Coronavirus N antigen nano antibody and application thereof

Abstract

The invention discloses a coronavirus N antigen nano antibody and application thereof. The nano antibody comprises a heavy chain variable region, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3. The invention also discloses a bivalent nano antibody containing the nano antibody, nucleic acid for coding the nano antibody or the bivalent nano antibody, a recombinant expression vector containing the nucleic acid, a transformant, a preparation method of the antibody, a pharmaceutical composition containing the nano antibody or the bivalent nano antibody, a chimeric antigen receptor, a chimeric antigen receptor, a chimeric antigen receptor and a chimeric antigen receptor. The invention relates to an antibody drug conjugate, a kit, a kit kit, a drug delivery device and a method for determining a coronavirus N antigen. The binding capacity of the nano antibody and an SARS-CoV-2N antigen reaches a picomole level, and the nano antibody has high specificity, high sensitivity, high affinity and broad spectrum, has extremely high application value in new coronavirus detection, and has an application theoretical basis in the aspects of scientific research or clinical industrial application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a coronavirus N antigen nano antibody and an application thereof. Background Art

[0002] The novel coronavirus 2 (SARS-CoV-2) and the virus that causes Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS-CoV-1) all belong to the beta coronavirus family. The overall shape of SARS-CoV-2 is round or oval, with a diameter of about 80-120nM. It is a positive-stranded single-stranded RNA virus with an envelope structure. The genome size is about 30Kb, and the related structural proteins are envelope protein (E protein), membrane protein (membrane, M protein), spike protein (spike, S protein) and nucleocapsid protein (nucleocapsid, N). Among them, S, E and M proteins together constitute the outer shell of the virus, and N protein combines with viral RNA to form the nucleocapsid of the virus. N protein is a phosphorylated protein composed of two domains, N-terminal domain (NTD) and C-terminal domain (CTD). It participates in and regulates RNA transcription, replication and protein translation. It is the structural protein with the most copies expressed during viral replication and is closely related to pathogenicity.

[0003] The diagnostic tests for the novel coronavirus 2 are mainly divided into three categories: nucleic acid testing, which is to detect the genetic material of the novel coronavirus, which is the gold standard for the current novel coronavirus testing and requires specific places, specially trained technicians, equipment and instruments, etc.; antibody testing, which is to detect the antibodies produced by the body in response to novel coronavirus infection. This test can often only detect the novel coronavirus antibodies IgM or IgG produced in the body for a period of time after infection, and often has a certain lag; antigen testing, which is the test for the novel coronavirus's own protein, is used to quickly diagnose (or exclude) active coronavirus infection, also known as rapid testing. The National Health Commission pointed out that nucleic acid testing is the basis for the diagnosis of novel coronavirus infection, and antigen testing can be used as a supplementary means for screening specific populations, which is conducive to improving early detection capabilities.

[0004] In addition, among different variants of the new coronavirus, unlike the S protein, which is prone to mutation, the N protein, especially the two domains of the N protein, NTD and CTD, mutate very little and are relatively conservative in structure; at the same time, compared with the N proteins of other coronaviruses, the new coronavirus N and its NTD and CTD domains maintain obvious specificity and can be identified and distinguished from other coronaviruses through specific binding detection. The N protein of the new coronavirus, with its conservative and specific characteristic patterns, as well as its high copy and rich content, has become a key target protein in the clinical diagnosis, epidemiological research, pathogenic infection mechanism and other studies of the new coronavirus. The development of high-affinity antibodies against the N protein of the new coronavirus is the key to the above studies.

[0005] Most of the existing antibodies against the SARS-CoV-2 N antigen are obtained through hybridomas or isolated from recovered patients infected with the SARS-CoV-2. Most of them are traditional monoclonal antibodies. Such traditional antibodies have high production costs and are expensive, which is not conducive to large-scale promotion and application in pandemic infectious diseases.

[0006] Nanobody (Nb) is a new type of antibody, which comes from the variable domain VHH of heavy chain antibody with natural light chain missing in camelids. Due to the natural lack of light chain and small molecular weight, it can be amplified and expressed in large quantities using prokaryotic and yeast systems, with relatively lower production costs, which is conducive to its promotion and application in infectious diseases such as pandemic viruses. Nb consists of four conserved framework regions (FR) and three complementarity-determining regions (CDR). The number of amino acids in its CDR region is generally longer than that of traditional human or mouse CDR3, which can form a convex loop structure (traditional antibodies usually have concave or flat antigen binding sites), with a large number of CDR loops exposed in the solvent, which improves the specificity and affinity of antigen binding (Vanlandschoot et al., 2011b; Desmyter et al., 2001). The four hydrophilic residues in the FR2 region of Nb replace the four hydrophobic residues in the FR2 of traditional antibodies, making it more soluble. The internal disulfide bonds make it more heat-resistant and acid-resistant than traditional antibodies, and its aggregation is reduced. It still has biological activity after long-term storage in high temperature environment or strong denaturation conditions, and has higher in vitro stability (Vanlandschoot et al., 2011b; Muyldermans et al., 1994; Hamers-Casterman et al., 1993). It can be amplified and expressed in large quantities using prokaryotic and yeast systems, with relatively lower production costs, which is more conducive to the development of antigen detection products that require room temperature conditions. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defects of the prior art in that the novel coronavirus N antigen-antibody binding specificity, broad spectrum, binding sensitivity and binding affinity are not high, and the production cost is high, the price is expensive, and it is not conducive to the large-scale promotion and application of pandemic infectious diseases. The SARS-CoV-2 wild-type and variant strain N proteins are cross-immunized with alpacas to detect and evaluate the titer of alpaca serum antibodies. After the immunization, alpaca peripheral blood is collected, and plasma and peripheral blood lymphocytes (PBMCs) are separated. PBMC total RNA is extracted and reversely transcribed into cDNA, and then alpaca nanoantibodies (VHHs) are amplified by multiple PCR, and the nanoantibody phage library is built. Then, by the method of multi-antigen cross-panning, SARS-CoV-2N nanoantibodies with high affinity and broad-spectrum binding activity are screened. The nanoantibody is subjected to multivalent modification and binding specificity, broad spectrum, binding sensitivity, binding affinity and other detection and evaluation.

