Bispecific nanobodies against sars-cov-2 and uses thereof

Abstract

The application provides a bispecific nanobody against SARS-CoV-2 and related nucleic acids, proteins, cell products and applications thereof. The bispecific nanobody is constructed by concatenating two specific nanobodies targeting different epitopes of the novel coronavirus in a specific manner, and exhibits excellent neutralization activity against each type of variant strain of the novel coronavirus, has the significant advantages of broad-spectrum and high-efficiency anti-novel coronavirus infection, and provides an effective treatment strategy for preventing or treating infection of the novel coronavirus and its variant strains in the clinic. In addition, the bispecific nanobody can also achieve sensitive and reliable detection of the novel coronavirus.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to bispecific nanobodies targeting SARS-CoV-2 and their applications. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) belongs to the genus β-coronavirus in the family Coronaviridae, and is a single-stranded, positive-sense RNA virus. One of the most notable characteristics of SARS-CoV-2 is its high mutation rate, particularly frequent mutations in the spike protein region. These mutations have led to the emergence of a series of variants, including Alpha, Delta, Omicron, and their subclades. Each variant exhibits varying degrees of difference in transmissibility, immune evasion characteristics, and resistance to existing treatments and preventative measures.

[0003] The diversity of viral variants poses a significant challenge to the efficacy of antibody drugs. While initially developed monoclonal antibodies exhibited good neutralizing ability against early strains, their potency has significantly decreased with the emergence of new variants. For example, many approved antibodies have almost completely lost their neutralizing ability against the spike protein of the Omeprone variant. This situation not only limits the widespread application of existing antibody drugs but also prompts researchers to continuously develop antibodies against new variants to address the rapid evolution of viruses.

[0004] Furthermore, the immune evasion capabilities of variant strains introduce uncertainty into the protective efficacy of vaccination. Although current mRNA vaccines (such as BNT162b2 and mRNA-1273) can provide some protection against variant strains with booster shots, their protective efficacy remains limited for immunocompromised individuals. Studies have shown that even after completing the full vaccination course, these individuals remain susceptible to infection with variant strains and the development of severe illness.

[0005] Purpose of the invention

[0006] The present invention aims to provide a bispecific nanobody against SARS-CoV-2, a fusion protein and an immunoconjugate thereof comprising the nanobody, a polynucleotide encoding the nanobody, a nucleic acid construct comprising the polynucleotide, an expression vector and a host cell, and a pharmaceutical composition comprising any one of the above products, and their use in the preparation of medicaments for the prevention and / or treatment of novel coronavirus, and in the preparation of reagents or kits for the detection of novel coronavirus or the diagnosis of novel coronavirus infection.

[0007] Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution.

[0009] In a first aspect, the present invention provides a bispecific nanobody against SARS-CoV-2, characterized in that the bispecific nanobody is a tandem assembly of VHH chains selected from two nanobody types, either directly tandemly connected from the N-terminus to the C-terminus or tandemly connected via linkers:

[0010] (1) Nanobody N235 and nanobody R218;

[0011] The nanobody N235 is a nanobody targeting a conserved epitope in the NTD region of the SARS-CoV-2 spike protein, and its VHH chain includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:3.

[0012] The nanobody R218 is a nanobody targeting a conserved epitope in the RBD region of the SARS-CoV-2 spike protein. Its VHH chain includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:4, CDR2 with an amino acid sequence as shown in SEQ ID NO:5, and CDR3 with an amino acid sequence as shown in SEQ ID NO:6.

[0013] (2) Nanobody N103 and nanobody S102;

[0014] The nanobody N103 is a nanobody targeting a conserved epitope in the NTD region of the SARS-CoV-2 spike protein. Its VHH chain includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:7, CDR2 with an amino acid sequence as shown in SEQ ID NO:8, and CDR3 with an amino acid sequence as shown in SEQ ID NO:9.

[0015] The nanobody S102 is a nanobody targeting a conserved epitope in the S2 region of the SARS-CoV-2 spike protein. Its VHH chain includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:10, CDR2 with an amino acid sequence as shown in SEQ ID NO:11, and CDR3 with an amino acid sequence as shown in SEQ ID NO:12.

[0016] (3) Nanobody N235 and nanobody S102;

[0017] The nanobody N235 is a nanobody targeting a conserved epitope in the NTD region of the SARS-CoV-2 spike protein, and its VHH chain includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:3.

[0018] The nanobody S102 is a nanobody targeting a conserved epitope in the S2 region of the SARS-CoV-2 spike protein, and its VHH chain includes the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO:10, CDR2 with the amino acid sequence shown in SEQ ID NO:11, and CDR3 with the amino acid sequence shown in SEQ ID NO:12; or,

[0019] (4) Nanobody R218 and nanobody S102;

[0020] The nanobody R218 is a nanobody targeting a conserved epitope in the RBD region of the SARS-CoV-2 spike protein. Its VHH chain includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:4, CDR2 with an amino acid sequence as shown in SEQ ID NO:5, and CDR3 with an amino acid sequence as shown in SEQ ID NO:6.

[0021] The nanobody S102 is a nanobody targeting a conserved epitope in the S2 region of the SARS-CoV-2 spike protein. Its VHH chain includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:10, CDR2 with an amino acid sequence as shown in SEQ ID NO:11, and CDR3 with an amino acid sequence as shown in SEQ ID NO:12.

