells per well, and cultured at 37°C in a 5% CO2 cell incubator for 24 hours; 2) the antibodies of the examples and the comparative examples are diluted in cell culture medium to different concentrations, mixed with an equal volume of 100 TCID50 of pseudovirus diluent, 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, a duplicate well is set up, and an antibody-free group, a virus-free group, and a positive serum control group are set up; 4) after 12 hours of culture, 150 μl of maintenance solution is added to each well, and the culture is continued at 37°C for 48 hours; 5) the cells are lysed and the luciferase activity of each well is detected using a luciferase assay kit (Luciferase Assay System, Promega Cat. #E1500), and the specific detection method is according to the kit instructions; the chemiluminescence RLU value of each well is detected using a multifunctional enzyme label instrument (Perkin Elmer); 6) the percentage of neutralization inhibition of the pseudovirus by the antibodies at different concentrations is calculated according to the ratio of the RLU values of the antibody and the virus control, and the half inhibitory dose IC50 of the antibody for the virus is calculated using PRISM7 software (GraphPad) (unit: μg / ml).

[0221] Experimental groups 1-4: antibodies of examples 1-4 of the present application are used;

[0222] Comparative group 1: IgG1 monoclonal antibody constructed using VL1 and VH1 described above (comparative example 1) is used;

[0223] Comparative group 2: IgG1 monoclonal antibody constructed using VL2 and VH2 described above (comparative example 2) is used;

[0224] Comparative group 3: an equal proportion mixture of comparative example 1 and comparative example 2 is used;

[0225] The detection results are shown in Table 1 below:

[0226] Table 1

[0227] Table 1 is the neutralization IC50 results of the antibodies of examples 1-4 and the control group antibodies for various new coronavirus mutant strains and other various coronaviruses.

[0228] As can be seen from Table 1:

[0229] 1) The double antibodies of Examples 1-4 have strong neutralization ability against pseudoviruses including major variants of SARS-CoV-2 Alpha, Beta, Delta, Omicron, etc. and SARS-CoV, bat coronavirus BtSL-CoVWIV-1, Rs3367; proving the broad spectrum of the antibodies of Examples 1-4 in neutralizing coronaviruses.

[0230] 2) It is surprisingly found that for the subvariant mutant strains of Omicron, Comparative Example 1 only has weak neutralization, and Comparative Example 2 has an IC50 value greater than 50 μg / mL for 12 strains of Omicron mutant strains, while the IC50 geometric median of the combined double antibody (Example 1) is 0.03 μg / mL, unexpectedly showing significantly strong and broad-spectrum neutralization ability. Although the double antibodies of Examples 2-4 are weaker than the double antibody of Example 1, they are still significantly better than Comparative Examples 1-3, which shows that the double antibody formed by the combination of the two antigen-binding units has a synergistic effect.

[0231] 3) The double antibody of Example 1 can still maintain strong neutralization activity against various mutant strains of the new coronavirus including the subvariant mutant strains of Omicron BQ.1.1, XBB.1.5, XBB.1.16, EG.5, BA.2.86, JN.1; even for the BA.2.86 and JN.1 mutant strains (compared to the BA.2 mutant strain, with 34 newly added amino acid mutations on the S protein), the double antibody of Example 1 of the present application unexpectedly shows significant neutralization ability, has excellent broad spectrum, and is significantly better than bebtelovimab antibody approved by FDA for clinical treatment of COVID-19.

[0232] Third, the prevention and treatment effect evaluation of the double antibody of Example 1 of the present application on mice infected with new coronavirus mutant strains XBB.1 and EG.5

[0233] The animal study has been approved by the Animal Experimental Committee of the First Affiliated Hospital of Guangzhou Medical University (Approval No.: 20230615). The prevention and treatment effects of the double antibody of Example 1 in animal studies were evaluated using 6-week-old human ACE2-K18 transgenic mice (n = 5 per group). Mice were intranasally infected with 10 5 The double antibody of Example 1 of the present application was administered by intraperitoneal injection (200 μg per mouse) (i.p.) or intranasal administration (20 μg per mouse) (i.n.) at a dose of 24 hours before infection or 24 hours after infection with the new coronavirus mutant strains XBB.1 or EG.5 virus. Two days after viral infection, lung samples were collected from mice for viral titer determination. The amount of virus per gram of lung tissue was determined 48 hours after viral infection; see Figure 4 for the operation process.

[0234] The mice treated with PBS were used as a control group, and the results are shown in FIG. 5 and FIG. 6. The lung virus load of the mice treated with the bispecific antibody of Example 1 by subcutaneous injection was reduced to the detection limit (undetectable), which was significantly better than that of the control mice, whether before (prophylactic group) or after (therapeutic group) the mice were infected with the XBB.1 or EG.5 virus. For the infection of the new crown mutant XBB.1, both prophylactic and therapeutic administration by nasal instillation can reduce the lung virus load of the mice to an undetectable level. In addition, the mice treated with the bispecific antibody of Example 1 had significantly reduced symptoms of respiratory tract infection, such as sneezing, runny nose, and increased wheezing, compared with the control mice.

[0235] These research findings also surprised and delighted the inventors, and they hope to promptly disclose the technical data and conduct clinical promotion to contribute to the prevention and control of the new round of epidemic situation caused by the Omicron mutant subtype.

