A full human single-domain antibody-based anti-novel coronavirus bispecific neutralizing antibody and application thereof

Abstract

The application belongs to the technical field of biology and relates to a full human dual-specific antibody for a novel coronavirus (SARS-CoV-2) and use thereof. Specifically, the application relates to a dual-specific antibody for binding to a novel coronavirus (SARS-CoV-2), a preparation method thereof and use thereof, and further relates to a vector for expressing the dual-specific antibody, a host cell, a preparation and purification method. The dual-specific antibody has broad binding and neutralizing capabilities, and the dual-specific antibody can be used for preparing a medicine for treating or preventing a disease caused by a novel coronavirus infection, and for preparing a product for detecting or diagnosing a novel coronavirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a bispecific antibody binding to SARS-CoV-2, a preparation method thereof, and uses thereof. BACKGROUND

[0002] It is reported that COVID-19, a pneumonia caused by SARS-CoV-2 infection, has most mild clinical manifestations, and severe cases can lead to death. At present, the SARS-CoV-2 strain of the source of the new crown pandemic has been gradually replaced by some SARS-CoV-2 variants, which have high infectivity and spread at a high speed, including B.1.1.7 (20I / 501Y.V1) in the United Kingdom, B.1.351 (20H / 501.V2) in South Africa, P.1 (20J / 501Y.V3) in Brazil, and B.1.617.1 (20A / S:154K) and B.1.617.2 (21A / S:478K) in India. Most importantly, these variants not only escape most neutralizing antibodies, but also weaken the protection ability of existing vaccines, thereby spreading extensively in various countries and regions around the world. Therefore, it is urgent to develop a therapeutic biological agent with broad spectrum against SARS-CoV-2 and its variants.

[0003] Among them, the bispecific antibody has obvious advantages in broad spectrum and neutralizing activity due to its targeting two different epitopes at the same time, and thus has great potential as a therapeutic drug.

[0004] Studies have shown that the new coronavirus invades cells by binding to the human angiotensin-converting enzyme 2 (ACE2) on the cell surface through the receptor binding domain (RBD) on the spike glycoprotein on the surface of the virus. Therefore, RBD is a key target for neutralizing antibodies. At present, most of the fully human monoclonal antibody drugs approved for marketing and in clinical research for SARS-CoV-2 are targeted at the RBD region. However, the molecular weight of the monoclonal antibody is too large (about 150 kDa), which leads to high production cost and high price of the monoclonal antibody drug, hindering its large-scale clinical application in the prevention and treatment of infectious diseases. For a long time, people have been exploring the use of "antibody fragments" with smaller molecular weights that can be expressed in prokaryotic cells to replace IgG-form monoclonal antibodies as a new generation of antibody drugs with low production cost and strong tissue penetration ability. In recent years, researchers have found a class of antibodies containing only heavy chain variable regions in llama serum, which is the smallest antibody fragment with antigen binding performance in nature and is called nanobody (VHH). Studies have shown that these nanobodies have high antigen affinity and specificity, similar to full-length IgG monoclonal antibodies, and are easier to modify and can be expressed in prokaryotic systems, so the preparation cost is very low. Due to the small molecular weight and excellent solubility, stability and other characteristics, nanobodies can be administered by inhalation, allowing nanobodies to reach the lungs directly, act quickly, and be easy to use, making them very suitable for use as pulmonary delivery formulations for respiratory system diseases.

[0005] Therefore, those skilled in the art hope to develop a broad-spectrum neutralizing antibody that can neutralize multiple coronavirus mutants and escape strains with high activity, effectively preventing or treating coronavirus infection. SUMMARY

[0006] The present application provides a bispecific neutralizing antibody against the new coronavirus to solve the above technical problems. Specifically, it relates to a bispecific antibody that binds to the new coronavirus (SARS-CoV-2), a method for preparing the same, and uses thereof.

[0007] The bispecific antibody molecule comprises two single-domain antibodies sdAb-1 and sdAb-2 that can bind to the S protein of the new coronavirus, wherein the amino acid sequence of sdAb-1 is shown in SEQ ID NO: 1, and the amino acid sequence of sdAb-2 is shown in SEQ ID NO: 2.

[0008] Preferably, the C-terminus of sdAb-1 is connected to the N-terminus of sdAb-2 through a peptide linker.

