Llama-derived nanobody r218 and applications thereof

Abstract

The present application relates to a llama-derived nanobody and its application, in particular to a llama-derived nanobody R218 or an antigen-binding fragment thereof binding to SARS-CoV-2 RBD and its application, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising 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. The nanobody R218 of the present application has great potential clinical application prospect in preventing, treating and detecting SARS-CoV-2 original strain and a series of variant strains and related coronavirus infections.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202111363957.9, filed on November 17, 2021, entitled “An Alpaca-Derived Nanobody R218 and Its Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedicine, specifically to an alpaca-derived nanobody R218 and its applications, and more specifically, to an alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 RBD, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a method for its preparation, transformed cells, and a pharmaceutical composition comprising the above, and their applications in the preparation of medicaments for the prevention, treatment, or detection of SARS-CoV-2 and / or related coronavirus infections. Background Technology

[0004] Since the end of 2019, the epidemic caused by the novel coronavirus (also known as SARS-CoV-2 or COVID-19) belonging to the family Coronaviridae has continued to spread globally, and newly emerging and re-emerging viruses pose a huge threat to global public health.

[0005] Neutralizing antibody drugs mainly work by binding to antigens on the surface of pathogenic microorganisms, preventing specific molecules expressed by the pathogenic microorganisms from binding to cell surface receptors, thus achieving a "neutralizing" effect.

[0006] Both SARS-CoV and SARS-CoV-2 viruses possess a glycosylated spike protein (S) on their surface. This S protein can interact with the host cell receptor protein ACE2 and trigger membrane fusion. Therefore, blocking the binding of the S protein to ACE2 is an effective approach to treating SARS-CoV-2 infection. The S protein contains two functional subunits, S1 and S2. The receptor-binding domain (RBD) located in the S1 subunit is mainly related to viral receptor recognition. Therefore, isolating and identifying neutralizing antibodies targeting the RBD region is crucial for the prevention and treatment of SARS-CoV-2 infection.

[0007] However, antibody strategies aimed at blocking viral interactions with host cell receptors still require further optimization and upgrading. On one hand, RNA viruses like SARS-CoV-2 are prone to mutation and immune evasion, making it difficult for single-specific antibodies to meet long-term therapeutic needs. On the other hand, conventional monoclonal antibodies also have certain limitations in practical applications due to their large molecular weight. Summary of the Invention

[0008] Purpose of the invention

[0009] The present invention aims to provide an alpaca-derived nanobody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a method for its preparation, transformed cells, and a pharmaceutical composition comprising the above, as well as their application in the preparation of drugs for the prevention or treatment of COVID-19. The alpaca-derived nanobody or its antigen-binding fragment of the present invention is a highly neutralizing nanobody with strong binding ability to the RBD protein of the original SARS-CoV-2 strain and its variants, as well as related coronaviruses, effectively inhibiting infection by the original SARS-CoV-2 strain and its series of variants, as well as related coronaviruses. This nanobody has advantages such as small molecular weight (~15kDa), low immunogenicity, better solubility and stability, and a longer CDR3 region, allowing for nebulized administration, direct delivery to the lungs, and faster onset of action, providing a potential therapeutic strategy for COVID-19 or other coronavirus infections.

[0010] Solution

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] In a first aspect, the present invention provides an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD, wherein the antibody comprises a heavy chain variable region, and the heavy chain variable region comprises the following CDRs:

[0013] The amino acid sequence is CDR1 as shown in SEQ ID NO:1 (i.e., GRPRSSYG).

[0014] The amino acid sequence is CDR2 as shown in SEQ ID NO:2 (i.e., ISLISDIT).

[0015] And CDR3 with an amino acid sequence as shown in SEQ ID NO:3 (i.e., NAAARIGWVG).

[0016] In a specific implementation, the heavy chain variable region further includes four frame regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

[0017] In a preferred embodiment, the amino acid sequences of FR1-4 are as shown in SEQ ID NO:4 (i.e., QVQLQESGGGLVQPGGSLRLSCLAS), SEQ ID NO:5 (i.e., MAWFRQAPGKERDFVAS), SEQ ID NO:6 (i.e., DYADSVKGRFTISRDYAKNTVYLQMNNLKPEDTAVYYC), and SEQ ID NO:7 (i.e., WGQGTQVTVSS).

[0018] In a preferred embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8:

[0019] The underlined parts represent the frame regions FR1-4, and the bolded parts represent the heavy chain variable regions CDR1, CDR2, and CDR3.

[0020] In a second aspect, the present invention provides a polynucleotide encoding an alpaca-derived nanobody or its antigen-binding fragment as described in the first aspect above.

[0021] Furthermore, the polynucleotide is DNA or mRNA.