[0236] In summary, based on the effect data of the bispecific antibodies of Examples 1, 2, 3, and 4, it is proved that the combination of the RBM motif-specific antibody and the RBM outer side-specific antibody has excellent broad-spectrum neutralization ability against coronaviruses. In particular, not only does it have strong and broad-spectrum neutralization ability against multiple Omicron mutant subtypes, but it also has strong and broad-spectrum neutralization ability against SARS-CoV-related sarbecoviruses.

[0237] Fourth, the rhinitis treatment effect of the antibody of Example 1

[0238] The method for constructing a mouse rhinitis model is as follows:

[0239] BALB / c mice, age: 6-W, 30 female mice;

[0240] Randomly divided into five groups: normal group (blank group), rhinitis group, Example 1 treatment group, hormone (budesonide) treatment group, and IgG treatment group; 6 mice per group;

[0241] Sensitization: Each model mouse was injected intraperitoneally with an OVA (1 mg / ml) aluminum hydroxide (20 mg / ml) solution, once every other day for 7 times from day 1 to day 14, to complete the sensitization process.

[0242] Challenge: From day 21 to day 27, each model mouse was given 50 μg OVA / normal saline solution by nasal instillation, 10 μL per side, once a day, for a total of 7 times, to complete the challenge process.

[0243] Method of administration:

[0244] The administration group (including the treatment group of Example 1, the hormone (budesonide) treatment group, and the IgG treatment group) was subjected to drug treatment (drug concentration 1 mg / ml) 30 min after each challenge, 20 μl per mouse, 10 μl per nostril; drug treatment was performed twice a day with an interval of 8 hours.

[0245] Observation: body weight: 2 times / week; observation score: 1 time / 2 days, challenge stage, within 15 min of OVA administration;

[0246] End point scheme: on day 8 after administration, all mice were euthanized by CO2, and sample collection (nasal mucosa, serum) was performed, and the whole operation process is shown in Figure 7.

[0247] Result evaluation:

[0248] (1) Observation index: ① body weight measurement 2 times / week; ② observation score: counting the number of sneezing of mice within 10 min after challenge on days 23, 25 and 27.

[0249] (2) Detection index:

[0250] ① Pathology: on day 8 after administration, all mice nasal mucosa was fixed with paraformaldehyde for pathological HE staining: inflammatory cells, eosinophils, mast cells, goblet cells, and pathological abnormalities were described;

[0251] ② Cytokines & immunoglobulins: on day 8 after administration, the levels of OVA-sIgE and histamine in the serum of all mice were detected by ELISA.

[0252] The detection results are shown in Figures 8-11 as follows:

[0253] The upper graph of Figure 8 shows the relationship between the body weight (weight) of mice in each group and time, and the lower graph of Figure 8 shows the relationship between the change % of mouse body weight and time. Compared with the other four groups, the body weight of the mice in the hormone (budesonide) treatment group decreased significantly (p<0.001) after 7 days of treatment of rhinitis mice, and the body weight decreased by more than 13.0%, indicating that budesonide had a significant inhibitory effect on the growth and development of mice. In contrast, the antibody treatment of Example 1 did not have a significant effect on the body weight and growth of mice in the same period, showing better safety and clinical feasibility.

[0254] Figure 9 shows the results of sneezing score of the mice with rhinitis after drug treatment. The average number of sneezing of the mice in the rhinitis group within 10 minutes was 5.1, indicating that the modeling was successful. After 7 days of budesonide treatment, the average number of sneezing of the mice in the hormone (budesonide) treatment group on the 5th, 6th and 7th day decreased to 2.8 (p=0.0031); and after treatment with the antibody of Example 1, the average number of sneezing of the mice on the corresponding three days was 2.7 (p=0.0013), indicating that the antibody can effectively relieve the symptoms of rhinitis and has an effect comparable to that of the hormone drug. In contrast, the IgG treatment group failed to significantly improve the sneezing symptoms.

[0255] IgE is considered as the "key to start" of the pathogenesis of allergic rhinitis, and its level is closely related to the occurrence, development and severity of the disease. When an allergic individual first contacts 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 receptor (FcεRI) on the surface of mast cells and basophils, forming a sensitized state (asymptomatic period). When the same allergen is encountered again, IgE binds to it, triggering mast cell degranulation, releasing histamine, leukotrienes and other inflammatory mediators, causing nasal mucosal blood vessels to dilate, glandular secretion to increase, and nerve endings to be sensitized, thereby triggering typical symptoms such as nasal itching, sneezing, clear rhinorrhea, and nasal congestion.

[0256] Figure 10 shows the changes in OVA-sIgE in the serum of the mice with rhinitis 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% close to the normal group level (11.3 ng / mL). The OVA-sIgE level of 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.

[0257] Histamine, as the "trigger factor" and "amplifier" of the symptoms of allergic rhinitis, plays a key role in the acute attack and the progression of chronic inflammation. It is mainly synthesized by mast cells and basophils and stored in the cell granules in a bound state. Once the nasal cavity contacts an allergen, IgE binds to mast cells to trigger the 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 the goblet cells and submucosal glands to increase the secretion of mucin; at the same time, it induces vasodilation and plasma extravasation, causing clear watery nasal discharge and mucosal edema, thereby aggravating the symptoms of nasal obstruction.