[0022] In feasible embodiments of the above-described bispecific nanobodies, the VHH chain of nanobody N235 has an amino acid sequence as shown in SEQ ID NO:13, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:13; and / or, the VHH chain of nanobody R218 has an amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:14; and / or, the VHH chain of nanobody N103 has an amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15; and / or, the VHH chain of nanobody S102 has an amino acid sequence as shown in SEQ ID NO:16, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15; The amino acid sequence shown in NO:16 has an amino acid sequence identity of at least 95%, 96%, 97%, 98%, or 99%.

[0023] Preferably, the amino acid sequence of the VHH chain of the nanobody N235 is shown in SEQ ID NO:13, the amino acid sequence of the VHH chain of the nanobody R218 is shown in SEQ ID NO:14, the amino acid sequence of the VHH chain of the nanobody N103 is shown in SEQ ID NO:15, and the amino acid sequence of the VHH chain of the nanobody S102 is shown in SEQ ID NO:16.

[0024] Preferably, the connector is (GGGGS)n, where n is an integer between 1 and 6, for example, n is 1, 2, 3, 4, 5 or 6.

[0025] Most preferably, the bispecific antibody comprises an amino acid sequence selected from the following: SEQ ID NO:19-22.

[0026] In a second aspect, the present invention provides a fusion protein comprising: a bispecific nanobody as described in the first aspect above, and optionally a signal peptide sequence and / or a tag sequence to assist in expression and / or purification.

[0027] In a feasible implementation, the signal peptide sequence is as shown in SEQ ID NO:17 or 18.

[0028] In a feasible implementation, the tag sequence is a histidine tag sequence, such as a hexahistidine tag.

[0029] Thirdly, the present invention provides a polynucleotide that encodes a bispecific nanobody as described in the first aspect above, or a somatic fusion protein as described in the second aspect above.

[0030] In a specific implementation, the polynucleotide is DNA or mRNA.

[0031] Preferably, the polynucleotide encodes a bispecific nanobody as described in any one of SEQ ID NO:19-22, and more preferably, the polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO:23-26.

[0032] Fourthly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the third aspect above, and at least one expression regulatory element, such as a histidine tag, a stop codon, etc., operably linked to the polynucleotide.

[0033] Fifthly, the present invention provides an expression vector comprising a polynucleotide as described in the third aspect above or a nucleic acid construct as described in the fourth aspect above.

[0034] In a sixth aspect, the present invention provides a host cell wherein it is transformed or transfected with the polynucleotides as described in the third aspect above, the nucleic acid constructs as described in the fourth aspect above, or the expression vectors as described in the fifth aspect above.

[0035] In a seventh aspect, the present invention provides an immunoconjugate comprising:

[0036] (1) The dual-specific nanoantibody as described in the first aspect above, or the fusion protein as described in the second aspect above; and

[0037] (2) Selected from the following conjugates: cytotoxins, small molecule drugs, radionuclides, photosensitizers, immunomodulatory factors, enzymes, nucleic acids, fluorescent markers, magnetic nanoparticles, liposomes, polymer nanoparticles or other biologically active molecules.

[0038] Eighthly, the present invention provides a pharmaceutical composition comprising a bispecific nanobody as described in the first aspect above, a fusion protein as described in the second aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, a host cell as described in the sixth aspect above, or an immunoconjugate as described in the seventh aspect above, and a pharmaceutically acceptable carrier and / or excipient.

[0039] In specific implementations, the pharmaceutical composition may be in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0040] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0041] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0042] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

[0043] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0044] In a ninth aspect, the present invention provides the use of the bispecific nanobody as described in the first aspect, the fusion protein as described in the second aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, the host cell as described in the sixth aspect, the immunoconjugate as described in the seventh aspect, or the pharmaceutical composition as described in the eighth aspect in the preparation of a medicament for the prevention and / or treatment of novel coronavirus infection.

[0045] In a specific implementation plan, the novel coronavirus may be the original SARS-CoV-2 strain and / or its variants.

[0046] In a tenth aspect, the present invention provides the use of the bispecific nanobody as described in the first aspect, the fusion protein as described in the second aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the expression vector as described in the fifth aspect, the host cell as described in the sixth aspect, the immunoconjugate as described in the seventh aspect, or the pharmaceutical composition as described in the eighth aspect in the preparation of reagents or kits for detecting the novel coronavirus or for diagnosing novel coronavirus infection.

[0047] In a specific implementation plan, the novel coronavirus may be the original SARS-CoV-2 strain and / or its variants.

[0048] In one aspect, the present invention provides a novel coronavirus detection kit, comprising a bispecific nanobody as described in the first aspect, a fusion protein as described in the second aspect, a polynucleotide as described in the third aspect, a nucleic acid construct as described in the fourth aspect, an expression vector as described in the fifth aspect, a host cell as described in the sixth aspect, an immunoconjugate as described in the seventh aspect, or a pharmaceutical composition as described in the eighth aspect.

[0049] In a twelfth aspect, the present invention provides a method for preventing and / or treating novel coronavirus infection, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of a bispecific nanobody as described in the first aspect above, a fusion protein as described in the second aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, a host cell as described in the sixth aspect above, an immunoconjugate as described in the seventh aspect above, or a pharmaceutical composition as described in the eighth aspect above.

[0050] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, the method of administration, the patient's age and weight, and the patient's symptoms.