[0009] Preferably, the linker peptide sequence is (Gly4Ser)4, as shown in SEQ ID NO: 3.

[0010] Preferably, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0011] Preferably, the single domain antibody is a heavy chain variable region (VH).

[0012] Preferably, the heavy chain variable region (VH) is fully human.

[0013] The present application also provides a fusion protein, characterized in comprising the bispecific antibody as described above, and a heterologous protein fusion.

[0014] Preferably, the heterologous protein is a human immunoglobulin Fc, more preferably the Fc of human IgG1.

[0015] The present application also provides a conjugate, characterized in that the conjugate comprises the bispecific antibody as described above, or any of the fusion proteins as described above, conjugated with an effector molecule.

[0016] Preferably, the effector molecule is a detectable label.

[0017] Preferably, the detectable label is a fluorescent label, a radioactive label, avidin, biotin, or an enzyme.

[0018] The present application provides a nucleic acid encoding the bispecific antibody as described above. The present application provides a vector comprising the nucleic acid, optionally operably linked to a regulatory sequence. The present application provides a host cell comprising the vector, and a method for producing and optionally recovering the bispecific antibody or antigen binding fragment. The host cell of the present application can be any prokaryotic or eukaryotic cell, including but not limited to bacterial cells (e.g. E. coli), insect cells (e.g. using baculovirus expression system), yeast or mammalian cells (e.g. CHO or BHK cell lines). Other suitable host cells are known to those skilled in the art. Preferably, the host cell is an E. coli cell, which has a short culture period and low production cost.

[0019] The bispecific antibody provided by the present application can specifically bind to corresponding antigens, which refer to all substances capable of inducing immune response of the body. The antigens include but are not limited to small molecule compounds. The antigens include but are not limited to the following listed proteins, subunits, domains, motifs and epitopes belonging to the following proteins: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, alpha-interferon, beta-interferon, gamma-interferon; TNF-alpha, TNF beta2, TNF c, TNF alpha beta, TNF-RI, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L, TRAIL receptor-1, A1 adenosine receptor, lymphotoxin beta receptor, TACI, BAFF-R, EPO;LFA-3, ICAM-1, ICAM-3, EpCAM, βl-integrin, β2-integrin, α4 / β7-integrin, α2-integrin, α3-integrin, α4-integrin, α5-integrin, α6-integrin, αv-integrin, αVβ3-integrin, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGF-βl, TGF-β2, eotaxin 1, BLyS (B-lymphocyte stimulator), complement C5, IgE, Factor VII, CD64, CBL, NCA90, EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), tissue factor, VEGF, VEGFR, endothelin receptor, VLA-4, hapten NP-capped or NIP-capped proteins, T cell receptor alpha / beta, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucin MUC1, MUC18, heparanase I, human cardiac myosin, tumor-associated glycoprotein-72 (TAG-72), tumor-associated antigen CA125, prostate-specific membrane antigen (PSMA), high molecular weight melanoma-associated antigen (HMW-MAA), cancer-associated antigen, Gcoprotein IIb / IIIa (GPIIb / IIIa), tumor-associated antigen expressing Lewis Y related carbohydrate, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HIV gp140, HCMV, respiratory syncytial virus FRSF, RSVF Fgp, VNR integrin, IL-8, cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpllbIIIa, HIV IIIB gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein, and Clostridium perfrmgens toxin, Programmed death-1 (PD-1).

[0020] It will be understood by those skilled in the art that the targets listed above refer not only to specific proteins and biomolecules, but also to the biochemical pathways or various pathways that comprise them. For example, when CTLA-4 is referred to as a target antigen, it is meant that the ligand and receptor make up the T cell costimulatory pathway, including CTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligands or receptors are also targets. Thus, as used herein, a target refers not only to a specific biomolecule, but also to a group of proteins that interact with the target and members of the biochemical pathway to which the target belongs. It will be further understood by those skilled in the art that any of the target antigens described above, the ligands or receptors that bind them, or other members of their respective biochemical pathways can be operably linked to a single-domain antibody or antigen-binding fragment of the application to produce a fusion. Thus, virtually any polypeptide, whether a ligand, receptor, or some other protein or protein domain, including but not limited to the targets described above and the proteins that make up their respective biochemical pathways, can be operably linked to a single-domain antibody or antigen-binding fragment of the application to form a fusion.