[0008] The present invention uses SARS-CoV-2 and its variants N antigen to cross-immunize alpacas to obtain high serum antibody immune titers, and then uses phage display technology to cross-pannounce and screen out nanoantibodies against the new coronavirus N antigen.

[0009] To solve the above technical problems, the present invention provides a technical solution: a nanobody targeting a coronavirus N antigen, comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR3 of the nanobody is as shown in SEQ ID NO: 3. In a specific embodiment of the present invention, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 9 or SEQ ID NO: 15.

[0010] Alternatively, the amino acid sequence of HCDR3 of the Nanobody is as shown in SEQ ID NO:6.

[0011] Alternatively, the amino acid sequence of HCDR3 of the Nanobody is as shown in SEQ ID NO:12.

[0012] As described in the Nanobody of the present invention, the Nanobody is a Nanobody whose amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, whose amino acid sequence of HCDR2 is shown in SEQ ID NO: 2 and whose amino acid sequence of HCDR3 is shown in SEQ ID NO: 3.

[0013] In a specific embodiment of the present invention, the Nanobody is a Nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO: 7, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 8 and an amino acid sequence of HCDR3 as shown in SEQ ID NO: 9, or the Nanobody is a Nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO: 13, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 14 and an amino acid sequence of HCDR3 as shown in SEQ ID NO: 15.

[0014] Alternatively, the Nanobody is a Nanobody whose amino acid sequence of HCDR1 is shown in SEQ ID NO:4, whose amino acid sequence of HCDR2 is shown in SEQ ID NO:5 and whose amino acid sequence of HCDR3 is shown in SEQ ID NO:6.

[0015] Alternatively, the Nanobody is a Nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO:10, an amino acid sequence of HCDR2 as shown in SEQ ID NO:11 and an amino acid sequence of HCDR3 as shown in SEQ ID NO:12.

[0016] The term "nanoantibody" in this article, also known as "single-domain antibody", contains only one heavy chain variable region VHH, with a molecular weight of about 15kDa and a diameter of about 10 nanometers. It retains all antigen binding ability and is the smallest fragment that retains complete antigen binding.

[0017] In the present invention, the amino acid sequences of the above-listed CDRs are shown in accordance with the IMGT definition rules. However, it is well known to those skilled in the art that the CDR of an antibody can be defined in the art by a variety of methods, such as Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be appreciated by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0018] Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of the present invention is based on the sequences shown in the IMGT definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of the present invention.

[0019] As described in the Nanobody of the present invention, the Nanobody is a Nanobody with an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 16; the Nanobody is a Nanobody with an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 17; the Nanobody is a Nanobody with an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 18; or, the Nanobody is a Nanobody with an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 19.

[0020] The term "variable region" refers to the domain of the antibody heavy chain that is involved in antibody binding to antigen. VH contains four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2 and HCDR3. Each VH is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0021] The four N antigen nanobodies involved in the present invention basically bind to the same epitope of the N antigen and are the same type of epitope-binding antibodies.

[0022] The present invention provides the biological activity characteristics of the above-mentioned nanoantibodies, including antibody binding specificity, binding broad spectrum, binding sensitivity, affinity dissociation equilibrium constant and other data. The invented new crown N nanoantibody can efficiently and specifically bind to the SARS-CoV-2N antigen. The above-mentioned N nanoantibody is used in SARS-CoV-2 detection and diagnosis, epidemiological research, pathogenic mechanism research, etc., which are all within the scope of protection of the present invention.

[0023] The N nanoantibodies involved in the present invention, regardless of the form of genetic engineering modification, including but not limited to the following modifications: small molecule structure modification, humanization, bispecific nanoantibody modification, bivalent or multivalent nanoantibody modification, coupling with other structural proteins or chemical reagents, etc., are all within the scope of protection of the present invention.

[0024] The term "specific binding" refers to an antibody that binds to an antigen or an epitope within the antigen with a higher affinity than it does to other antigens or epitopes. -9 M or less (e.g., about 1×10 -10 M or smaller, about 1×10 -11 M or smaller, about 1×10 -12 M or smaller, about 1×10 -13 M or smaller, or about 1×10 -14The equilibrium dissociation constant (KD) of a molecule binding to an antigen or an epitope within an antigen is 2 M or less. KD can be measured using standard procedures, for example, by direct coupling (Biacore) testing.

[0025] To solve the above technical problems, the present invention provides a technical solution: a bivalent nanobody targeting coronavirus N antigen, wherein the bivalent nanobody comprises the nanobody as described in the present invention, and preferably the bivalent nanobody further comprises a heavy chain constant region.

[0026] In a preferred embodiment of the present invention, the heavy chain constant region is rabbit IgG Fc.

[0027] In a more preferred embodiment of the present invention, the amino acid sequence of the rabbit IgG Fc is shown in SEQ ID NO:20.

[0028] As for the bivalent Nanobody of the present invention, the amino acid sequence of the bivalent Nanobody is as shown in SEQ ID NO: 21; the amino acid sequence of the bivalent Nanobody is as shown in SEQ ID NO: 22; the amino acid sequence of the bivalent Nanobody is as shown in SEQ ID NO: 23; or, the amino acid sequence of the bivalent Nanobody is as shown in SEQ ID NO: 24.

[0029] The bivalent nanobody described in the present invention is a new type of antibody molecule consisting only of heavy chains. Although it lacks light chains compared to ordinary antibodies, it still retains the ability to bind to antigens.