[0051] In a thirteenth aspect, the present invention provides a method for detecting or diagnosing novel coronavirus infection, the method comprising using a bispecific nanobody as described in the first aspect above, a fusion protein as described in the second aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, a host cell as described in the sixth aspect above, an immunoconjugate as described in the seventh aspect above, or a pharmaceutical composition as described in the eighth aspect above.

[0052] Beneficial effects

[0053] This invention creatively constructs a bispecific nanobody against SARS-CoV-2 by tandemly constructing two specific nanobodies targeting different antigenic epitopes of the SARS-CoV-2 virus in a carefully designed manner. The bispecific nanobody exhibits excellent neutralizing activity against various variants of the SARS-CoV-2 virus, demonstrating significant advantages in broad-spectrum and highly effective anti-SARS-CoV-2 infection.

[0054] Notably, compared to using either of the two nanobodies constituting this bispecific nanobody alone, or a simple mixture of the two nanobodies, the virus-neutralizing activity of this bispecific nanobody is significantly enhanced. In particular, when dealing with some currently circulating strains, the neutralizing activity is increased by hundreds of times, highlighting the significant synergistic effect between the two nanobodies. This characteristic makes this bispecific nanobody exhibit excellent broad-spectrum activity and high activity in the prevention and treatment of COVID-19 infection. Furthermore, this bispecific nanobody can also achieve sensitive and reliable detection of the novel coronavirus. Attached Figure Description

[0055] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0056] Figure 1 This is a schematic diagram of the structure of the bispecific nanobody of the present invention.

[0057] Figure 2 This is the molecular sieve chromatography elution curve of the bispecific nanobody of the present invention.

[0058] Figure 3 This is an SDS-PAGE identification result of the bispecific nanobody of the present invention.

[0059] Figure 4 This is a graph showing the experimental results of the neutralizing activity of the bispecific nanobody of the present invention against XBB pseudovirus; where the horizontal axis represents antibody concentration (log(μg / mL)) and the vertical axis represents virus inhibition rate (%).

[0060] Figure 5 This is a graph showing the experimental results of the neutralizing activity of the bispecific nanobody of the present invention against the JN.1 pseudovirus; where the horizontal axis represents the antibody concentration (log(μg / mL)) and the vertical axis represents the virus inhibition rate (%).

[0061] Figure 6 This is a graph showing the experimental results of the neutralizing activity of the bispecific nanobody of the present invention against KP.2 pseudovirus; where the horizontal axis represents antibody concentration (log(μg / mL)) and the vertical axis represents virus inhibition rate (%). Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0064] The term "nanobody" used in this article refers to "heavy chain single-domain antibody," which contains only one variable domain of heavy chain (VHH) and naturally lacks the light chain compared to other antibodies.

[0065] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0066] Example 1: Construction, expression and purification of bispecific nanobodies

[0067] To construct a bispecific nanobody that simultaneously recognizes different conserved epitopes of the novel coronavirus spike protein, this embodiment employs a homologous recombination strategy, connecting the VHH domains of two nanobodies in tandem with a flexible linker peptide.

[0068] Specifically, this embodiment designs the following four types of bispecific nanobodies of the present invention, the structural schematic diagrams of which are shown below. Figure 1 :

[0069] Bispecific nanobody I (abbreviated as N235-R218), wherein VHH1 is the VHH chain of nanobody N235 (its amino acid sequence is shown in SEQ ID NO:13), VHH2 is the VHH chain of nanobody R218 (its amino acid sequence is shown in SEQ ID NO:14), and the linker is (GGGGS)4; the complete amino acid sequence of this bispecific nanobody is shown in SEQ ID NO:19;

[0070] Bispecific nanobody II (abbreviated as N103-S102), wherein VHH1 is the VHH chain of nanobody N103 (its amino acid sequence is shown in SEQ ID NO:15), VHH2 is the VHH chain of nanobody S102 (its amino acid sequence is shown in SEQ ID NO:16), and the linker is (GGGGS)4; the complete amino acid sequence of this bispecific nanobody is shown in SEQ ID NO:20;

[0071] Bispecific nanobody III (abbreviated as N235-S102), wherein VHH1 is the VHH chain of nanobody N235 (its amino acid sequence is shown in SEQ ID NO:13), VHH2 is the VHH chain of nanobody S102 (its amino acid sequence is shown in SEQ ID NO:16), and the linker is (GGGGS)4; the complete amino acid sequence of this bispecific nanobody is shown in SEQ ID NO:21;

[0072] Bispecific nanobody IV (abbreviated as R218-S102), wherein VHH1 is the VHH chain of nanobody R218 (its amino acid sequence is shown in SEQ ID NO:14), VHH2 is the VHH chain of nanobody S102 (its amino acid sequence is shown in SEQ ID NO:16), and the linker is (GGGGS)4; the complete amino acid sequence of this bispecific nanobody is shown in SEQ ID NO:22.

[0073] All nanobodies used in the above-mentioned bispecific nanobodies were previously constructed and patented by the applicant. Among them, nanobodies N235 and N103 are described in patent application CN202210935778.6, nanobodies R218 are described in patent application CN202111363957.9, and nanobodies S102 are described in patent application CN 202211433689.8.