[0021] More preferably, the antigens of interest include, but are not limited to, cancer, infectious diseases (e.g., viral, bacterial, fungal, parasitic infections, etc.), autoimmune diseases, inflammatory disorders.

[0022] The antigen can be from a virus. Viruses contemplated by the present application, for example, are from one of the following families: Retroviridae (e.g., human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV)); Picornaviridae (e.g., poliovirus, hepatitis A virus, hepatitis C virus, enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses, foot-and-mouth disease virus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, yellow fever virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, and other encephalitis viruses); Coronaviridae (e.g., coronavirus, severe acute respiratory syndrome (SARS) virus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV)); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hantaan virus, Sin Nombre virus, Rift Valley fever virus, bunya viruses, phleboviruses, and Nairo viruses); Arenaviridae (e.g., hemorrhagic fever virus, Machupo virus, Junin virus); Reoviridae (e.g., reoviruses, orbiviurses, and rotaviruses); Birnaviridae; Hepadnaviridae (e.g., hepatitis B virus); Parvoviridae (e.g., parvovirus); Papovaviridae (e.g., papillomavirus, polyomavirus, BK virus); Adenoviridae (e.g., most adenoviruses, such as aden-associated viruses); Herpesviridae (e.g., herpes simplex virus (HSV-1 and HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), and other herpes viruses, including HSV-6); Poxviridae (e.g., variola virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); Filoviridae (e.g., Ebola virus, Marburg virus); Caliciviridae (e.g., Norwalk virus) and unclassified viruses (e.g., the agent of spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of the hepatitis B virus), and astroviruses).

[0023] The antigen can be from a bacterium. Bacteria contemplated by the present application are, for example, Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Menigococcus, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenza, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Hog cholera virus, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides species, Clostridium sphenoides, Streptomyces madurae, Treponema pallidum, Treponema pertenue, Leptospira or Actinomyces israelli.

[0024] The antigen can be from a fungus. Fungi contemplated by the present application are, for example, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis or Candida albicans.

[0025] The antigen can be from a parasite. Parasites contemplated by the present application are, for example, Plasmodium falciparum or Toxoplasma gondii.

[0026] The antigen can be a cancer antigen, such as a solid tumor or a blood-borne cancer antigen. The solid tumors contemplated by the present application are sarcomas or carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or another sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary duct carcinoma, choriocarcinoma, nephroblastoma, cervical cancer, testicular tumor, bladder carcinoma, or a central nervous system tumor (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, angioma, melanoma, neuroblastoma, or retinoblastoma). The blood-borne cancers contemplated by the present application are leukemias, such as acute leukemias (e.g., acute lymphatic leukemia, acute myelocytic leukemia, acute granulocytic leukemia and myeloblasts, promyelocytcs, myelomonocytic, monocytic and erythroleukemia); chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia, chronic granulocytic leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia or myelodysplasia. Tumor antigens are well known in the art and include, for example, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin (β-HCG), alpha-fetoprotein (AFP), lectin-reactive AFP, (AFP-L3), thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase (hTERT), RU1, RU2 (AS), intestinal carboxyl esterase, muthsp70-2, M-CSF, prostein, prostate specific membrane antigen (PSMA), Her2 / neu, survivin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), melanoma-associated antigen (MAGE), ELF2M, neutrophil elastase, ephrinB2 and CD22. It can also be any cancer-related protein, such as IGF-I, IGF-II, IGR-IR or mesothelin.

[0027] The present application provides a pharmaceutical composition prepared by mixing an effective prophylactic or therapeutic dose of the bispecific antibody, fusion protein, conjugate, nucleic acid molecule for the bispecific antibody, vector comprising the nucleic acid molecule for the bispecific antibody, and physiologically or pharmaceutically acceptable carriers, excipients, or stabilizers, including but not limited to a lyophilized dosage form, an aqueous solution dosage form, a liposome, or a capsule dosage form, etc. The concentration of the bispecific antibody, fusion protein, conjugate, nucleic acid molecule for the bispecific antibody, vector comprising the nucleic acid molecule for the bispecific antibody, according to the present application, can vary from about 0.1% to 100% by weight.

[0028] In order that the application can be more completely understood, some definitions are set forth below. The above definitions are intended to encompass grammatical equivalents.

[0029] As used herein, "bispecific antibody" means an artificial antibody containing two specific antigen binding sites.