[0030] To solve the above technical problems, the present invention provides a technical solution: an isolated nucleic acid, which encodes the nanobody as described in the present invention or the bivalent nanobody as described in the present invention.

[0031] As known in the art, "nucleic acid" in the present invention refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0032] In order to solve the above technical problems, the present invention provides a technical solution: a recombinant expression vector comprising the isolated nucleic acid as described in the present invention;

[0033] In a preferred embodiment of the present invention, the backbone of the recombinant expression vector is a plasmid, a phage or a viral vector, for example, the backbone of the recombinant expression vector is pMECS or pCMV.

[0034] The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of mRNA, protein, polypeptide or peptide by a host cell when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is in contact with a cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell. The vectors disclosed herein are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring internucleotide connections, or both types of connections. In exemplary aspects, altered nucleotides or non-naturally occurring internucleotide connections do not hinder transcription or replication of the vector.

[0035] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, which can be used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the gene or sequence in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0036] In order to solve the above technical problems, the present invention provides a technical solution: a transformant, which comprises the isolated nucleic acid as described in the present invention or the recombinant expression vector as described in the present invention.

[0037] In a preferred embodiment of the present invention, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell;

[0038] In a more preferred embodiment of the present invention, the prokaryotic cell is Escherichia coli, or the eukaryotic cell is a mammalian cell;

[0039] In a further preferred embodiment of the present invention, the Escherichia coli is E. coli TG1 or E. coli WK6, or the mammalian cell is HEK293F cell or CHO cell.

[0040] As used herein, the term "host cell" refers to any type of cell that can contain a nucleic acid or vector described herein. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae; or the host cell can be a prokaryotic cell, such as a bacterium or a protozoan.

[0041] The expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transposon (Sleeping Beauty or PiggyBAC), gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cell is cultivated in a culture medium and screened for suitable activity. The method and conditions for cultivating the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the present specification and the methods known in the prior art, according to the specific expression vector used and the host cell change or optimization. In addition, one or more markers of the host cell transfected can be selected by introducing, and the cell that has been stably incorporated into its chromosome by DNA can be selected. The marker can, for example, provide prototrophy, biocidal resistance (for example, antibiotics) or heavy metal (such as copper) resistance to auxotrophic hosts. Selectable marker genes can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

[0042] To solve the above technical problems, the present invention provides a technical solution: a method for preparing a nanobody or a bivalent nanobody targeting a coronavirus N antigen, the method comprising culturing a transformant as described in the present invention, obtaining the nanobody or the bivalent nanobody from the culture, and the culturing method is conventional in the art.

[0043] To solve the above technical problems, the present invention provides a technical solution: a pharmaceutical composition, comprising the nanobody as described in the present invention, and / or the bivalent nanobody as described in the present invention, and a pharmaceutically acceptable carrier.

[0044] In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present invention comprises suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used in the present invention, "pharmaceutically acceptable carriers" or "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents and absorption delay agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of wetting agent or emulsifier, or pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may include standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. The pharmaceutical preparation or pharmaceutical composition comprising the present invention may be prepared by mixing the antibody or antigen-binding fragment thereof of the present invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activity that will not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral active agents, small molecule drugs or immunomodulators, etc. The active ingredient is suitably combined in an amount effective for the purpose of use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies of the present invention or their Fabs, and the matrix is ​​a formed article, such as a film or microcapsule form.

[0045] To solve the above technical problems, the present invention provides a technical solution: a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the nanobody as described in the present invention, and / or the bivalent nanobody as described in the present invention.

[0046] Chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of antigen-specific antibodies with T cell receptor functions. In general, CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of CAR comprises an antigen recognition region.

[0047] To solve the above technical problems, the present invention provides a technical solution: an antibody-drug conjugate, which comprises a cytotoxic agent or a label, and the nanobody as described in the present invention, and / or the bivalent nanobody as described in the present invention.

[0048] To solve the above technical problems, the present invention provides a technical solution: the use of the nanoantibody as described in the present invention, the bivalent nanoantibody as described in the present invention, the pharmaceutical composition as described in the present invention, the chimeric antigen receptor as described in the present invention and / or the antibody-drug conjugate as described in the present invention in the preparation of drugs for diagnosing, preventing and / or treating diseases caused by the new coronavirus SARS-CoV-2 or a variant strain of the new coronavirus.

[0049] In a preferred embodiment of the present invention, the variants of the new coronavirus include the British alpha mutant, the South African beta mutant, the Brazilian gamma mutant, the Indian delta mutant and the South African omicron mutant.

[0050] To solve the above technical problems, the present invention provides a technical solution: a kit, comprising the nanobody as described in the present invention, the bivalent nanobody as described in the present invention, the pharmaceutical composition as described in the present invention, the chimeric antigen receptor as described in the present invention and / or the antibody-drug conjugate as described in the present invention.

[0051] In order to solve the above technical problems, the present invention provides a technical solution: a set of medicine boxes, the set of medicine boxes comprising medicine box A and medicine box B, wherein:

[0052] The drug kit A contains the nanobody as described in the present invention, the bivalent nanobody as described in the present invention, the pharmaceutical composition as described in the present invention, the chimeric antigen receptor as described in the present invention and / or the antibody-drug conjugate as described in the present invention.

[0053] The medicine kit B contains other anti-new coronavirus antibodies or pharmaceutical compositions containing the other anti-new coronavirus antibodies, and / or other anti-new coronavirus drugs.

[0054] To solve the above technical problems, the present invention provides a technical solution: a drug delivery device, which comprises the nanobody as described in the present invention, the bivalent nanobody as described in the present invention, the pharmaceutical composition as described in the present invention, the chimeric antigen receptor as described in the present invention and / or the antibody-drug conjugate as described in the present invention.