[0258] Figure 11 shows the change of histamine level in serum of rhinitis mice after drug treatment. After 8 days of antibody treatment of Example 1, the concentration of histamine in the serum of mice decreased from 7.2 ng / mL to 3.5 ng / mL (p < 0.0001), with a decrease of 51%, which was close to the concentration (3.8 ng / mL) of the normal group. The concentration of histamine in the hormone (budesonide) treatment group was 4.3 ng / mL (p < 0.0001). The IgG treatment group failed to significantly reduce the concentration of histamine in the serum. Thus, the antibody of Example 1 is superior to budesonide in inhibiting the release of histamine and can more effectively alleviate the symptoms related to rhinitis, especially the frequency of sneezing.

[0259] Figure 12 is the pathological HE staining of the nasal mucosa of the rhinitis group of mice after drug treatment. Compared with the normal group, the comprehensive pathological performance of the rhinitis group of mice is the destruction of the epithelial layer (loss of cilia, cell necrosis), interstitial edema, vascular dilation, and gland hyperplasia with retention of secretions. A large number of inflammatory cell infiltrates can be seen in the lamina propria and submucosa of the nasal mucosa. Inflammatory cells are distributed in patches or foci around blood vessels and glands, suggesting chronic inflammatory reaction. The number of goblet cells significantly increases, showing multifocal hyperplasia, and squamous epithelial metaplasia occurs in some areas. The hyperplasia of goblet cells leads to hypersecretion of mucus, forming purulent secretions, which is related to the symptoms of nasal congestion and rhinorrhea. After treatment with hormones (budesonide) or the antibody described in Example 1, the pathological changes are significantly improved. The nasal mucosa structure of the treatment group of mice tends to be complete, and only a small amount of inflammatory cell infiltration can be seen in the lamina propria and submucosa. The hyperplasia of goblet cells is significantly reduced, and there is no obvious mucus retention or epithelial metaplasia. The above results show that the hormone budesonide and the antibody in Example 1 both have obvious anti-inflammatory effects and can effectively alleviate the chronic inflammatory reaction in the rhinitis model.

[0260] In summary, the antibody of Example 1 can effectively control the symptoms of allergic rhinitis by reducing the level of OVA-sIgE and inhibiting the release of histamine from mast cells, and has a similar mechanism of action to the hormone (budesonide) treatment group. However, the hormone budesonide has adverse effects on the growth and development of mice, while the antibody does not exhibit similar side effects. Therefore, the antibody of Example 1 is more secure and has potential for clinical application in the treatment of allergic rhinitis, especially in children.

[0261] Fifth, the asthma treatment effect of the antibody of Example 1

[0262] The method for constructing a mouse rhinitis model is as follows:

[0263] BALB / c mice, age: 6-W, 35 female;

[0264] Randomly divided into five groups: normal group (blank group), asthma group, Example 1 treatment group, hormone (budesonide) treatment group, IgG treatment group; 7 in each group;

[0265] Sensitization: OVA-V (20 μg / mouse); 1 mg of aluminum hydroxide was dissolved in normal saline (100 μl / mouse), and the solution was mixed using a three-way valve. Mixing was continued for 30 minutes to allow aluminum hydroxide to effectively adsorb the antigen. The sensitized mice were injected intraperitoneally on days 0, 7, and 14 of the experiment to complete the sensitization process. Mice in the blank control group were treated with normal saline in the same manner.

[0266] Challenge: 3% (w / v) OVA-II (30 g / L) was prepared and dissolved in normal saline. After nebulization, it was passed into a closed nebulizer box for mice to inhale, once a day for 30 minutes each time, and nebulized on days 21-27 of the experiment; mice in the blank control group were treated with normal saline in the same way.

[0267] Dosing: 30 minutes after each challenge, the drug group received drug treatment (drug concentration 1 mg / ml), 20 μl per animal, 10 μl per nostril. Drug treatment was administered twice daily, eight hours apart. The normal (blank) and asthma groups were treated with saline intranasally. Treatment lasted a total of seven days. See Figure 13 for the complete protocol.

[0268] Detection indicators:

[0269] 1. Body weight: During the sensitization phase, the animals were weighed and recorded twice a week; during the provocation phase, the animals were weighed and recorded on days 20, 22, 24, 26, and 28.

[0270] 2. Symptom observation: 30 minutes after the administration of the drug during the stimulation phase, clinical symptoms were recorded: arched back, curled up, and accelerated and deepened breathing.

[0271] 3. Serum retention: On the 8th day after administration, all mice were euthanized with CO2, and blood was collected. After standing at room temperature for 0.5 hours, the blood was centrifuged in a centrifuge at 4°C / 3000 rpm / 15 min. After centrifugation, the serum was separated and stored in separate devices at -80°C for subsequent ELISA testing (OVA-sIgE, IL-4, IL-5, IL-13, histamine).

[0272] 4. Bronchoalveolar lavage fluid collection: PBS was pre-cooled and ready to use. The trachea of the mouse was opened with a syringe needle or microscissors. The lavage needle was inserted into the trachea and fixed with a ligature. The left lung of the mouse was lavaged by connecting a 1 ml syringe. 0.3 ml of pre-cooled PBS was drawn into the syringe and slowly injected into the left lung through the lavage needle. The syringe was slowly drawn back and forth 3 times to fully lavage the left lung. The fluid in the lung was recovered and placed in a sterile 1.5 ml centrifuge tube and placed on ice. Each mouse was lavaged 3 times (0.3+0.3+0.4=1 ml PBS), and the fluid recovery rate was about 70%. Centrifugation at 4°C / 2000 rpm for 10 minutes, and the supernatant was aliquoted and stored at -80°C for subsequent ELISA detection (OVA-sIgE, IL-4, IL-5, IL-13, histamine).