[0030] As used herein, "antibody molecule" means a protein consisting of one or more domains encoded by substantially all or a portion of an accepted immunoglobulin gene. The accepted immunoglobulin genes, for example in humans, include the kappa (K), lambda (λ), and heavy chain loci, which contain a multitude of variable region genes, and the constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), alpha (α) that encode the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. Antibody herein is meant to include full-length antibodies, individual chains thereof, and all portions, domains or fragments thereof, as well as natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes as further specified below. The term "antibody" includes antibody fragments, which are well known in the art, such as Fab, Fab', F(ab')2, Fv, scFv, or antigen binding domains of antibodies (e.g., VHH domains or VH / VL domains), or those produced by modification of whole antibodies or resynthesized using recombinant DNA techniques. The term "antibody" includes monoclonal as well as polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing antibodies, or inhibitory antibodies, or stimulatory antibodies. Antibodies of the present application can be non-human antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0031] Specifically included in the definition of "antibody" are aglycosylated antibodies. Preferably, as used herein, "aglycosylated antibody" means an antibody that lacks the attachment of a carbohydrate at position 297 in the Fc region, wherein numbering is according to the EU system of Kabat. The aglycosylated antibody can be a deglycosylated antibody, which is an antibody from which the Fc carbohydrate has been removed, for example, by chemical or enzymatic means. Alternatively, the aglycosylated antibody can be an aglycosylated or unglycosylated antibody, which is an antibody that does not express an Fc carbohydrate, for example, by mutating the glycosylation site(s) encoding residue(s) or by expression in an organism that does not attach carbohydrate to proteins, such as bacteria.

[0032] "IgG" as used herein means a polypeptide belonging to the class of antibodies that are essentially encoded by the immunoglobulin gamma gene. In humans, this class includes IgGl, IgG2, IgG3, and IgG4. In mice, this class includes IgGl, IgG2a, IgG2b, IgG3. "Immunoglobulin (Ig)" as used herein means a protein consisting of one or more polypeptides essentially encoded by an immunoglobulin gene. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have many structural forms, including but not limited to full-length antibodies, antibody fragments, and individual immunoglobulin domains. The Ig domains VH, Cyi, Cy2, Cy3, VL, and CL known in the IgG class of antibodies.

[0033] "Antigen" as used herein means a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including a composition injected or absorbed into an animal, which can be a protein, carbohydrate, lipid, or other pathogen.

[0034] "Single domain antibody" as used herein means an immunoglobulin variable domain that is capable of specifically binding to an epitope of an antigen without pairing with other immunoglobulin variable domains.

[0035] "Variable region of heavy chain (VH)" as used herein means the region of an immunoglobulin heavy chain near the N-terminal amino acid sequence that varies more greatly than the constant region of the heavy chain in a conventional 4-chain antibody. It is particularly used to distinguish VHH antibodies of the Camelidae family.

[0036] "Identity" as used herein means the similarity between nucleotide or amino acid sequences, or is referred to as sequence identity. Sequence identity is typically measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants will have a relatively high degree of sequence identity when aligned using standard methods. Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv Appl. Math., 2:482, 1981; Needlema and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; and Altschul et al., Nature Genet., 1994, 6:119-129.

[0037] NCBI Basic Local Alignment Search Tool (BLAST™) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet at the address: www.ncbi.nlm.nih.gov / BLAST / . The program blastp, blastn, blastx, tblastn and tblastx are used for sequence analysis.

[0038] "Fully human" as used herein means that the antibody is encoded entirely by human antibody genes.

[0039] "Fusion protein" as used herein means the expression product of a fused gene, or two or more proteins fused by biological or chemical means.

[0040] "Coupled" as used herein means that the antibody or protein molecule is linked to another small or large molecule by biological or chemical means.

[0041] As used herein, "amino acid" means one of the 20 naturally occurring amino acids or any unnatural analog thereof, which can be located at a specifically prescribed position. As used herein, "protein" means at least two covalently linked amino acids, which includes proteins, polypeptides, oligopeptides, and peptides. A protein can be composed of naturally occurring amino acids and peptide bonds, or of synthetic peptidomimetic structures, i.e., "analog." Thus, as used herein, "amino acid" or "peptide residue" means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline, and norleucine are considered amino acids for purposes of the present application. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain can be in the (R) or (S) configuration. In preferred embodiments, the amino acids are present in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substitutions can be used, e.g., to prevent or delay in vivo degradation.