[0022] Furthermore, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:9:

[0023] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCTTGTCTAGCCTCTGGACGACCCCGCAGTAGCTATGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGACTTTGTCGCATCTATTAGTTTGATTAGTGATATTACAGA CTATGCAGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGATTACGCTAAGAACACGGTGTATCTGCAAATGAACAACCTGAAAACCTGAGGACACGGCCGTCTATTACTGTAATGCCGCTGCCAGAATTGGATGGGTCGGCTGGGGCCAGGGGACCCAGGTGACCGTGAGCTCT.

[0024] Thirdly, the present invention provides a nucleic acid construct comprising the polynucleotides described in the second aspect above.

[0025] More preferably, the nucleic acid construct further comprises at least one expression regulatory element operatively linked to the polynucleotide, such as a histidine tag, a stop codon, etc.

[0026] Fourthly, the present invention provides an expression vector comprising the nucleic acid construct as described in the third aspect above.

[0027] Fifthly, the present invention provides a transformed cell comprising the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0028] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, or transformed cells as described in the fifth aspect above, and a pharmaceutically acceptable carrier and / or excipient.

[0029] Preferably, the pharmaceutical composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation.

[0030] More preferably, the nasal spray is selected from aerosols, sprays, and powders.

[0031] More preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0032] More preferably, the tablet is a sublingual tablet;

[0033] More preferably, the granules are fine granules;

[0034] More preferably, the powder is a granule;

[0035] More preferably, the pills are small pills.

[0036] More preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; even more preferably, the injectable preparation is a push-in preparation.

[0037] In a seventh aspect, the present invention provides the use of an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, or a transformed cell as described in the fifth aspect above, or a pharmaceutical composition as described in the sixth aspect above, in the preparation of a medicament for the prevention, treatment, or detection of SARS-CoV-2 and / or related coronavirus infections.

[0038] Preferably, the novel coronavirus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

[0039] More preferably, the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and / or Delta (B.1.617.2) variant strain of SARS-CoV-2.

[0040] And / or, the relevant coronavirus is SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017 or GD / 1 / 2019.

[0041] Eighthly, the present invention provides a method for preventing or treating SARS-CoV-2 or related coronaviruses, comprising: administering to a subject in need a preventive or therapeutically effective amount of an alpaca-derived nanobody or antigen-binding fragment thereof that is conjugated to SARS-CoV-2 RBD as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, or a transformed cell as described in the fifth aspect above, or a pharmaceutical composition as described in the sixth aspect above.

[0042] Preferably, the relevant coronavirus is SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, or GD / 1 / 2019.

[0043] In a ninth aspect, the present invention provides a method for detecting SARS-CoV-2 or related coronaviruses, comprising using an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD as described in the first aspect above.

[0044] Preferably, the novel coronavirus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

[0045] More preferably, the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and / or Delta (B.1.617.2) variant strain of SARS-CoV-2.

[0046] Preferably, the related coronavirus is SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, or GD / 1 / 2019.

[0047] 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.

[0048] Beneficial effects

[0049] This invention develops nanobody drugs targeting the novel coronavirus. By immunizing alpacas with SARS-CoV-2 RBD and NTD proteins, constructing an antibody library, and using phage display technology to screen for specific nanobodies, a nanobody that specifically binds to SARS-CoV-2 RBD with high affinity was screened and named nanobody R218 in this paper. The inventors of this invention have confirmed through surface plasmon resonance (SPR) technology that the nanobody R218 of this invention can bind with high affinity to the original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Kappa, Delta, and related coronaviruses SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, and GD / 1 / 2019RBD. Furthermore, in virus neutralization tests (including both true and false virus neutralization tests), it can neutralize the original SARS-CoV-2 strain and its series of variants with high neutralizing activity. These findings indicate that the nanobody R218 is a high-affinity alpaca-derived nanobody with high neutralizing activity against the original SARS-CoV-2 strain and its variants, as well as related coronaviruses.

[0050] This invention provides potential nanobody drugs for the clinical prevention, treatment and detection of the original strain of the novel coronavirus and its variant strains, as well as related coronavirus infections. Attached Figure Description

[0051] 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.

[0052] Figure 1 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 WT RBD-his protein described in Example 1 of this invention;

[0053] Figure 2 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 WT NTD-his protein described in Example 1 of this invention;

[0054] Figure 3 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 WT RBD-hFc protein described in Example 1 of this invention;

[0055] Figure 4 This is a schematic diagram illustrating the effect of the nanobody R218 on SARS-CoV-2 WT pseudovirus infection as measured in Example 7 of the present invention. Detailed Implementation

[0056] 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. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0057] 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.

[0058] The present invention will now be described in detail.

[0059] definition

[0060] "Nanobodies," also known as "single-domain antibodies," contain only one variable domain of heavy chain (VHH) and, unlike other antibodies, naturally lack the light chain.