[0055] In a preferred embodiment of the present invention, the drug delivery device further comprises a component for administering the Nanobody or the bivalent Nanobody, the chimeric antigen receptor, the antibody drug conjugate or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

[0056] To solve the above technical problems, the present invention provides a technical solution: a method for immunodetection or determination of coronavirus N antigen, the method comprising using a nanobody as described in any one of the present invention, and / or a bivalent nanobody as described in the present invention.

[0057] In a preferred embodiment of the present invention, the method is for non-diagnostic and / or therapeutic purposes, such as detecting the presence of the new coronavirus in the laboratory; or screening other anti-new coronavirus antibodies as a positive antibody; or competing with other anti-new coronavirus antibodies for binding, detecting whether there is competition between antibodies, that is, whether the antigenic epitopes are the same or similar, and other application scenarios.

[0058] To solve the above technical problems, the present invention provides a technical solution: a method for diagnosing, treating and / or preventing diseases or conditions mediated by the new coronavirus SARS-CoV-2 or a variant of the new coronavirus, the method comprising administering a therapeutically effective amount of the nanoantibody as described in the present invention, the bivalent nanoantibody as described in the present invention, the pharmaceutical composition as described in the present invention, the chimeric antigen receptor as described in the present invention and / or the antibody-drug conjugate as described in the present invention to a patient in need, or using the kit as described in the present invention to treat a patient in need.

[0059] As used herein, the term "effective amount" means the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal or human that is sought, for example, by a researcher or clinician. In addition, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention or alleviation of a disease, condition or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that is effective to enhance normal physiological function.

[0060] To solve the above technical problems, the present invention provides a technical solution: a combination therapy, which comprises administering to patients in need thereof a nanobody as described in the present invention, a bivalent nanobody as described in the present invention, a pharmaceutical composition as described in the present invention, a chimeric antigen receptor as described in the present invention and / or an antibody-drug conjugate as described in the present invention, and a second therapeutic agent, respectively.

[0061] In a preferred embodiment of the present invention, the second therapeutic agent comprises other anti-novel coronavirus antibodies or a pharmaceutical composition comprising such other anti-novel coronavirus antibodies, and / or other anti-novel coronavirus drugs.

[0062] To solve the above technical problems, the present invention provides a technical solution: using the nanoantibody as described in the present invention, the bivalent nanoantibody as described in the present invention, the pharmaceutical composition as described in the present invention, the chimeric antigen receptor as described in the present invention and / or the antibody-drug conjugate as described in the present invention for the diagnosis, prevention and / or treatment of related diseases caused by the new coronavirus SARS-CoV-2 or a variant strain of the new coronavirus.

[0063] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0064] The reagents and raw materials used in the present invention are commercially available.

[0065] The positive and progressive effects of the present invention are:

[0066] The present invention utilizes in vitro passive cross-immunization of camelids, combined with phage display technology, to develop a high-affinity nano-antibody for the N antigen of the new coronavirus (SARS-CoV-2). The antibody can specifically bind to the N antigen of the new coronavirus, and does not bind to other respiratory viruses, such as the N antigen of influenza virus, showing the characteristics of high antigen binding specificity. At the same time, the nano-antibody can broadly recognize the N antigens of different new coronavirus variants, and can avoid the phenomenon of "missed detection" in the detection of different new coronavirus variants (Alpha new coronavirus mutant strain (B.1.1.7) in the UK, Beta new coronavirus mutant strain (B.1.351) in South Africa, Gamma new coronavirus mutant strain (P.1) in Brazil, Delta new coronavirus mutant strain (B.1.617.2) in India, and omicron new coronavirus mutant strain (B.1.1.529) in South Africa, etc.).

[0067] The novel coronavirus N nanoantibody of the present invention can be efficiently expressed through prokaryotes, which has lower production costs than traditional antibody eukaryotic mammalian cell expression and is more conducive to promotion and application in such infectious diseases. The novel coronavirus N nanoantibody of the present invention has a picomolar binding ability with SARS-CoV-2N antigen and has strong specificity, which is superior to commercial detection antibodies.

[0068] In summary, the new coronavirus nanoantibody in the present invention is a broad-spectrum new coronavirus N antibody with high specificity, high sensitivity and high affinity. It has extremely high application value in the detection of new coronavirus and has an application theoretical basis both in scientific research and clinical industry applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The purified nanoantibody was detected by SDS-PAGE electrophoresis, wherein lane 1: VHH1, lane 2: VHH2, lane 3: VHH3, lane 4: VHH4, and lane 5: protein ladder.

[0070] Figure 2 For electrophoresis detection of bivalent nanobodies, "reduction" refers to reducing conditions; "non-reduction" refers to non-reducing conditions; the electrophoresis conditions are 140V, 50min.

[0071] Figure 3 It is a specific detection of the binding of the new coronavirus N antigen.

[0072] Figure 4 It is a broad-spectrum detection of the new coronavirus N antigen.

[0073] Figure 5 It is a sensitivity test for antibody binding to the new coronavirus N antigen. DETAILED DESCRIPTION

[0074] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0075] Example 1 Multi-antigen cross-immunization

[0076] 200 μg of wild-type SARS-CoV-2N full-length antigen (Synopsis) was mixed with an equal volume of Freund's complete adjuvant (Sigma) and injected subcutaneously at multiple points near the forelimbs of the alpaca's neck for the first immunization, and then immunized every 2 weeks. The second and third immunizations were repeated with wild-type SARS-CoV-2N full-length antigen, and the immune adjuvant was changed from the first Freund's complete adjuvant (Sigma) to Freund's incomplete adjuvant (Sigma); the fourth immunization was performed with the two domains of the N antigen, NTD and CTD, and the fifth immunization was performed with different mutant strains of N antigen (Synopsis). Seven days after the end of immunization, 20-30 ml of venous peripheral blood was collected from the alpaca, and plasma and PBMC samples were separated for subsequent immune library construction.