[0042] As used herein, "polypeptide" means a polymer in which the monomers are amino acid residues linked via amide bonds. When the amino acids are alpha-amino acids, L- optical isomers or D-optical isomers can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences, e.g., glycoproteins. The term "polypeptide" is specifically intended to encompass naturally occurring proteins, as well as recombinantly or synthetically produced proteins.

[0043] As used herein, "nucleic acid" means a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include synthetic non-naturally occurring analogs, such as, but not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. For example, these polynucleotides can be synthesized using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, typically no more than about 50 nucleotides. It will be appreciated that, when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" is substituted for "T."

[0044] Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5' end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcriptional direction. DNA sequences encoding the same sequence of RNA as the mRNA are referred to as coding strands.

[0045] As used herein, "vector" means a vehicle that is artificially constructed on the basis of natural plasmids for adaptation to laboratory manipulation. A nucleic acid molecule can be introduced into a host cell to produce a transformed host cell. The vector can include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication, and can also include one or more selectable marker genes and other genetic elements known in the art.

[0046] As used herein, "host cell" also referred to as a recipient cell, means a host cell that receives and reproduces a foreign gene in transformation and transduction (infection).

[0047] As used herein, "pharmaceutically acceptable carrier" means a conventional pharmaceutically acceptable carrier. Remington's Pharmaceutical Sciences, E W Martin, Mack Publishing Co., Easton, Pa., 15th Ed. (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more antibodies, and additional agents.

[0048] As used herein, "effective prophylactic or therapeutic dose" means an amount of a particular agent that is sufficient to achieve a desired effect in a subject treated with the agent. The precise dose will depend upon the purpose of the treatment, and can be determined by one of skill in the art using well-known techniques. The dose range can be 0.01-100 mg / kg body weight or more, such as 0.1, 1, 10, or 50 mg / kg body weight, preferably 1-10 mg / kg. Adjustments can be necessary for antibody or Fc fusion degradation, systemic or local delivery, and the rate of new protease synthesis, as well as age, body weight, general health, sex, diet, time of administration, drug interactions, and the severity of the condition, as is well known in the art, and can be determined by one of skill in the art through routine experimentation. Ideally, a therapeutically effective amount of an antibody is an amount sufficient to prevent, treat, or ameliorate an infection or disease. A therapeutically effective amount of an agent for preventing, ameliorating, and / or treating a subject will depend on the subject being treated, the type and severity of the affliction, and the manner of administration of the therapeutic composition.

[0049] “Encoding” as used herein is intended to mean the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having the determined sequence of nucleotides, or to serve as templates for synthesis of other sequences of nucleotides and proteins having a determined sequence of amino acids resulting from biological processes. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene results in a protein in a cell or other biological system. Both the coding strand, which has the same nucleotide sequence as the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, which is the actual template for transcription, serve as templates for synthesis of mRNA, genes, and cDNAs. Unless otherwise indicated, “a nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence. Nucleotide sequences which encode proteins and RNA can include introns.

[0050] 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 disclosure belongs. Except where otherwise expressly indicated, all singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term “comprising” means “including.” All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including the BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 Neutralization activity of single domain antibodies n3113.4 and n3130.6 after affinity maturation on SARS-CoV-2 pseudovirus.

[0052] Figure 2 Structure illustration of bispecific antibodies against SARS-CoV-2.

[0053] Figure 3 SDS-PAGE to detect the purity of bispecific antibodies.

[0054] Figure 4 ELISA to detect the binding ability of bispecific antibodies to SARS-CoV-2 variant RBD protein.

[0055] Figure 5 ForteBio to detect the binding affinity of bispecific antibodies to SARS-CoV-2 variant B1.617 RBD protein.

[0056] Figure 6 Neutralization activity of single domain antibody n3113.4, n3130.6 and double antibody against SARS-CoV-2 original strain and variant B1.617 using pseudovirus system, with single antibody S309 as positive control. DETAILED DESCRIPTION

[0057] The standard recombinant DNA techniques and molecular cloning techniques used in the embodiments of the present application are well known in the art (Ausubel, F. M et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience), and the materials and methods suitable for microbial growth are well known in the art. The main chemicals and biological reagents are purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, ThermoFisher Scientific, OMEGA bio-tek, etc.