[0061] Due to their inherent biophysical advantages, nanobodies can be easily atomized and delivered directly to the lungs via inhalers to treat viral respiratory infections, making them a highly promising antibody drug.

[0062] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction of the nanobody of the present invention to the SARS-CoV-2 RBD protein, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0063] The reagents, enzymes, culture media, antibiotics, and milk used in the following examples of the present invention are all commercially available products. For example, TRIzol was purchased from Invitrogen, and the Superscript II First-Strand Synthesis System for RT-PCR kit was purchased from Invitrogen.

[0064] Some commonly used biological materials, such as competent cells, vectors, helper phages, and cells to be transformed, are also commercially available products. For example, the pCAGGS vector was purchased from MiaoLingPlasmid, and 293F cells and HEK293T cells were purchased from ATCC; electrocompetent E. coli TG1 cells were purchased from Lucigen, VCSM13 helper phages were purchased from StrataGene, and plasmid pMES4 was purchased from Addgene; protein A microarrays were purchased from GE Healthcare; and Vero cells were purchased from ATCC CCL81.

[0065] Some synthetic biological materials, such as primers and sequences, which require artificial synthesis, are outsourced to synthetic companies. For example, the primers (SED ID NO: 15-20) in this invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0066] Example 1: Expression and purification of RBD-hFc protein of SARS-CoV-2 original strain (WT) RBD-his, SARS-CoV-2 WT NTD-his and SARS-CoV-2 WT and its variants, as well as related coronaviruses.

[0067] The coding sequence of the signal peptide (as shown in SEQ ID NO:11) was linked to the 5' end of the SARS-CoV-2 WT RBD protein coding sequence (as shown in SEQ ID NO:10), and the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA were linked to the 3' end. The sequence was constructed into the pCAGGS vector through the restriction endonuclease sites EcoRI and XhoI, and transfected into 293F cells for expression of SARS-CoV-2 WT RBD-his protein.

[0068] Similarly, the coding sequence of the signal peptide (as shown in SEQ ID NO:13) was linked to the 5' end of the SARS-CoV-2 WT NTD protein coding sequence (as shown in SEQ ID NO:12), and the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA were linked to the 3' end. The pCAGGS vector was constructed using EcoRI and XhoI restriction endonuclease sites and transfected into 293F cells to express the SARS-CoV-2 WT NTD-his protein.

[0069] Similarly, the coding sequence of the signal peptide (as shown in SEQ ID NO:11) was linked to the 5' end of the SARS-CoV-2 WT RBD protein coding sequence (as shown in SEQ ID NO:10), and the coding sequence of the human Fc tag (hFc) (as shown in SEQ ID NO:14) and the translation stop codon TGA were linked to the 3' end. The pCAGGS vector was constructed by linking EcoRI and XhoI, and transfected into 293F cells to express the SARS-CoV-2 WT RBD-hFc protein. After further purification and identification as described below, the protein was used for surface plasmon resonance analysis.

[0070] Cell culture medium containing the target protein was subjected to nickel ion affinity chromatography (HisTrap). TM Excel (GE Healthcare) and gel filtration chromatography (Superdex) TM After purification using a 200 Increase 10 / 300 GL column (GE Healthcare), a relatively pure target protein can be obtained. The SDS-PAGE analysis of the SARS-CoV-2 WT RBD-his protein showed a size of approximately 30 kDa, as shown in the results. Figure 1 The SDS-PAGE analysis of the SARS-CoV-2 WT NTD-his protein revealed a size of approximately 60 kDa, as shown in the results. Figure 2The SDS-PAGE analysis of the SARS-CoV-2 WT RBD-hFc protein revealed a size of approximately 130 kDa, as shown in the results. Figure 3 .

[0071] Using the same method as described above for preparing SARS-CoV-2 WTRBD-hFc protein, RBD-hFc proteins of SARS-CoV-2 variant strains Alpha, Beta, Gamma, Kappa, Delta, and related coronaviruses SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, and GD / 1 / 2019 were prepared for surface plasmon resonance analysis. The DNA coding sequences of the RBD proteins of the above viral strains are available in the public database NCBI.