[0273] 5. Inflammatory factor detection: Enzyme-linked immunosorbent assay was used to detect the concentrations of inflammatory factors (IL-4, IL-5, IL-13, histamine) in the serum and bronchoalveolar lavage fluid of all mice, and the concentrations of OVA-sIgE and histamine in the serum. The experimental process was carried out according to the manufacturer's instructions

[0274] 6. Pathological indicators: The right lower lobe was washed with PBS, fixed with 4% paraformaldehyde, dehydrated, embedded and sectioned for subsequent staining experiments (H&E staining to observe lung tissue pathological changes and evaluate lung tissue inflammation; PAS staining to evaluate the proliferation of airway goblet cells).

[0275] The detection results are shown in Figures 14-19 as follows:

[0276] Figure 14 shows the trend of the change in the weight of the mice. Compared with the other four groups, the weight of the mice in the hormone (budesonide) treatment group decreased significantly (p<0.0001) after the mice were treated with the hormone drug budesonide for 7 days, and the weight loss was more than 10.6%, indicating that the hormone budesonide had a significant inhibitory effect on the growth and development of the mice. In contrast, the weight of the mice did not change significantly after the mice were treated with the bispecific antibody in Example 1 for 7 days, indicating that the treatment had no adverse effects on the growth of the mice, and had better safety and clinical feasibility.

[0277] Figure 15 shows the symptom score of the asthmatic mice after drug intervention, including arching the back, curling up and rapid breathing, etc. Each symptom was scored 1 point. The score of the asthma model group was 2.6, indicating that the asthma modeling was successful. In the hormone (budesonide) treatment group, the asthma symptom score decreased to 1.1 (p=0.0064) after 7 days of treatment with budesonide. After treatment with the antibody of Example 1, the symptom score also decreased to 1.1 (p=0.0042), indicating that the antibody can significantly alleviate the symptoms related to asthma. The IgG in the IgG treatment group did not show obvious therapeutic effect, indicating that it had no significant improvement on the symptoms of asthma.

[0278] The study first detected the effect of drug intervention on the level of OVA-specific IgE (OVA-sIgE) in the bronchoalveolar lavage fluid (BALF) and serum of asthmatic mice. As shown in the upper and lower panels of Figure 16, 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 antibody treatment of Example 1, the OVA-sIgE concentration in BALF was significantly reduced from 29.2 ng / mL to 0.7 ng / mL (p < 0.0006), with a decrease of 97%, close to the normal group of 0 ng / mL. In the hormone (budesonide) treatment group, after budesonide treatment, it was only reduced to 4.9 ng / mL (p < 0.006), and the IgG treatment group did not show significant therapeutic effect. The above results show that the antibody of Example 1 is superior to hormone drugs in reducing OVA-sIgE.

[0279] IL-4 (interleukin-4) plays a key role in Th2 cell differentiation and asthma pathogenesis. In asthmatic patients, inhaled allergens (such as dust mites, pollen) are captured by antigen-presenting cells (such as dendritic cells), which induce the differentiation of initial CD4+ T cells to Th2 phenotype, which 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 response, and promote B cells to undergo antibody class switching to generate IgE antibodies. IgE binds to the FcεRI receptor on the surface of mast cells and basophils, and initiates the degranulation reaction when it comes into contact with allergens again, releasing mediators such as histamine and leukotrienes, leading to bronchial constriction and mucus secretion, etc. acute symptoms of asthma. Then we evaluated the regulatory effect of the antibody of Example 1 on IL-4 level (see the upper and lower panels of Figure 17). After 8 days of treatment, the antibody of Example 1 can significantly reduce the IL-4 concentration in BALF and serum: the IL-4 in serum is reduced from 50.0 pg / mL to 13.1 pg / mL (p < 0.0002), with a decrease of 73.8%, even lower than the normal group of 27.7 pg / mL; the IL-4 concentration in BALF is also significantly reduced from 16.7 pg / mL to 6.8 pg / mL (p < 0.0023). In contrast, budesonide treatment can significantly reduce the IL-4 concentration in BALF (to 3.1 pg / mL, p < 0.0001), but has no significant effect on serum IL-4, suggesting that its effect is more locally targeted. The antibody of Example 1 achieves more extensive inflammation control by simultaneously regulating systemic (serum) and local (lung) IL-4 levels.

[0280] In addition, IL-5 is an important cytokine secreted by Th2 cells, which can promote the differentiation and proliferation of eosinophils in bone marrow. In the process of rhinitis and asthma, IL-5 induces a large number of eosinophils to gather in the nasal mucosa and respiratory tract, releases inflammatory mediators (such as ECP, MBP, etc.), initiates tissue damage, increases vascular permeability and mucus secretion, and causes typical symptoms such as nasal congestion, runny nose, etc.

[0281] We further detected the effect of the antibody of Example 1 on the level of IL-5 (see the upper and lower graphs of FIG. 18), 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, close to the normal group of 11.6 pg / mL. Budesonide can significantly reduce the concentration of IL-5 in BALF and serum (both p<0.0001), showing that it has a more comprehensive inhibitory effect on the IL-5 pathway.