[0074] The nucleic acid coding sequences of the above-mentioned bispecific nanoantibodies were codon-optimized to obtain codon-optimized DNA coding sequences (as shown in SEQ ID NO:23, 24, 25, and 26, respectively). A signal peptide coding sequence (the signal peptide sequence used for bispecific nanoantibodies I, II, and III is shown in SEQ ID NO:17, and the signal peptide sequence used for bispecific nanoantibodies IV is shown in SEQ ID NO:18) was attached to the 5' end, along with a coding sequence for a 6-histidine tag and a translation stop codon TGA at the 3' end. The sequences were then constructed into the pCAGGS vector (purchased from Invitrogen) using the restriction endonuclease sites EcoRI and XhoI to obtain the recombinant expression plasmids for each bispecific nanoantibody.

[0075] For expression, transient transfection was performed using the 293F cell line (purchased from Invitrogen) in suspension culture. Each construct was constructed independently into an expression system. Specifically, the recombinant expression plasmid and polyethyleneimine (PEI, linear, MW 25,000) were separately dissolved in 150 mM NaCl solution, mixed at a 1:3 mass ratio, and incubated for 15 minutes to form a complex. This complex was then added to a solution with a density of 2.0 × 10⁻⁶ cells / mL. 6Cells were cultured in a 293F cell culture system at 37°C, 8% CO2, and 120 rpm for 5 days without changing the culture medium. Cell status and suspension homogeneity were observed periodically.

[0076] After culture, the cell supernatant was collected, centrifuged at 8000 rpm for 30 minutes to remove cell debris, and then filtered through a 0.22 μm PES (Millipore) membrane to obtain a clear culture supernatant for protein purification. For purification, one-step affinity purification was performed using Prism A affinity chromatography media (GE Healthcare). The specific method is as follows: an overnight-loaded affinity column was added to an AKTApure25 protein purification system at a flow rate of 1 mL / min. Non-specific impurities were washed away by equilibrating with 5 column volumes (CV) of Prism A buffer A (pH 7.4). The target protein was then eluted with buffer B (pH 3.0), and immediately neutralized with 1M Tris-HCl (pH 9).

[0077] To further improve protein purity and remove any possible aggregates or degradation products, the eluted target protein was concentrated to 1–2 mL and then loaded onto a Superdex 75 Increase 10 / 300GL molecular sieve column (GE Healthcare) for gel filtration chromatography (PBS as mobile phase, flow rate 0.5 mL / min). The molecular sieve chromatography elution curves for each bispecific nanobody are shown below. Figure 2 .Depend on Figure 2 As can be seen, the target protein with a purity greater than 90% can be obtained through the above expression and purification procedures, and its molecular sieve chromatography pattern shows a single main peak.

[0078] The main single-peak fraction was collected, which is the purified product. The purified product was subjected to SDS-PAGE gel electrophoresis to analyze whether the protein bands met expectations and to assess disulfide bond formation and polymeric state. Results are shown below. Figure 3 ; Figure 3 The SDS-PAGE electrophoresis showed clear bands, and the band sizes were consistent with the theoretical molecular weights of each bispecific nanobody.

[0079] The above results indicate that purified bispecific nanobodies N235-R218, N103-S102, N235-S102 and R218-S102 were successfully obtained through the above procedure.

[0080] Example 2: Packaging of SARS-CoV-2 variant pseudoviruses

[0081] 1) The last 18 amino acids of the S protein encoding the SARS-CoV-2 variants XBB, JN.1, and KP.2 were removed, and the remaining S protein sequences were synthesized (synthesis services were provided by Suzhou Genewiz), resulting in the truncated S protein gene sequences of each strain.

[0082] 2) The gene sequences obtained in 1) are cloned into the pCAGGS vector to obtain their recombinant expression plasmids.

[0083] 3) Packaging of SARS-CoV-2 variants XBB, JN.1, and KP.2 pseudoviruses: The specific packaging steps are as follows:

[0084] a. Cell preparation: Seed HEK293T cells (purchased from ATCC CRL-3216) in 10cm cell culture dishes and allow the cell confluence to reach approximately 80% by the second day. The culture medium is DMEM containing 10% FBS.

[0085] b. Transfection: Take the recombinant expression plasmids of each S protein from step 2) above, and transfect 30 μg plasmid / 10 cm cell culture dish with PEI. Mix the target plasmid and PEI at a ratio of 1:3 before transfection. Change the culture medium (DMEM medium containing 10% FBS) after 4-6 hours and incubate at 37℃ for 24 hours.

[0086] c. Virus addition: The pseudovirus packaging backbone virus G*VSV-delG (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) was added to the above-transfected HEK293T cells, incubated at 37°C for 2 hours, the culture medium was changed (DMEM medium containing 10% FBS), and VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank) was added. The cells were then cultured in an incubator for another 30 hours.

[0087] d. Collection of the virus: Collect the supernatant, centrifuge at 3000 rpm for 10 min, filter through a 0.45 μm sterile filter in a laminar flow hood to remove cell debris, aliquot, and freeze at -80℃.

[0088] Pseudoviruses of SARS-CoV-2 variants XBB, JN.1, and KP.2 were obtained respectively.