[0077] Example 2 Plasma titer detection

[0078] 100 ng of N antigen was coated on ELISA plate at 4°C overnight. Washed with PBST (0.05%) 3 times. 200 μl of 2% BSA was added to each well at room temperature for 2 h. Washed with PBST 3 times. The plasma before and after immunization in Example 1 was diluted 10 3 , 10 4 , 10 5 , 10 6 and 10 7 The cells were washed 5 times with PBST, and then added to the corresponding ELISA plate for 1 hour at room temperature. The cells were washed 5 times with PBST, and 100 μl of 2500-fold diluted anti-alpaca H&L IgG HRP (Apac) was added to each well and placed in the dark at room temperature for 1 hour. The cells were washed 5 times with PBST, and 100 μl of TMB colorimetric solution (abcam) was added for 10 minutes. An equal volume of TMB stop buffer (abcam) was added to terminate the colorimetric OD450 reading.

[0079] The results of ELISA test are shown in Table 1. The plasma titer after immunization increased significantly. 6 The OD450 of antibody detection after immunization was 3.38 times that before immunization, and the titer of new crown N antigen plasma reached 10 6 , indicating that after immunization, the alpaca has acquired rich new coronavirus N antigen antibodies.

[0080] Table 1 Alpaca immune plasma potency determination

[0081]

[0082] Example 3 Obtaining Nanobodies

[0083] Blood samples of immunized alpacas were collected and lymphocytes were isolated from the alpacas peripheral blood using lymphocyte separation solution (GE, 17-1440-02) and density gradient centrifugation. RNA was extracted from lymphocytes according to the instructions of Trizol reagent (INVITROGEN, 15596-018). Then 20 μg of RNA was taken and purified according to SuperScript TMIII First-Strand Synthesis SuperMix (Invitrogen) kit and process to synthesize cDNA. Then, the reverse transcription product cDNA was used as a template, and the library was constructed according to the phage library construction and screening process of Vincke C et al. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs (doi: 10.1007 / 978-1-61779-974-7_8. PMID: 22907350, in which the phage display and recombinant expression vector backbone was pMECS, the host cell used for phage library display was the amber stop codon suppressed strain E. coli TG1, and the host cell used for antibody expression and purification was the non-amber stop codon suppressed strain E. coli WK6) to obtain a library capacity of about 10 8 , a clone library with an abundance greater than 94%. The phage library was enriched with the novel coronavirus antigen N for 2-3 rounds of panning, and clones were selected from the affinity-enriched library for antigen affinity clone ELISA testing.

[0084] 100 ng of N antigen was coated on the ELISA plate as the experimental group, and an equal number of wells were left without antigen as the negative control. Incubate at 4°C overnight; randomly pick single clones from the plate screened in the third round of the phage library in 1 ml of culture medium, culture at 37°C to the logarithmic phase, and add 1 mM IPTG for induction overnight; the next day, collect the bacterial pellet by centrifugation, break it, centrifuge it at 5,000g for 15 minutes, and collect the supernatant; at the same time, add 2% BSA to the ELISA plate and block it at room temperature for 1 hour; add the supernatant of single clone breakage to each well of the experimental group and negative control group, incubate at room temperature for 2 hours; wash 10 times with PBST, add anti HA-HRP label antibody (abcam), and incubate at room temperature for 1 hour; wash 5 times with PBST, add substrate colorimetric agent, react for 10-20 minutes, add terminator, and read the absorbance value on the microplate reader; when the absorbance value is greater than 2.1 compared with the control well, it is determined to be a positive clone. In this example, a total of 4 nanobodies (VHH1-VHH4) with specificity and affinity for N antigen were screened, and the amino acid sequences of their CDR regions are shown in Table 2 (determined according to the IMGT definition rules), and the full-length amino acid sequences are shown in Table 3.

[0085] Table 2 Amino acid sequences of the CDR region of SARS-CoV-2N antigen nanoantibodies

[0086]

[0087]

[0088] In the VHH2 & VHH4 consensus sequences in Table 2, X 1 A or S, X 2 T or N, X 3 T or S, X 4 I or T, X 5 V or I, X 6 A or T, X 7 M or Q.

[0089] Table 3 Full-length amino acid sequence of SARS-CoV-2N antigen nanoantibody

[0090]

[0091] Example 4 Induced expression and purification of nanobodies

[0092] (1) Inducible expression of nanobodies.

[0093] The four nanobody monoclonal strains in Example 3 were selected and inoculated into 10 ml of culture medium containing ampicillin, and cultured overnight at 37°C, 220 rpm. The next day, 2 ml of overnight culture was inoculated into 200 ml of culture medium containing ampicillin, and cultured at 37°C, 220 rpm until the logarithmic phase (OD 600 The pH value was 0.6-0.8), and IPTG was added to induce the expression of nanobodies overnight. The next day, the bacterial precipitate was collected, and the bacterial cells were broken by hypotonic method, and the supernatant was collected by high-speed centrifugation for subsequent protein purification.

[0094] (2) Purification of nanobodies.

[0095] Purified nanoantibodies were obtained using affinity purification His-nickel filler (referred to as Ni filler, BioRad). The Ni filler was loaded into the column, first washed with ultrapure water, and then washed with equilibration buffer PBS; the above-mentioned supernatant was added to the purification column at a flow rate of 1 ml / min; impurities were washed with an appropriate volume of PBS until OD280 was below 0.0001; and the target protein was eluted with 10 times the volume of eluent (150 mM imidazole). The target protein was purified, and an equal volume was taken and 12% SDS-PAGE was used to detect the expression and purification of the nanoantibody ( Figure 1 ).from Figure 1 It can be seen that the size of the nanobody band is around 15KD, the purity is >95%, and all four nanobodies are successfully and correctly expressed.