[0282] FIG. 19 is the pathological HE staining of the nasal mucosa of the asthma mice after drug treatment. Compared with the normal group, a large number of inflammatory cells infiltrate the lung interstitium and the peribronchial area of the asthma group mice, and the inflammatory cells are distributed in a sheet or focal manner around the bronchi, blood vessels and alveolar septum, indicating chronic inflammatory reaction. The number of goblet cells increases significantly, showing multifocal hyperplasia, and squamous epithelial metaplasia appears in some areas. The hyperplasia of goblet cells leads to hypersecretion of mucus, forming purulent secretion, which is related to airway obstruction and coughing. Alveolitis, alveolar cavity exudation, inflammatory cells fill the alveolar cavity, and "hemophagocytosis" (red cell extravasation) occurs in some areas. Interstitial edema, vascular dilation, and alveolar septum thickening. The pathological HE staining of the 2IgG treated asthma mice in the control group is similar to that of the asthma group. After the hormone positive drug and the antibody in Example 1 treat the asthma mice, the pathological HE staining is similar, only a small amount of inflammatory cells gather around the bronchi, a small amount of exudation in the alveolar cavity, and the hyperplasia of goblet cells is not obvious.

[0283] In summary, the hormone budesonide mainly regulates the level of IL-5 to inhibit the eosinophil-mediated inflammatory response at both the lung and systemic levels, thereby relieving the symptoms of asthma. The antibody of Example 1 more significantly regulates the level of IL-4, inhibits the activity of Th2 cells and the immune response triggered thereby, and blocks the class switching of B cells to IgE, thereby reducing the asthma reaction, and has a different immune regulation mechanism from budesonide. The difference in the anti-inflammatory pathway between the two provides an important reference for clinical joint or alternative treatment strategies. Although the overall efficacy of the two is comparable, compared with the adverse effects on the development of the body that the hormone drugs may cause, the treatment of the antibody of Example 1 shows better safety in mice, showing good clinical application prospects.

[0284] Six, the atopic dermatitis treatment effect of the antibody of Example 1 of the present application

[0285] Mouse dermatitis model construction:

[0286] BALB / c mice, age: 6-W, 40 female;

[0287] Randomly divided into five groups: normal group (blank group), dermatitis group, Example 1 treatment group, hormone (dexamethasone) treatment group, IgG treatment group; 8 in each group;

[0288] All mice were adaptively fed for one week, and after the mice were anesthetized with tri- bromoethanol, the hair on the back was removed with depilatory cream and a shaving razor, with an area of about 2 cm*3 cm. Then the mice were randomly divided into groups, except for the normal group (blank group), 200 μL of 0.5% DNCB (dissolved in a mixture of acetone: olive oil (3:1)) was evenly applied to the shaved area to sensitize the mice on the first to third days of the test. Next, on the 11th, 14th, 17th, 20th, 23rd, 26th, 29th, and 30th days of the test, 20 μL, 100 μL of 1% DNCB was applied to each ear and the dorsal skin, respectively. At the same time, on the 11th to 30th days of the test, the mice in the treatment groups were given daily drug treatment.

[0289] Dosing method: 1 hour after the 11th to 30th day of the challenge, the drug was applied in a volume of 50 μL, twice a day, for a total of 20 times. The entire procedure is shown in Figure 20.

[0290] Detection index:

[0291] 1) Body weight measurement: body weight monitoring three times a week

[0292] Ear and dorsal skin thickness measurement: images were taken with a camera every week to record the clinical symptoms of the ears and dorsal skin. The ear and dorsal skin thickness of each mouse was measured every week and recorded.

[0293] 2) Lesion score:

[0294] The mice were scored for lesion severity during the test, including 4 aspects: erythema / hemorrhage, edema / exudation, dryness / scaling, and epidermal exfoliation. The score represents the severity, and is divided into four grades: none (0), mild (1), moderate (2), and severe (3). The final score is the sum of the 4 symptoms, with a score of 0-12. (Note: the scoring time points are the 1st, 11th, 13th, 16th, 19th, 22nd, 25th, 28th, and 30th days)

[0295] End point sampling on the 30th day:

[0296] (1) Peripheral blood (Blood): 1 hour after drug administration, peripheral blood was collected after the animal was anesthetized, and serum was obtained by centrifugation at room temperature for 4-5 hours, and stored at -80°C;

[0297] (2) Spleen: the spleen was weighed and the spleen mass coefficient was calculated;

[0298] (3) Ear, back skin: After the animals were euthanized, the back skin tissue and ear skin tissue of each group of mice were fixed with paraformaldehyde solution. (The ear skin was left after being fixed)

[0299] Detection index:

[0300] (1) ELISA: The contents of total TNF-a, IL-1b, IL-13 and IgE in the serum of each group of mice were detected by using ELISA kit;

[0301] (2) Pathological histological analysis: After the fixed back skin tissue was dehydrated and fully immersed in wax, paraffin embedding, sectioning, baking, HE staining, toluidine blue staining, neutral gum sealing were performed, and finally observed under a microscope, read and analyzed, and photographed (field magnification 100X, 200X each one).

[0302] Data analysis

[0303] In this experiment, the data were analyzed by using scientific research statistics and drawing software GraphPad Prism 8.4.3. Single factor 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 two-tailed, and the statistical level was set at p≤0.05.