[0089] Example 3: Detection of the ability of bispecific nanobodies to neutralize pseudovirus infection

[0090] The high-purity bispecific nanobody obtained in Example 1, its constituent monomeric nanobody, and a simple mixture of the two nanobody solutions (mixed at a 1:1 mass ratio) were serially diluted 6-fold starting from 100 μg / mL using DMEM as the diluent, resulting in nine antibody concentration gradients: 100 μg / mL, 16.67 μg / mL, 2.78 μg / mL, 0.463 μg / mL, 0.077 μg / mL, 0.0129 μg / mL, 0.00215 μg / mL, and 0.000358 μg / mL, with a minimum final concentration of 10.24 pg / mL. Each concentration of antibody solution was then mixed with an equal volume of virus at a dose of 2 × 10⁻⁶. 4 TCID 50 Pseudoviruses (SARS-CoV-2 variants XBB, JN.1, and KP.2) were incubated at 37°C for 1 hour to form antibody-virus complexes. A positive control group (pseudovirus + no antibody) was set up to assess the maximum infection level, and a negative control group (no virus) was set up to assess background signal. Subsequently, the mixture was added to pre-seeded Vero E6 cells (ATCC CRL-1587) at a density of approximately 1 × 10⁶ cells per well. 4 Cells were incubated in 96-well plates at 37°C and 5% CO2 for 18–20 hours. GFP fluorescence signals in each well were then directly scanned using a CQ1 confocal quantitative imaging cell analysis system. The number of GFP-positive cells in each well was identified to reflect the degree of viral infection and served as the basis for subsequent calculations of antibody neutralizing activity. To evaluate the inhibitory effect of the antibody, the number of GFP-positive cells in each antibody treatment group and the number of GFP-positive cells in the positive control group (i.e., pseudovirus + no antibody) were standardized, and the viral infection inhibition rate (%) was calculated using the formula: Inhibition rate (%) = [1 - (number of GFP-positive cells in antibody treatment group / number of GFP-positive cells in positive control group)] × 100, which represents the degree of inhibition of viral infection at that antibody concentration. Subsequently, a four-parameter logistic regression model was used to fit the "antibody concentration - inhibition rate" data, and the antibody concentration required to reduce the viral infection rate by 50% was calculated from the fitted curve, which is the IC50 value, expressed in μg / mL. The results are shown in Table 1 and [Table data would be inserted here]. Figure 4-6 .

[0091] Table 1. Neutralizing effect of various nanobodies against SARS-CoV-2 pseudovirus (IC50) 50 value)

[0092]

[0093]

[0094] Depend on Figure 4-6As can be seen from Table 1, the IC50 values ​​of the bispecific nanobodies N235-R218, N103-S102, N235-S102, and R218-S102 of this invention against SARS-CoV-2 pseudoviruses are significantly higher than those of individual nanobodies or simple mixtures of two nanobodies. 50 The values ​​all decreased significantly.

[0095] To more intuitively compare the pseudovirus neutralization effect of the bispecific nanobody of the present invention with that of the individual nanobody constituting it, the inventors further calculated the fold change of neutralization capacity of each bispecific nanobody compared with that of the individual nanobody constituting it, and the results are shown in Table 2 below.

[0096] Table 2. Neutralization effect gain of bispecific nanobodies compared to single nanobodies

[0097]

[0098] As shown in Table 2, compared with using the two nanobodies constituting the bispecific nanobodies alone, the bispecific nanobodies N235-R218, N103-S102, N235-S102 and R218-S102 of the present invention have a significantly improved overall ability to neutralize SARS-CoV-2 pseudovirus. In particular, when targeting some variant strains, their neutralizing ability can be increased by hundreds of times (for example, the neutralizing ability of bispecific antibody N235-S102 against XBB pseudovirus is more than 300 times higher than that of nanobody N235), showing an unexpected synergistic effect.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0100] The sequences involved in this article:

[0101] SEQ ID NO:1 (CDR1 of nanobody N235)

[0102] GLIISRYD

[0103] SEQ ID NO:2 (CDR2 of nanobody N235)

[0104] TPIPAARP

[0105] SEQ ID NO:3 (CDR3 of nanobody N235)

[0106] NLGPESQDHNYNY

[0107] SEQ ID NO:4 (CDR1 of nanobody R218)

[0108] GRPRSSYG

[0109] SEQ ID NO:5 (CDR2 of nanobody R218)

[0110] ISLISDIT

[0111] SEQ ID NO:6 (CDR3 of nanobody R218)

[0112] NAAARIGWVG

[0113] SEQ ID NO:7 (CDR1 of nanobody N103)

[0114] VNLFIGAT

[0115] SEQ ID NO:8 (CDR2 of nanobody N103)

[0116] IGAGGTT

[0117] SEQ ID NO:9 (CDR3 of nanobody N103)

[0118] NYGSMSNPRTSGPNAF

[0119] SEQ ID NO:10 (CDR1 of nanobody S102)

[0120] GFTFSSYA

[0121] SEQ ID NO:11 (CDR2 of nanobody S102)

[0122] IGSFVTNY

[0123] SEQ ID NO:12 (CDR3 of nanobody S102)

[0124] RRVQVERSEY

[0125] SEQ ID NO:13 (VHH chain of nanobody N235)

[0126] QVQLQESGGGLVQPGGSLRLSCAASGLIISRYDMSWYRQAPGKERELVATTPIPAARPQYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCNLGPESQDHNYNYWGQGTQVTVSS

[0127] SEQ ID NO:14 (VHH chain of nanobody R218)

[0128] QVQLQESGGGLVQPGGSLRLSCLASGRPRSSYGMAWFRQAPGKERDFVASISLISDITDYADSVKGRFTISRDYAKNTVYLQMNNLKPEDTAVYYCNAAARIGWVGWGQGTQVTVSS

[0129] SEQ ID NO:15 (VHH chain of nanobody N103)