[0096] Example 5 SPR analysis of the binding affinity of nanoantibodies to the new coronavirus antigen N

[0097] Take the CM5 chip, couple an appropriate amount of SARS-CoV-2N antigen, and then dilute the nanobody protein solution in Example 4 with HBS-P buffer to a suitable concentration gradient. In this example, the starting concentration is about 0.6μg / ml (40nM) or 0.3μg / ml (20nM), and then the gradient is diluted by 2 times in sequence to dilute 6 concentration gradients. Each injection is 120s, dissociation is 180s, the flow rate is 30 / min, and then regenerated with 10mM PH2.0 Gly-HCL, and the cycle is repeated until all concentration gradient injections are completed. After the program is completed, the Biacore T200 (GE) instrument's built-in analysis program is used for fitting analysis to obtain the nanobody affinity constant results (Table 4). The results show that in this example, the affinity binding relay constants (referred to as KD values) of the four nanoantibodies with the new crown N antigen are all within 10 -10 M level and antibody affinity were both high.

[0098] Table 4 Binding affinity constants of novel coronavirus N antigen nanoantibodies

[0099]

[0100] Example 6 Identification of binding epitopes of nanobodies using the SPR method

[0101] Take the CM5 chip coupled with the new crown N antigen in Example 5, add about 3μg / ml VHH4 antibody diluted with HBS-P buffer, inject and bind for 180s, flow rate 10μl / min, so that VHH4 is nearly saturated with the N antigen on the chip, occupying the epitope of the VHH4 antibody itself binding to the N antigen; then add VHH1, VHH2, VHH3 nanoantibodies in turn, inject for 90s, flow rate 30μl / min, dissociate for 60s, flow rate 30μl / min. Finally, regenerate with 10mM PH2.0 Gly-HCL, and repeat the cycle until three samples are injected. After the program was run, the analysis program provided by the Biacore T200 (GE) instrument was used to analyze the readings. The results in Table 5 show that the binding values ​​of VHH1, VHH2 and VHH3 to the N antigen on the chip were between 148.9-187.5RU. When the N antigen on the chip was nearly saturated with VHH4, the N antigen was occupied by the binding epitope of VHH4, so that VHH1, VHH2 and VHH3 could only bind to 27.1-40RU, which was close to the residual binding value of VHH4 of 36.3. Through calculation and analysis, the epitopes of VHH1, VHH2 and VHH3 were close to those of VHH4, reaching 103.3%, 91.5% and 91.0% respectively. The four antibodies bound to the same epitope region of the N antigen. The commercially available positive antibody AB0046-1 (Biacore Biotechnology) and VHH4 have different binding epitopes, with only 12.5% ​​similarity. The four nanobodies in the present invention are another type of N antigen binding epitope nanobodies that are different from the epitopes of commercially available positive antibodies.

[0102] Binding rate = binding value with N antigen complex bound to VHH4 / binding value with chip N antigen; VHH4 epitope similarity = 1-(binding rate of the antibody to be tested-binding rate of VHH4 antibody)

[0103] Table 5 Nanobody binding epitope detection

[0104]

[0105] Note: AB0046-1 is a commercially available positive antibody (Baikang Biotechnology)

[0106] Example 7 Monovalent Nanobody Bivalent Transformation, Eukaryotic Expression and Purification

[0107] The affinities of the four nanoantibodies in the above examples are all at the sub-nanomolar level, with high antigen-binding affinity, and different binding epitopes from commercially available antibodies. Further structural modifications can be performed to enhance the ability of nanoantibodies to bind to the new coronavirus N antigen.

[0108] First, the above four nanobodies were fused with rabbit IgG Fc amino acid sequence (SEQ ID NO: 20) for gene synthesis (amino acid sequence see VHH1-FC to VHH4-FC, SEQ ID NO: 21 to SEQ ID NO: 24), and the synthesized gene sequence was cloned by seamless PCR technology (for specific operation, refer to Hieff plus One Step Cloning Kit Instructions) was subcloned into the customized expression vector pCMV (Sino-Qiao). The recombinantly constructed single-domain antibody VHH-FC fusion protein plasmid was transfected into HEK293F cells for expression. The recombinant expression plasmid was diluted with PBS, and the PEI (polyethylenimine) solution required for transformation was added. After mixing, it was added to the HEK293F cell suspension and placed in an incubator at 37°C and 8% CO 2 , relative humidity ≥ 80%, 150rpm. After 5-6 days of culture, the transient expression culture supernatant was collected. The supernatant was used for subsequent protein purification, and was purified by protein A affinity chromatography to obtain 4 bivalent nanobodies, which were detected by reducing and non-reducing electrophoresis ( Figure 2 The results showed that the size of the purified bivalent nanobody band was about 76KD (under non-reducing conditions), and the size of the single chain under reducing conditions was about 38KD, which was consistent with the theoretical size, with a purity of more than 90%, and the transient expression yield was at the level of 500mg / L, which was extremely high.