[0304] The detection results are shown in Figures 21-26 as follows:

[0305] Figure 21 shows the change trend of the body weight of the dermatitis mice during treatment. In the hormone (dexamethasone) treatment group, after 20 days of continuous treatment with the hormone drug dexamethasone, the body weight of the mice decreased significantly (p<0.0001), with an average weight loss of more than 10%, indicating that dexamethasone had a significant inhibitory effect on the growth and development of mice. In contrast, the antibody of Example 1 was treated for 20 days, and the body weight of the mice did not change significantly, suggesting that the treatment had no adverse effects on growth, and had better safety and clinical feasibility.

[0306] In the mouse dermatitis model, the spleen mass coefficient (spleen weight / body weight) is often used as an important indicator to assess the degree of immune system activation. In the models of allergic contact dermatitis (ACD) and atopic dermatitis (AD), the increase in the spleen mass coefficient is often closely related to the enhancement of inflammatory response. The results of Figure 22 show that the average spleen mass coefficient of the mice in the dermatitis group is 0.46, which is significantly higher than that of the normal group (blank group) mice (0.36, p<0.0019), verifying the effectiveness of the model construction. In terms of treatment effect, the spleen mass coefficient of the IgG treatment group, i.e., the mice receiving IgG treatment, is 0.44, which is also significantly higher than that of the normal group (p<0.0033), indicating that the treatment fails to effectively inhibit the inflammatory response. In contrast, the spleen mass coefficient of the hormone (dexamethasone) treatment group mice significantly decreases from 0.46 to 0.154 (p<0.0001) after receiving dexamethasone treatment for 20 days. Such a dramatic decrease is often related to the damage of spleen function or structure, indicating that dexamethasone may be accompanied by spleen damage and other adverse reactions while strongly inhibiting the immune response. The spleen mass coefficient of the antibody treatment group mice of Example 1 is 0.42, which is still higher than that of the normal group (p=0.045), but compared with the hormone (dexamethasone) treatment group and the IgG treatment group, the value is closer to the normal level, indicating that the antibody can effectively inhibit the inflammatory response while having less impact on the immune system. In addition, the antibody treatment does not cause obvious body weight changes or spleen function damage within the same period, showing good safety and potential clinical application value.

[0307] Figure 23 shows the dermatitis symptom scores of the mice in each group, including erythema / bleeding, edema / exudation, dryness / desquamation, and epidermal shedding, etc., each scored 1-3 points according to the severity. The total dermatitis score of the model group mice can be as high as 7 points, and still maintains at 5.6 points at the end of the experiment, further verifying the stability and effectiveness of the dermatitis model construction. After 20 days of treatment, the hormone (dexamethasone) treatment group significantly reduces the dermatitis score from the highest 4.3 points to 1.1 points (p=0.0064), showing its good anti-inflammatory effect. After 20 days of treatment of the antibody of Example 1, the score decreases from the highest 4.8 to 2.4 points (p=0.0042), also showing a significant symptom relief effect. In contrast, the IgG treatment group does not show obvious score improvement, indicating that the IgG antibody has no effective intervention effect on the dermatitis symptoms. In summary, the antibody treatment of Example 1 has good effect in relieving the dermatitis symptoms, showing its potential therapeutic value.

[0308] Figure 24 is a comparison chart of the back skin conditions of the mice in each group after 20 days of treatment. The skin improvement degree from good to bad is in the order of: normal group> hormone (dexamethasone) treatment group> Example 1 treatment group> IgG group, which is consistent with the skin score results.

[0309] In acute irritant dermatitis, TNF-a and IL-1b can activate mast cells and macrophages, induce the release of histamine and proteases, and thus trigger erythema, edema and exudation. TNF-a can also promote abnormal proliferation and apoptosis of keratinocytes, leading to thickening of the epidermis, and stimulate fibroblasts and endothelial cells to secrete matrix metalloproteinases (MMPs), exacerbating tissue damage.

[0310] The present study first evaluated the regulatory effect of each treatment on the level of TNF-a in serum. As shown in Figure 25, after 20 days of antibody treatment in Example 1, the concentration of TNF-a decreased significantly from 728 pg / mL to 499.3 pg / mL (p = 0.0001), even lower than the normal group of 586 pg / mL. The IgG treatment group failed to reduce the concentration of TNF-a. The TNF-a concentration of the hormone (dexamethasone) treatment group decreased to 410.3 pg / mL (p < 0.0001), showing that the hormone (dexamethasone) was comparable to the effect of antibody treatment in Example 1, indicating that the antibody can effectively inhibit the expression of inflammatory factors and thus relieve the symptoms of dermatitis.

[0311] In addition, IL-1b can enhance mast cell degranulation by activating NLRP3 inflammasome, release inflammatory mediators such as histamine and chymotrypsin, and induce fibroblasts to secrete MMP-1 and MMP-9, causing damage to the epidermal barrier and collagen degradation. Therefore, we further detected the changes in the level of IL-1b in serum. As shown in Figure 26, the concentration of IL-1b in the dermatitis group 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-1b decreased significantly from 217.6 pg / mL to 128.5 pg / mL (p = 0.0028), slightly lower than the normal group of 143.2 pg / mL; the IgG treatment group failed to reduce the concentration of IL-1b; while the level of IL-1b was 193.9 pg / mL after dexamethasone treatment, no significant decrease was observed, suggesting that the antibody of Example 1 was superior to dexamethasone in inhibiting IL-1b.