[0130] QVQLQESGGGLVQPGGSLRLSCAASVNLFIGATMAWYRQAPGNQRELVATIGAGGTTNYADSVKGRFTASRDDAKKTIYLQMNSLTPEDTAVYYCNYGSMSNPRTSGPNAFWGRGTQVTVSS

[0131] SEQ ID NO:16 (VHH chain of nanobody S102)

[0132] QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMAWYRQAPGKEREGVAVIGSFVTNYADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCHARRVQVERSEYWGQGTQVTVSS

[0133] SEQ ID NO:17 (Signal peptide)

[0134] METDTLLLWVLLLWVPGSTG

[0135] SEQ ID NO:18 (Signal peptide)

[0136] MHSSALLCCLVLLTGVRA

[0137] SEQ ID NO:19 (Amino acid sequence of bispecific nanobody N235-R218)

[0138] QVQLQESGGGLVQPGGSLRLSCAASGLIISRYDMSWYRQAPGKERELVATTPIPAARPQYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCNLGPESQDHNYNYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCLASGRPRSSYGMAWFRQAPGKERDFVASISLISDITDYADSVKGRFTISRDYAKNTVYLQMNNLKPEDTAVYYCNAAARIGWVGWGQGTQVTVSS

[0139] SEQ ID NO:20 (Amino acid sequence of bispecific nanobody N103-S102)

[0140] QVQLQESGGGLVQPGGSLRLSCAASVNLFIGATMAWYRQAPGNQRELVATIGAGGTTNYADSVKGRFTASRDDAKKTIYLQMNSLTPEDTAVYYCNYGSMSNPRTSGPNAFWGRGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMAWYRQAPGKEREGVAVIGSFVTNYADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCHARRVQVERSEYWGQGTQVTVSS

[0141] SEQ ID NO:21 (Amino acid sequence of bispecific nanobody N235-S102)

[0142] QVQLQESGGGLVQPGGSLRLSCAASGLIISRYDMSWYRQAPGKERELVATTPIPAARPQYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCNLGPESQDHNYNYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMAWYRQAPGKEREGVAVIGSFVTNYADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCHARRVQVERSEYWGQGTQVTVSS

[0143] SEQ ID NO:22 (Amino acid sequence of bispecific nanobody R218-S102)

[0144] QVQLQESGGGLVQPGGSLRLSCLASGRPRSSYGMAWFRQAPGKERDFVASISLISDITDYADSVKGRFTISRDYAKNTVYLQMNNLKPEDTAVYYCNAAARIGWVGWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMAWYRQAPGKEREGVAVIGSFVTNYADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCHARRVQVERSEYWGQGTQVTVSS

[0145] SEQ ID NO:23 (Nucleic acid coding sequence of bispecific nanobody N235-R218)

[0146] CAGGTGCAGCTGCAGGAGTCCGGAGGAGGGCTGGTGCAGCCCGGAGGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGACTGATTATCAGCAGATATGACATGAGCTGGTATAGACAGGCCCCTGGCAAAGAAAGGGAGCTGGTGGCAACCACCCCCATCCCTGCTGCCCGGCCTCAGTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCGTGAGCCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTCTACTACTGCAACCTGGGCCCCGAAAGCCAGGACCACAACTACAACTACTGGGGCCAGGGAACCCAGGTGACCGTGAGCTCCGGCGGCGGTGGTAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCTCAGGCGGCGGTGGCAGCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCTTGTCTAGCCTCTGGACGACCCCGCAGTAGCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGACTTTGTCGCATCTATTAGTTTGATTAGTGATATTACAGACTATGCAGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGATTACGCTAAGAACACGGTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCCGCTGCCAGAATTGGATGGGTCGGCTGGGGCCAGGGGACCCAGGTGACCGTGAGCTCT

[0147] SEQ ID NO:24 (Nucleic acid coding sequence of bispecific nanobody N103-S102)

[0148] CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGGCTGAGCTGCGCCGCCAGCGTGAACCTGTTCATCGGCGCCACCATGGCCTGGTACCGGCAGGCCCCCGGCAACCAGCGGGAGCTGGTGGCCACCATCGGCGCCGGCGGCACCACCAACTACGCCGACAGCGTGAAGGGCCGGTTCACCGCCAGCCGGGACGACGCCAAGAAGACCATCTACCTGCAGATGAACAGCCTGACCCCCGAGGACACCGCCGTGTACTACTGCAACTACGGCAGCATGAGCAACCCCCGGACCAGCGGCCCCAACGCCTTCTGGGGCCGGGGCACCCAGGTGACCGTGAGCAGCGGCGGCGGTGGTAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCTCAGGCGGCGGTGGCAGCCAGGTGCAGCTGCAGGAGTCCGGCGGCGGACTGGTGCAGCCTGGAGGATCCCTGAGGCTGTCCTGTGCCGCCAGCGGCTTCACATTCTCCTCCTACGCCATGGCCTGGTACAGACAGGCCCCTGGCAAGGAGAGAGAGGGAGTGGCCGTGATCGGCAGCTTCGTGACAAACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACAACGCCAAGAACATGGTGTACCTGCAGATGAATAGCCTGAAGCCCGAGGACACAGCCGTGTACTACTGTCACGCCAGGAGAGTGCAGGTGGAGAGGAGCGAGTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC

[0149] SEQ ID NO:25 (Nucleic acid coding sequence of bispecific nanobody N235-S102)