[0109] The amino acid sequence of rabbit IgG Fc is (SEQ ID NO: 20):

[0110] APSTCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIE KTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0111] The amino acid sequences of VHH1-FC to VHH4-FC are (the underlined region is VHH):

[0112] VHH1-FC (SEQ ID NO: 21):

[0113] QVQLQESGGGLVQPGGSLRLSCAASGFTADYYTIGWFRQAPGKGREGVSCISGSGAGGETVYADSVKGR FTISRDSAKNTVYLQMSSLKPDDTAVYTCAASFRPNYMCYPGGGPDDYGYWGQGTQVTVSSAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0114] VHH2-FC(SEQ ID NO:22):

[0115] QVQLQESGGGLVQSGGSLRLSCAASGFRLGFYAIGWFRQAPGKEREGVACISNTGGTTNHADSVKGRFT ISTDNAKNTVYLQMNSLKPEDTAIYYCAADFKPSISVAQPTCVMTGYDHWGQGTQVTVSS APSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0116] VHH3-FC(SEQ ID NO:23):

[0117] QVQLQESGGGLVQPGGSLILSCAASGRTLDYYIIGWFRQAPGKEREGVACITGGGGSTEYADSVKGRFT ISRDNAKNTVYLQMNNLKPEDTAIYSCAVDFRPTGQPMCVVTSTVGYAYWGQGTQVTVSS APSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0118] VHH4-FC(SEQ ID NO:24):

[0119] QVQLQESGGGLVQTGGSLRLSCAASGFRLGFYSIGWFRQAPGKEREGVSCISNNGGSTNYADSVKGRFT ISSDNAKNTVYLQMNSLKPEDTAVYYCAADFKPSTSITQPTCVQTGYDHWGQGTQVTVSSAPSTCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIE KTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0120] Example 8 SPR method to determine the affinity constant of bivalent nanoantibodies with the new crown N antigen

[0121] Take the CM5 chip, couple it with the SARS-CoV-2N antigen, and then dilute the bivalent antibody in Example 7 with HBS-P buffer into a suitable concentration gradient. In this case, the starting concentration is about 0.64μg / ml (8nM) or 0.32μg / ml (4nM), and the gradient is diluted by 2 times in sequence to dilute 6 concentration gradients. Each injection is 120s, dissociation is 180s, the flow rate is 30 / min, and then regenerated with 10mM PH2.0 Gly-HCL, and the cycle is repeated until all concentration gradient injections are completed. After the program is completed, the Biacore T200 (GE) instrument's built-in analysis program is used for fitting analysis to obtain the nanobody affinity constant results (Table 6). The results show that in this example, after the 4 nanoantibodies were transformed into fusion IgG-FC bivalent nanoantibodies, the affinity of the antibody to the new crown N antigen was increased to the pM level, and the affinity was 10 -12 and 10 -14 Between, the superiority is higher.

[0122] Table 6 Binding affinity constants of bivalent nanoantibodies to the SARS-CoV-2 N antigen

[0123]

[0124] Example 9 Specificity Detection

[0125] 50ng of the new crown N antigen (Yi Qiao) and other viral N antigens, in this example, different subtypes of influenza A and B N antigens (Yi Qiao) were coated in the ELISA plate, 50μl per well; washed 3 times with PBST, blocked with 2% BSA, and incubated at room temperature for 2 hours. Washed 3 times with PBST, added 50μl of the 4 antibodies in Example 7, repeated for 2 wells, and incubated at room temperature for 1 hour. Washed 5 times with PBST, added 50μl of 5000-fold diluted anti-rabbit IgG FC HRP (Yi Qiao), and protected from light at room temperature for 1 hour. Washed 5 times with PBST, added 50μl of TMB solution to develop color in the dark for 10 minutes, and then added 50μl of TMB stop buffer (abcam) to stop the color development. Finally, the ELISA plate was placed in an ELISA reader (BioTek) for reading. Figure 3 The results showed that the four new coronavirus N antibodies in this example only specifically bind to the new coronavirus N antigen, and do not bind to multiple different subtypes of influenza N antigens, with high binding specificity.

[0126] Example 10 Broad spectrum detection

[0127] 50ng of different mutant COVID-19 N antigen (Synopsis) was coated in the ELISA plate, 50μl per well; washed 3 times with PBST, blocked with 2% BSA, and incubated at room temperature for 2 hours. Washed 3 times with PBST, added 50μl of the 4 VHH-FC antibodies in Example 7, repeated for 2 wells, and incubated at room temperature for 1 hour. Washed 5 times with PBST, added 50μl of 5000-fold diluted anti-rabbit IgG FC HRP (Synopsis), and protected from light at room temperature for 1 hour. Washed 5 times with PBST, added 50μl of TMB solution to develop color in the dark for 10 minutes, and then added 50μl of TMB stopbuffer (abcam) to stop the color development. Finally, the ELISA plate was placed in an ELISA reader (BioTek) for reading. Figure 4 The results showed that the four new coronavirus N antibodies in this example bound to the wild-type, British alpha mutant, South African beta mutant, Brazilian gamma mutant, Indian delta mutant and South African omicron mutant N antigens, and broadly bound to N antigens of various different subtypes of new coronavirus mutant strains, and are suitable for the detection of constantly mutating new coronavirus strains.

[0128] Example 11 Sensitivity Detection

[0129] The four strains of VHH-FC antibodies in Example 7 were coated in ELISA plates, 50 μl per well; washed 3 times with PBST, blocked with 2% BSA, washed at room temperature for 2 hours, and washed 3 times with PBST. The new crown N antigen was diluted 2-fold from 3000 pg / ml, diluted 6 gradients, and the diluted new crown N antigen was added to the ELISA plate wells, 50 μl per well, and each dilution concentration was repeated 3 wells at room temperature for 1 hour. Washed 5 times with PBST, added 50 μl of 3000-fold diluted anti-His HRP (abcam), and kept away from light at room temperature for 1 hour. Washed 5 times with PBST, added 50 μl of TMB solution to develop color for 10 minutes in the dark, and then added 50 μl of TMB stop buffer (abcam) to stop the color development. Finally, the ELISA plate was placed in an ELISA reader (BioTek) for reading.

[0130] Figure 5 The results showed that the four new coronavirus N antibodies in this example were linearly bound to the new coronavirus N antigen between 93.75-3000pg / ml, and the new coronavirus N antigen could be detected to the pg level, with extremely high detection sensitivity.