[0312] In summary, this study first confirmed that the antibody of Example 1 can achieve precise intervention on the inflammatory cascade by simultaneously neutralizing TNF-a and IL-1b, effectively block the activation of mast cells and macrophages and the release of their inflammatory mediators, and thus relieve the symptoms of dermatitis such as erythema, edema and exudation. In contrast, dexamethasone mainly relies on the inhibition of TNF-a to exert anti-inflammatory effect, and the mechanism is relatively single. Although the overall efficacy of the two is comparable, the antibody of Example 1 is more precise in mechanism, and is expected to become an important basis for the new generation of immunoregulatory treatment strategies. More importantly, compared to the possible kidney damage and adverse effects on the development of the body caused by hormone drugs, the antibody treatment in mice showed better safety, showing good prospects for clinical application.

[0313] On the basis of the disclosure and spirit of the present application, one of ordinary skill in the art can make some simple adjustments on the basis of the bispecific antibody sequence of Example 1, for example, on the basis of the bispecific antibody sequence of Example 1, the order of the light chain and the heavy chain is exchanged to obtain an antibody 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.

[0314] On the basis of the disclosure and spirit of the present application, one of ordinary skill in the art can make some simple adjustments on the basis of the bispecific antibody sequence of Example 1, for example, on the basis of the bispecific antibody sequence of Example 1, the order of the light chain and the heavy chain is exchanged to obtain an antibody 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.

[0315] On the basis of the disclosure and spirit of the present application, one of ordinary skill in the art can make some simple adjustments on the basis of the bispecific antibody sequence of Example 1, for example, on the basis of the bispecific antibody sequence of Example 1, the order of the light chain and the heavy chain is exchanged to obtain an antibody 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.

[0316] On the basis of the disclosure and spirit of the present application, one of ordinary skill in the art can make some simple adjustments on the basis of the bispecific antibody sequence of Example 1, for example, on the basis of the bispecific antibody sequence of Example 1, the order of the light chain and the heavy chain is exchanged to obtain an antibody 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.

[0317] It can be reasonably speculated that they all have similar effects to the bispecific antibodies of Examples 1, 2, 3, and 4; these equivalent replacement schemes all fall within the protection scope of the present application.

[0318] The skilled in the art can also make routine equivalent substitutions to the linker peptide sequence, Fc domain sequence and hinge peptide sequence on the basis of the bispecific antibody sequences of embodiments 1, 2, 3 and 4 of the present application; the skilled in the art can also make amino acid insertion, substitution or deletion treatment which does not affect the overall effect of the antibody on the basis of the bispecific antibody sequences of the present application; these equivalent substitution schemes all fall within the protection scope of the present application.

[0319] On the basis of the skilled in the art obtaining the sequence of the scFv-scFv bispecific antibody scheme and its antigen binding module of the present application, the existing bispecific antibody technology can be used to transform it into a Fab-Fab combination, Fab-Fv combination or other Fv-Fv combination bispecific / multispecific antibody.

[0320] As can be seen from the above, the multispecific antibody or its antigen binding molecule of the present application for neutralizing coronavirus has excellent broad-spectrum, strong neutralization ability for coronavirus, and can effectively block the infection of multiple coronaviruses. Therefore, on the basis of knowing this technical content, the 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 respiratory inflammatory reactions (such as rhinitis, asthma, etc.) or inflammatory diseases such as dermatitis.

[0321] Application Example

[0322] The present application example describes the method for preventing or treating respiratory infection diseases or respiratory inflammation or dermatitis caused by viruses (including coronaviruses) by using the bispecific antibody of embodiments 1-4 of the present application.

[0323] Although specific methods of administration, dosages, and modes are provided, those skilled in the art will appreciate that changes can be made without materially affecting the treatment. Based on the guidance disclosed herein, viral (including coronavirus) induced respiratory infection diseases or respiratory inflammation or dermatitis can be treated or prevented by administering a therapeutically effective amount of the antibodies described herein.

[0324] Specific methods of administration are as follows:

[0325] 1) Pretreatment of the subject: In specific embodiments, the subject is pretreated before administration of a therapeutic agent comprising one or more drug therapies for viral induced respiratory infection or respiratory inflammation known to the skilled in the art. However, such pretreatment is not always required and can be determined by the skilled clinician.

[0326] 2) Administration of the therapeutic composition

[0327] After the subject is selected, a therapeutically effective amount of the antibody of the application, as described above, is administered to the patient. Additional agents, such as antiviral agents, can be administered to the subject concurrently with, prior to, or following administration of the disclosed agents. Administration is achieved by any method known in the art, such as injection (including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinous, epidural, and sternal injection and infusion), or by non-parenteral routes, e.g., topical, epidermal, or mucosal routes of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical administration, etc. The amount of the composition administered to prevent, reduce, inhibit, and / or treat a condition in a subject depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic agent to the subject. Ideally, a therapeutically effective amount of the agent is one that is sufficient to prevent, reduce, and / or inhibit, and / or treat a condition in a subject without causing substantial cellular toxicity effects in the subject. Effective amounts can be readily determined by one of ordinary skill in the art, e.g., by routine testing establishing dose response curves. Also, the compositions can be formulated with an inert diluent or a pharmaceutically acceptable carrier. In a particular example, the antibody is administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks, depending on the particular stage of 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 chronically (e.g., for a period of months or years).

[0328] 3) Evaluation

[0329] After administration of the one or more therapies, the patient is monitored for a reduction in viral levels, or a reduction in one or more associated clinical symptoms, or a reduction in respiratory distress symptoms. In particular examples, the subject is analyzed one or more times beginning 2 days after treatment. The subject is monitored using any method known in the art. For example, a biological sample from the subject, including a throat swab, can be obtained and evaluated for changes in viral levels.