[0150] CAGGTGCAGCTGCAGGAGTCCGGAGGAGGGCTGGTGCAGCCCGGAGGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGACTGATTATCAGCAGATATGACATGAGCTGGTATAGACAGGCCCCTGGCAAAGAAAGGGAGCTGGTGGCAACCACCCCCATCCCTGCTGCCCGGCCTCAGTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCGTGAGCCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTCTACTACTGCAACCTGGGCCCCGAAAGCCAGGACCACAACTACAACTACTGGGGCCAGGGAACCCAGGTGACCGTGAGCTCCGGCGGCGGTGGTAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCTCAGGCGGCGGTGGCAGCCAGGTGCAGCTGCAGGAGTCCGGCGGCGGACTGGTGCAGCCTGGAGGATCCCTGAGGCTGTCCTGTGCCGCCAGCGGCTTCACATTCTCCTCCTACGCCATGGCCTGGTACAGACAGGCCCCTGGCAAGGAGAGAGAGGGAGTGGCCGTGATCGGCAGCTTCGTGACAAACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACAACGCCAAGAACATGGTGTACCTGCAGATGAATAGCCTGAAGCCCGAGGACACAGCCGTGTACTACTGTCACGCCAGGAGAGTGCAGGTGGAGAGGAGCGAGTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC

[0151] SEQ ID NO:26 (Nucleic acid coding sequence of bispecific nanobody R218-S102)

[0152] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCTTGTCTAGCCTCTGGACGACCCCGCAGTAGCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGACTTTGTCGCATCTATTAGTTTGATTAGTGATATTACAGACTATGCAGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGATTACGCTAAGAACACGGTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCCGCTGCCAGAATTGGATGGGTCGGCTGGGGCCAGGGGACCCAGGTGACCGTGAGCTCTGGCGGCGGTGGTAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCTCAGGCGGCGGTGGCAGCCAGGTGCAGCTGCAGGAGTCCGGCGGCGGACTGGTGCAGCCTGGAGGATCCCTGAGGCTGTCCTGTGCCGCCAGCGGCTTCACATTCTCCTCCTACGCCATGGCCTGGTACAGACAGGCCCCTGGCAAGGAGAGAGAGGGAGTGGCCGTGATCGGCAGCTTCGTGACAAACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACAACGCCAAGAACATGGTGTACCTGCAGATGAATAGCCTGAAGCCCGAGGACACAGCCGTGTACTACTGTCACGCCAGGAGAGTGCAGGTGGAGAGGAGCGAGTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC

Claims

1. A bispecific nanobody targeting SARS-CoV-2, characterized in that, The bispecific nanobody is a tandem assembly of VHH chains selected from the following two nanobody types, linked together by a linker in order from the N-terminus to the C-terminus: (1) Nanobody N235 and nanobody R218; wherein, the VHH chain of nanobody N235 includes the following CDRs: CDR1 with amino acid sequence as shown in SEQ ID NO: 1, CDR2 with amino acid sequence as shown in SEQ ID NO: 2, and CDR3 with amino acid sequence as shown in SEQ ID NO: 3; the VHH chain of nanobody R218 includes the following CDRs: CDR1 with amino acid sequence as shown in SEQ ID NO: 4, CDR2 with amino acid sequence as shown in SEQ ID NO: 5, and CDR3 with amino acid sequence as shown in SEQ ID NO: 6; (2) Nanobody N103 and nanobody S102; wherein, the VHH chain of nanobody N103 includes the following CDRs: CDR1 with amino acid sequence as shown in SEQ ID NO: 7, CDR2 with amino acid sequence as shown in SEQ ID NO: 8, and CDR3 with amino acid sequence as shown in SEQ ID NO: 9; the VHH chain of nanobody S102 includes the following CDRs: CDR1 with amino acid sequence as shown in SEQ ID NO: 10, CDR2 with amino acid sequence as shown in SEQ ID NO: 11, and CDR3 with amino acid sequence as shown in SEQ ID NO: 12; (3) Nanobody N235 and nanobody S102; wherein, the VHH chain of nanobody N235 includes the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO: 1, CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and CDR3 with the amino acid sequence shown in SEQ ID NO: 3; the VHH chain of nanobody S102 includes the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO: 10, CDR2 with the amino acid sequence shown in SEQ ID NO: 11, and CDR3 with the amino acid sequence shown in SEQ ID NO: 12; or, (4) Nanobody R218 and nanobody S102; wherein, the VHH chain of nanobody R218 includes the following CDRs: CDR1 with amino acid sequence as shown in SEQ ID NO: 4, CDR2 with amino acid sequence as shown in SEQ ID NO: 5, and CDR3 with amino acid sequence as shown in SEQ ID NO: 6; the VHH chain of nanobody S102 includes the following CDRs: CDR1 with amino acid sequence as shown in SEQ ID NO: 10, CDR2 with amino acid sequence as shown in SEQ ID NO: 11, and CDR3 with amino acid sequence as shown in SEQ ID NO:

12.