[0058] The present application will be described in detail below with specific examples.

[0059] Example 1 Method for affinity maturation of single domain antibody against SARS-CoV-2

[0060] The single domain antibodies sdAb1 and sdAb2 contained in the bispecific antibody adopt the heavy chain variable region sequences of single domain antibodies n3113 and n3130 (patent application No. 202010239592.8), and then based on the above-mentioned single domain antibodies, an affinity maturation method is used to improve the binding capacity to the new coronavirus. The specific method is as follows: first, the amino acids in the 3 CDR regions of the single domain antibody VH are subjected to site-directed mutagenesis by PCR, and each amino acid is subjected to 20 kinds of amino acid saturation mutation. Then, the mutated fragments are constructed into the phage display vector pComb3x (Addgene, product No: 63891) using homologous recombination. The homologous recombination product is electroporated into E. coli electrocompetent TG1 (purchased from Lucigen), and single colonies are picked from the ampicillin overnight growth plate. Single colony ELISA identification is performed, and positive clones are sent for sequencing to obtain the sequence. The positive clone plasmid is transformed into HB2151 competent cells, and single colonies are picked from the ampicillin overnight growth plate. SB bacterial culture solution is inoculated, and expression is induced at 30 degrees IPTG for 12-14 hours. The bacteria are harvested and the single domain antibodies are purified from them using Ni-NTA (purchased from GE Healthcare). The neutralization ability of these antibodies to SARS-CoV-2 is determined by the pseudovirus neutralization system. The results show that after affinity maturation of n3113, the neutralization ability of n3113.4 is greatly improved, and the IC50 value is reduced from 10.96 μg / ml to 1.82 μg / ml. After affinity maturation of n3130, the best neutralization activity is n3130.6, and the IC50 value is 0.7 μg / ml. The neutralization IC50 of the parent single domain antibody n3130 is 4 μg / ml (data from patent application No. 202010239592.8).

[0061] Example 2 Synthesis of bispecific antibody gene

[0062] The nucleic acid sequence encoding the affinity-matured single domain antibody n3113.4 against the new coronavirus is shown as SEQ ID NO: 4, and the nucleic acid sequence encoding the affinity-matured single domain antibody n3130.6 against the new coronavirus is shown as SEQ ID NO: 5. The two sequences are fused using a peptide linker (GGGGS)4 (shown as SEQ ID NO: 6), i.e. n3113.4-(GGGGS)-n3130.6. The structure is shown in Figure 2 .

[0063] Example 3 Construction of bispecific antibody expression vector

[0064] The genes of the synthesized bispecific antibodies were cloned into the prokaryotic expression vector pComb3x (Addgene, product number: 63891) by homologous recombination. The homologous recombination product was transformed into E. coli DH5a competent cells, and the correct sequence of the bispecific antibody expression vector was obtained by sequencing a single colony.

[0065] Example 4 Expression and purification of bispecific antibodies

[0066] The bispecific antibody expression vector was transformed into E. coli HB2151 competent cells, a single colony was picked from an overnight grown ampicillin plate, inoculated into SB bacterial culture, and expressed for 12-14 hours under 30 degree IPTG induction conditions. The bacteria were harvested and the bispecific antibodies were purified from them using Ni-NTA (purchased from GE Healthcare).

[0067] Example 5 Polyacrylamide gel electrophoresis (SDS-PAGE) for detecting the purity of bispecific antibodies

[0068] 10 μg of purified bispecific antibodies were added to the loading buffer at a volume ratio of 1:6, boiled in boiling water for 10 min, and centrifuged briefly after cooling. Electrophoresis was performed at a constant voltage of 120 V for 1.5 h. After electrophoresis, the gel was gently removed and stained in an appropriate amount of 0.25% Coomassie brilliant blue staining solution for 30 min, and then decolorized in double distilled water until clear bands were shown. If Figure 3 It was shown that the molecular weight of the bispecific antibody was about 30 kD, the band was single, and the purity was very good.