[0330] 4) Additional Treatment

[0331] In particular embodiments, if the subject is stable or has a small, mixed, or partial response to treatment, additional treatment can be performed after reevaluation with the same regimen and material formulation as they were previously administered for a desired period of time.

[0332] It should be understood that although the specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

[0333] The above detailed description merely illustrates preferred and possible non-limiting implementations of the application, and is not intended to limit the scope of the application. Other equally efficacious implementations or changes thereof, which do not depart from the spirit of the application, shall be encompassed by the scope of the application.

Claims

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

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

12.

3. The multispecific antibody or antigen-binding molecule thereof according to claim 2, wherein: 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 more than 80% sequence homology 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 shown in SEQ ID NO. 16, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.

16.

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

5. The multispecific antibody or antigen-binding molecule thereof according to claim 4, wherein: The first antigen binding moiety is a single-chain antibody fragment scFv-1; the second antigen binding moiety is a single-chain antibody fragment scFv-2; The scFv-1 comprises, from N-terminus to C-terminus, the light chain variable region VL-1, the first linker peptide, and the heavy chain variable region VH-1; or, the scFv-1 comprises, from N-terminus to C-terminus, the heavy chain variable region VH-1, the first linker peptide, and the light chain variable region VL-1; The scFv-2 comprises, from N-terminus to C-terminus, the light chain variable region VL-2, the second linker peptide, and the heavy chain variable region VH-2; or, the scFv-2 comprises, from N-terminus to C-terminus, the heavy chain variable region VH-2, the second linker peptide, and the light chain variable region VL-2; 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, GGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; Wherein, the value of n falls within the range of 1 to 10.

6. The multispecific antibody or antigen-binding molecule according to any one of claims 2 to 5, wherein: The C-terminus of the scFv-1 is connected to the N-terminus of the scFv-2 via 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 via 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, GGGGSGGGSGGSGGG, 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 antigen-binding molecule according to any one of claims 2 to 5, wherein: The multispecific antibody or antigen-binding molecule thereof comprises a constant region; preferably, the constant region is a heavy chain constant region and / or a light chain constant region of a human immunoglobulin; 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.

8. The multispecific antibody or antigen-binding molecule thereof according to claim 7, wherein: The C-terminus of the scFv-1 is connected to the N-terminus of the scFv-2 via a third linker peptide, and the C-terminus of the scFv-2 is connected to the Fc domain of human IgG1 via a hinge peptide; or The C-terminus of the scFv-2 is connected to the N-terminus of the scFv-1 via a third linker peptide, and the C-terminus of the scFv-1 is connected to the Fc domain of human IgG1 via a hinge peptide; or The C-terminus of the scFv-1 is connected to the N-terminus of the Fc domain of human IgG1 via 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 via a fifth linker peptide; or The C-terminus of the scFv-2 is connected to the N-terminus of the Fc domain of human IgG1 via 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 via a fifth linker peptide; 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, GGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n , where 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 can 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 antigen-binding molecule thereof according to claim 8, wherein: The Fc domain of human IgG1 comprises, from N-terminus to C-terminus, a heavy chain constant region CH2 and a heavy chain constant region CH3; Preferably, the sequence of the heavy chain constant region CH2 is shown in SEQ ID NO.17; Preferably, the sequence of the heavy chain constant region CH3 is shown as SEQ ID NO.

18.

10. A multispecific antibody or a homodimer of its antigen-binding molecule, characterized in that: The homologous dimer of the multispecific antibody or antigen-binding molecule thereof is: when the multispecific antibody or antigen-binding molecule thereof according to any one of claims 7 to 9 is expressed in a host cell, the domains of the heavy chain constant region undergo homologous dimerization to form a homologous dimer.

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

12. A vector comprising the nucleic acid molecule as claimed 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 comprising the vector according to claim 12; preferably, the host cell is a mammalian cell.

14. A method for producing the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 9, or the homodimer according to claim 10, characterized in that: The vector or its vector system as claimed in claim 12 is used to transfect host cells and produce the vector; preferably, a lentiviral vector or its vector system comprising the nucleic acid molecule as claimed in claim 11 is used; preferably, an adeno-associated viral vector or its vector system comprising the nucleic acid molecule as claimed in claim 11 is used.

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

16. An immunoconjugate comprising the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 9, or the homodimer according to claim 10; preferably, one or more heterologous molecules are conjugated to the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 9, or the homodimer according to 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 antigen-binding molecule thereof according to any one of claims 1 to 9, or the homodimer according to claim 10, or the nucleic acid molecule according to claim 11, or the vector according to claim 12, or the host cell according to claim 13, or the recombinant protein according to claim 15, or the immunoconjugate according to claim 16, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is in the form of an injection solution or a dosage form for application to the mucosa; more preferably, the pharmaceutical composition is in the form of an intraperitoneal injection solution, an intravenous injection solution, or an intramuscular injection solution; more preferably, the pharmaceutical composition is in the form of a nasal spray solution, a nasal drop solution, an atomized inhalation solution, a nasal lavage solution solution, an oral liquid solution, or a mouthwash solution; more preferably, the pharmaceutical composition is in the form of a tablet, capsule, or powder; more preferably, the pharmaceutical composition is in the form of an external ointment or cream solution, an external gel solution, an external lotion, or an external spray.

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

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

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