2. The dual-specific nanobody according to claim 1, characterized in that, The VHH chain of the nanobody N235 has an amino acid sequence as shown in SEQ ID NO:13, or an amino acid sequence that has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

13. And / or, the VHH chain of the nanobody R218 has an amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence that has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:14; And / or, the VHH chain of the nanobody N103 has an amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence that has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:15; And / or, the VHH chain of the nanobody S102 has an amino acid sequence as shown in SEQ ID NO:16, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

3. The dual-specific nanobody according to claim 1, characterized in that, The VHH chain of the nanobody N235 has an amino acid sequence as shown in SEQ ID NO:13, or an amino acid sequence that has at least 97% sequence identity with the amino acid sequence shown in SEQ ID NO:

13. And / or, the VHH chain of the nanobody R218 has an amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence that has at least 97% sequence identity with the amino acid sequence shown in SEQ ID NO:14; And / or, the VHH chain of the nanobody N103 has an amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence that has at least 97% sequence identity with the amino acid sequence shown in SEQ ID NO:15; And / or, the VHH chain of the nanobody S102 has an amino acid sequence as shown in SEQ ID NO:16, or an amino acid sequence having at least 97% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

4. The dual-specific nanobody according to claim 1, characterized in that, The VHH chain of the nanobody N235 has an amino acid sequence as shown in SEQ ID NO:13, or an amino acid sequence that has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:

13. And / or, the VHH chain of the nanobody R218 has an amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence that has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:14; And / or, the VHH chain of the nanobody N103 has an amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence that has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:15; And / or, the VHH chain of the nanobody S102 has an amino acid sequence as shown in SEQ ID NO:16, or an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

5. The dual-specific nanobody according to claim 1, characterized in that, The VHH chain of the nanobody N235 has an amino acid sequence as shown in SEQ ID NO:13, or an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

13. And / or, the VHH chain of the nanobody R218 has an amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:14; And / or, the VHH chain of the nanobody N103 has an amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15; And / or, the VHH chain of the nanobody S102 has an amino acid sequence as shown in SEQ ID NO:16, or an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

6. The bispecific nanobody according to any one of claims 1-5, characterized in that, The connector is (GGGGS)n, where n is an integer between 1 and 6.

7. The bispecific nanobody according to any one of claims 1-5, characterized in that, The bispecific nanobody comprises an amino acid sequence selected from the following: SEQ ID NO:19-22.

8. A fusion protein comprising: a bispecific nanobody as described in any one of claims 1 to 7, and a signal peptide sequence and / or tag sequence for assisting in expression and / or purification.

9. A polynucleotide encoding a bispecific nanobody as described in any one of claims 1 to 7 or a fusion protein as described in claim 8.

10. The polynucleotide according to claim 9, characterized in that, The polynucleotide is DNA or mRNA.

11. The polynucleotide according to claim 10, characterized in that, The polynucleotide encodes the bispecific nanobody as described in claim 7.

12. The polynucleotide according to claim 10, characterized in that, The polynucleotide includes a nucleotide sequence as shown in one of SEQ ID NO:23-26.

13. A nucleic acid construct comprising a polynucleotide as described in any one of claims 9-12, and at least one expression regulatory element operatively linked to said polynucleotide.

14. An expression vector comprising a polynucleotide as described in any one of claims 9-12 or a nucleic acid construct as described in claim 13.

15. A host cell wherein it is transformed or transfected with the polynucleotide of any one of claims 9-12, the nucleic acid construct of claim 13, or the expression vector of claim 14.

16. An immunoconjugate, characterized in that, The term includes: (1) The bispecific nanobody as described in any one of claims 1 to 7, or the fusion protein as described in claim 8; and (2) Selected from the following coupling components: radionuclides, photosensitizers, enzymes, fluorescent markers, magnetic nanoparticles, liposomes, and polymer nanoparticles.

17. A pharmaceutical composition comprising a bispecific nanobody as described in any one of claims 1 to 7, a fusion protein as described in claim 8, a polynucleotide as described in any one of claims 9 to 12, a nucleic acid construct as described in claim 13, an expression vector as described in claim 14, a host cell as described in claim 15, or an immunoconjugate as described in claim 16, and a pharmaceutically acceptable carrier and / or excipient.

18. The pharmaceutical composition according to claim 17, characterized in that, The pharmaceutical composition is in the form of a nasal spray or a parenteral preparation.

19. The pharmaceutical composition according to claim 17, characterized in that, The nasal spray is selected from aerosols, sprays, and powders.

20. The pharmaceutical composition according to claim 17, characterized in that, The parenteral preparation is an injectable or bolus-type preparation.

21. The use of the bispecific nanobody as described in any one of claims 1 to 7, the fusion protein as described in claim 8, the polynucleotide as described in any one of claims 9 to 12, the nucleic acid construct as described in claim 13, the expression vector as described in claim 14, the host cell as described in claim 15, the immunoconjugate as described in claim 16, or the pharmaceutical composition as described in any one of claims 17 to 20 in the preparation of a medicament for the prevention and / or treatment of novel coronavirus infection.

22. The use of the bispecific nanobody as described in any one of claims 1 to 7, the fusion protein as described in claim 8, the polynucleotide as described in any one of claims 9 to 12, the nucleic acid construct as described in claim 13, the expression vector as described in claim 14, the host cell as described in claim 15, the immunoconjugate as described in claim 16, or the pharmaceutical composition as described in any one of claims 17 to 20 in the preparation of reagents or kits for detecting the novel coronavirus or for diagnosing novel coronavirus infection.

23. A novel coronavirus detection kit comprising a bispecific nanobody as described in any one of claims 1 to 7, a fusion protein as described in claim 8, a polynucleotide as described in any one of claims 9 to 12, a nucleic acid construct as described in claim 13, an expression vector as described in claim 14, a host cell as described in claim 15, an immunoconjugate as described in claim 16, or a pharmaceutical composition as described in any one of claims 17 to 20.

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