[0069] Example 6 Binding ability of bispecific antibodies to SARS-CoV-2 variant RBD proteins

[0070] 200 ng of SARS-CoV-2 variant RBD proteins (all purchased from Yiqiao) were coated on an ELISA plate at 4 degrees overnight, and dilutions of bispecific antibodies were added to incubate. Anti-FLAG-HRP antibody (purchased from SIGMA) was used to detect the binding ability of the antibody to SARS-CoV-2 RBD protein. The results showed that the specific antibody could very strongly bind to SARS-CoV-2 RBD protein Figure 4 ), and the binding ability to each variant was comparable to that of the original strain, indicating that the bispecific antibody had good broad-spectrum.

[0071] Example 7 Kinetics detection of bispecific antibodies binding to SARS-CoV-2 variant B1.617 RBD protein

[0072] The binding activity of bispecific antibodies to the SARS-CoV-2 variant B1.617 RBD protein was determined using BLI technology (Cell Host Microbe. 2020; 27(6):891-898.e5). B1.617-RBD-his protein (purchased from Yiqiao, product number: 40592-V08H88) was immobilized on an amino-coupled sensor (AR2G biosensor) (Pall Fortebio), and serially diluted antibody solutions were used as analytical samples. The procedure was as follows: RBD protein was diluted to 10 μg / ml with sodium acetate solution (pH 5.0), then immobilized on the AR2G biosensor, and bound to 3-fold serially diluted antibody solutions (concentration range 500–6 nM) in running buffer (PBST). No antibody was added to the blank control wells. The program settings were: Association, 420s; Dissociation, 420s, temperature set at 37℃. The data were analyzed using ForteBio Data Analysis 10.1 software. The corresponding blank control wells were used as controls to subtract background. The curves for each concentration were fitted using a 1:1 binding mode to obtain the kinetic analysis results and curves. Figure 5 The results showed that the affinity of the double antibody for the mutant strain B1.617 RBD protein was 0.69 nM.

[0073] Example 8: Detection of neutralizing activity of bispecific antibodies using a pseudovirus system

[0074] 293T cells were co-transfected with an HIV-1 backbone plasmid carrying the luciferase reporter gene and a recombinant plasmid containing the SARS-CoV-2 S protein or a variant S protein. The supernatant was harvested after 48 hours. n3113.4, n3130.6, and bispecific antibodies were diluted with cell culture medium containing 10% serum. Monoclonal antibody S309 (Nature. 2020; 583(7815):290-295.), which has broad-spectrum neutralizing activity against coronaviruses, was used as a positive control. 50 μl of the diluted antibody was mixed with 50 μl of virus and added to Huh-7 cells. After 12 hours of incubation at 37°C, the medium was replaced with fresh medium, and the cells were cultured for another 48 hours. The relative fluorescence values ​​in the cell lysate were detected using the Promega Luciferase Assay Kit. The results showed that the bispecific antibody exhibited stronger neutralizing activity against both the original and variant strains of SARS-CoV-2 than the single-domain antibody alone, and its neutralizing activity against the variant strain was stronger than that of the broad-spectrum antibody S309. Figure 6 ).