Claims

1. A nanobody targeting a coronavirus N antigen, comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, characterized in that: The amino acid sequence of HCDR3 of the Nanobody is as shown in SEQ ID NO: 3, preferably as shown in SEQ ID NO: 9 or SEQ ID NO: 15; The amino acid sequence of HCDR3 of the Nanobody is shown in SEQ ID NO: 6; or, The amino acid sequence of HCDR3 of the Nanobody is shown in SEQ ID NO:

12.

2. The Nanobody according to claim 1, characterized in that The Nanobody is a Nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO: 1, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 2, and an amino acid sequence of HCDR3 as shown in SEQ ID NO: 3; the Nanobody is preferably a Nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO: 7, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 8, and an amino acid sequence of HCDR3 as shown in SEQ ID NO: 9, or the Nanobody is preferably a Nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO: 13, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 14, and an amino acid sequence of HCDR3 as shown in SEQ ID NO: 15; The Nanobody is a Nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO:4, an amino acid sequence of HCDR2 as shown in SEQ ID NO:5 and an amino acid sequence of HCDR3 as shown in SEQ ID NO:6; or, The nanobody is a nanobody having an amino acid sequence of HCDR1 as shown in SEQ ID NO: 10, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 11 and an amino acid sequence of HCDR3 as shown in SEQ ID NO:

12.

3. The Nanobody according to claim 1 or 2, characterized in that The Nanobody is a Nanobody whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16; The Nanobody is a Nanobody whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; The Nanobody is a Nanobody whose amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 18; or, The nanobody is a nanobody whose amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

19.

4. A bivalent nanobody targeting coronavirus N antigen, characterized in that: The bivalent Nanobody comprises the Nanobody according to any one of claims 1 to 3, preferably the bivalent Nanobody further comprises a heavy chain constant region; Preferably, the heavy chain constant region is rabbit IgG Fc; More preferably, the amino acid sequence of the rabbit IgG Fc is as shown in SEQ ID NO:

20.

5. The bivalent Nanobody according to claim 4, characterized in that The amino acid sequence of the bivalent nanobody is shown in SEQ ID NO: 21; The amino acid sequence of the bivalent nanobody is shown in SEQ ID NO: 22; The amino acid sequence of the bivalent Nanobody is shown in SEQ ID NO: 23; or, The amino acid sequence of the bivalent nanobody is shown in SEQ ID NO:

24.

6. An isolated nucleic acid, characterized in that The nucleic acid encodes the Nanobody according to any one of claims 1 to 3 or the bivalent Nanobody according to claim 4 or 5.

7. A recombinant expression vector, characterized in that: It comprises the isolated nucleic acid according to claim 6; Preferably, the backbone of the recombinant expression vector is a plasmid, a phage or a viral vector, for example, the backbone of the recombinant expression vector is pMECS or pCMV.

8. A transformant, characterized in that It comprises the isolated nucleic acid according to claim 6 or the recombinant expression vector according to claim 7; Preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; More preferably, the prokaryotic cell is Escherichia coli, or the eukaryotic cell is a mammalian cell; Further preferably, the Escherichia coli is E. coli TG1 or E. coli WK6, or the mammalian cell is HEK293F cell or CHO cell.

9. A method for preparing a nanobody or a bivalent nanobody targeting a coronavirus N antigen, characterized in that: The method comprises culturing the transformant according to claim 8, and obtaining the Nanobody or the bivalent Nanobody from the culture.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody according to any one of claims 1 to 3, and / or the bivalent Nanobody according to claim 4 or 5, and a pharmaceutically acceptable carrier.

11. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the Nanobody according to any one of claims 1 to 3, and / or the bivalent Nanobody according to claim 4 or 5.

12. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate comprises a cytotoxic agent or a label, and the Nanobody according to any one of claims 1 to 3, and / or the bivalent Nanobody according to claim 4 or 5.

13. Use of the Nanobody according to any one of claims 1 to 3, the bivalent Nanobody according to claim 4 or 5, the pharmaceutical composition according to claim 10, the chimeric antigen receptor according to claim 11 and / or the antibody-drug conjugate according to claim 12 in the preparation of a drug for diagnosing, preventing and / or treating a disease caused by the new coronavirus SARS-CoV-2 or a variant of the new coronavirus; Preferably, the variants of the new coronavirus include the British alpha mutant, the South African beta mutant, the Brazilian gamma mutant, the Indian delta mutant and the South African omicron mutant.

14. A kit, characterized in that: The kit comprises the Nanobody according to any one of claims 1 to 3, the bivalent Nanobody according to claim 4 or 5, the pharmaceutical composition according to claim 10, the chimeric antigen receptor according to claim 11 and / or the antibody-drug conjugate according to claim 12.

15. A medicine kit, characterized in that: The kit comprises kit A and kit B, wherein: The kit A contains the Nanobody according to any one of claims 1 to 3, the bivalent Nanobody according to claim 4 or 5, the pharmaceutical composition according to claim 10, the chimeric antigen receptor according to claim 11 and / or the antibody-drug conjugate according to claim 12; The medicine kit B contains other anti-new coronavirus antibodies or pharmaceutical compositions containing the other anti-new coronavirus antibodies, and / or other anti-new coronavirus drugs.

16. A drug delivery device, characterized in that: The drug delivery device comprises the Nanobody according to any one of claims 1 to 3, the bivalent Nanobody according to claim 4 or 5, the pharmaceutical composition according to claim 10, the chimeric antigen receptor according to claim 11 and / or the antibody drug conjugate according to claim 12; Preferably, the drug delivery device further comprises a component for administering the Nanobody or the bivalent Nanobody, the chimeric antigen receptor, the antibody drug conjugate or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

17. A method for immunodetection or determination of coronavirus N antigen, characterized in that: The method comprises using the Nanobody as claimed in any one of claims 1 to 3, and / or the bivalent Nanobody as claimed in claim 4 or 5; Preferably, the method is for non-diagnostic and / or therapeutic purposes.

Images