[0075] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. The above description is only preferred embodiments of the application. It is understood that numerous modifications and additions can be made by those skilled in the art without departing from the spirit and scope of the application. These modifications and additions are intended to fall within the scope of the application. It will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents. SEQUENCE LISTING <110> Fudan University <120> A fully human single-domain antibody-based anti-SARS-CoV-2 bispecific neutralizing antibody and application thereof <130> 20210819 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 118 <212> PRT <213> n3113.4 variable heavy chain <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Ser Ser Phe Tyr Asp Tyr 20 25 30 Glu Met Ser Trp Val Arg Gln Val Pro Gly Lys Thr Pro Glu Trp Ile 35 40 45 Gly Ser Met Tyr Pro Ser Gly Arg Thr Tyr Ile Asn Pro Ser Leu Lys 50 55 60 Ser Leu Val Thr lie Ser Arg Asp Asn Ser Glu Asn Met Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Val 85 90 95 Ser Asn Trp Ala Ser Gly Ser Thr Gly Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 2 <211> 120 <212> PRT <213> n3130.6 variable heavy chain <400> 2 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Phe Tyr Phe Asp Tyr Tyr 20 25 30 Glu Met Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Val 35 40 45 Ser Thr lie Ser Gly Leu Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr Arg Ser Pro Phe Gly Asp Tyr Ala Phe Ser Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 20 <212> PRT <213> Peptide linker <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 4 <211> 354 <212> DNA <213> n3113.4 variable heavy chain <400> 4 gaagttcagc tggttgaatc tggtggtggt ctggtacagc caggtggtag cctgcgtctg 60 agctgtgcag cgagcgatag cagcttctat gactatgaaa tgtcctgggt acgtcaagtt 120 ccaggtaaga ccccggaatg gatcggcagc atgtacccga gcggccgtac ttacatcaac 180 ccgtctctga aatccctggt taccatcagc cgtgataact ctgaaaacat gctgtatctg 240 ccgtctctga aatccctggt taccatcagc cgtgataact ctgaaaacat gctgtatctg 240cagatgaact ccctgcgcgc tgaagatacc gcaatgtatt actgcgtgag caactgggca 300 tccggctcta ccggtgacta ctggggtcag ggcaccctgg tcactgtatc ttct 354 <210> 5 <211> 360 <212> DNA <213> n3130.6 variable heavy chain <400> 5 gaagtgcagc tggtagaatc tggcggcggt ctggttcaac caggcggttc tctgcgcctg 60 tcttgtgctg cttctgactt ctactttgat tactacgaaa tgagctgggt tcgtcaggct 120 ccaggtcagg gtctggaatg ggtatctact atctccggtc tgggcggcgc tacctattac 180 gcggatagcg ttaaaggccg tttcacgatc agccgtgaca actctaaaaa cactctgtac 240 ctgcagatga acagcctgcg tgcagaagac actgcactgt attactgtgc tacccgtagc 300 ccgtttggtg attacgcgtt ctcctactgg ggccagggca ctctggttac cgttagcagc 360 <210> 6 <211> 60 <212> DNA <213> Peptide linker <400> 6 ggtggcggtg gttctggtgg tggcggttct ggtggtggtg gttctggtgg tggtggtagc 60

Claims

1. A bispecific antibody that binds to the novel coronavirus (SARS-CoV-2), characterized in that, The bispecific antibody comprises two single-domain antibodies, sdAb-1 and sdAb-2, which can bind to the novel coronavirus S protein, wherein the amino acid sequence of sdAb-1 is shown in SEQ ID NO:1 and the amino acid sequence of sdAb-2 is shown in SEQ ID NO:

2.

2. The bispecific antibody as described in claim 1, characterized in that, The C-terminus of sdAb-1 is connected to the N-terminus of sdAb-2 via a peptide linker.

3. The bispecific antibody as described in claim 2, characterized in that, The linker peptide sequence is (GGGGS)4, as shown in SEQ ID NO:

3.

4. A fusion protein, characterized in that, The invention comprises a bispecific antibody according to any one of claims 1-3, and a heterologous protein fused together, wherein the heterologous protein is human immunoglobulin Fc.

5. The fusion protein as described in claim 4, characterized in that, The heterologous protein mentioned therein is the Fc of human IgG1.

6. A coupling agent, characterized in that, A bispecific antibody according to any one of claims 1-3, or a fusion protein according to claim 4 or 5, conjugated to an effector molecule, wherein the effector molecule is a detectable marker.

7. The coupling as described in claim 6, characterized in that, The detectable marker is a fluorescent marker, a radioactive marker, avidin, biotin, or an enzyme.

8. A nucleic acid molecule, characterized in that... Encoding the bispecific antibody of any one of claims 1-3, or the fusion protein of claim 4 or 5.

9. A carrier, characterized in that... It includes the nucleic acid molecule as described in claim 8.

10. A host cell, characterized in that... It comprises the nucleic acid molecule of claim 8 or the vector of claim 9.

11. A pharmaceutical composition, characterized in that, The bispecific antibody containing an effective preventive or therapeutic dose of any one of claims 1-3, or the fusion protein of claim 4 or 5, or the conjugate of claim 6 or 7, and a pharmaceutically acceptable carrier.

12. Use of the bispecific antibody of any one of claims 1-3, or the fusion protein of claim 4 or 5, or the conjugate of claim 6 or 7, or the nucleic acid molecule of claim 8, or the carrier of claim 9, or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating and / or preventing diseases caused by novel coronavirus infection.

13. Use of the bispecific antibody of any one of claims 1-3, or the fusion protein of claim 4 or 5, or the conjugate of claim 6 or 7, or the nucleic acid molecule of claim 8, or the carrier of claim 9, or the pharmaceutical composition of claim 11 in the preparation of diagnostic or detection products for detecting and / or diagnosing novel coronavirus infection.

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