[0072] Example 2: Construction of an alpaca immune and antibody library

[0073] In Example 1, 200 μg each of the SARS-CoV-2 WT RBD and NTD proteins with six histidine tags were prepared, diluted to a final volume of 1 mL with PBS, emulsified with 1 mL of complete Freund's adjuvant for 5 min, and administered via subcutaneous injection at multiple sites for immunization. Immunization was then performed every two weeks. On day 12 after the fourth immunization, 50-60 mL of blood was collected, and PBMCs (peripheral blood mononuclear cells) were isolated. The isolated PBMCs were added to 1 mL of TRIzol, and total RNA was extracted according to the manufacturer's instructions. Using the extracted total RNA as a template, the Superscript II First-Strand Synthesis System for RT-PCR kit was used with random primers oligo-dT... 12-18 cDNA was synthesized using primers. Using the cDNA as a template, PCR was performed using specific primers CALL001 and CALL002 (primer sequences shown in Table 1). The 700 bp band was excised from the gel and recovered. The purified DNA was then used as a template for nested PCR using nested primers VHH-BACK and PMCF to amplify the nanobody (VHHs) sequence. The purified VHHs sequence, approximately 400 bp in size, was recovered.

[0074] The VHHs fragment was ligated into plasmid pMES4 using a double restriction enzyme digestion method via restriction enzyme sites PstⅠ and BstEⅡ. The purified cloning vector was mixed with electrocompetent E. coli TG1 cells, and the cloning vector was transformed into electrocompetent E. coli TG1 cells using a BIO-RAD MicroPulser electroporator. All cells were plated on selective medium containing ampicillin and incubated overnight at 37°C. All colonies were then collected in LB medium, centrifuged, and the supernatant was discarded. The cells were resuspended in LB medium to obtain the antibody library.

[0075] Table 1. Reaction primers

[0076]

[0077] Example 3: Screening for specific nanobodies using phage display technology

[0078] Take E. coli TG1 cells transfected with the recombinant plasmid from Example 2, add VCSM13 helper phage at a ratio of approximately 20 multiplicity of infection (MOI), incubate overnight, centrifuge at 4000 rpm, collect the supernatant, filter through a 0.22 μm membrane, add PEG6000 / NaCl at a volume ratio of 1:4, mix, incubate at 4°C for at least 1 hour, centrifuge at 8000×g for 30 min, discard the supernatant, resuspend the precipitate in PBS, and the collected phage particles are obtained. Determine the phage titer.

[0079] 2×10 11 The collected phages were mixed with an equal volume of 5% (w / v) skim milk and added to a 96-well plate coated with SARS-CoV-2 WT RBD-his antigen. After incubation at room temperature for 1 hour, the specific phages were eluted with 0.2M glycine and neutralized with Tris-HCl (pH 9.1). E. coli TG1 cells were then infected with this phage, and the phages were amplified. A second round of panning was performed on 96-well plates coated with SARS-CoV-2 WT RBD-his antigen to enrich phages expressing specific nanobodies. A total of three rounds of panning were conducted. After each round of selection, different single colonies were randomly selected from agar plates containing bacterial colonies and cultured in a shaker at 37°C. Then, VCSM13 helper phage was added for overnight amplification. The culture medium was centrifuged the next day, and the phage supernatant was used for ELISA experiments (using SARS-CoV-2 WT RBD-his protein as the coating antigen). When OD... 450nMWhen the result is >0.2, it is considered a positive reaction. The corresponding clone is then taken, and the plasmid is sequenced using specific primers MP57 and GⅢ (primer sequences are shown in Table 2) to obtain the sequence encoding VHHs in the plasmid. The core coding sequence of R218 is obtained through sequencing.

[0080] Table 2. Reaction Primers

[0081]

[0082] Example 4: Expression and purification of nanobody R218

[0083] To make the heavy chain variable region of R218 more complete, the coding sequence of QVQLQ (CAGGTGCAGCTGCAG, SEQ ID NO:23) was added to the 5' end of the core coding sequence of R218 obtained in Example 3, and the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT, SEQ ID NO:24) was added to the 3' end, resulting in the nucleotide sequence as shown in SEQ ID NO:9, which is the coding sequence of the nanobody R218 of this application. Then, a signal peptide (SED ID NO:21) was added before it, and the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA were added after it. The sequence was constructed into the pCAGGS vector through the restriction enzyme sites EcoRI and XhoI, transfected into 293F cells, and cultured for 5 days. The supernatant was collected, centrifuged at 5000 rpm for 30 min, filtered through a 0.22 μm filter membrane, and then subjected to nickel ion affinity chromatography (HisTrap). TM Excel (GE Healthcare) and gel filtration chromatography (Superdex) TM After purification (using a 75 Increase 10 / 300GL column (GE Healthcare)), a relatively pure target protein was obtained. The target peak was identified by SDS-PAGE, yielding the purified nanobody R218.

[0084] Example 5: Detection of antibody binding ability to the RBD of SARS-CoV-2 original strain, its variants, and related coronaviruses using surface plasmon resonance technology.

[0085] Surface plasmon resonance analysis was performed using a Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0086] A protein A chip (purchased from GE Healthcare) was used. Based on the affinity of protein A chip for hFc, the RBD-hFc proteins of the original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Kappa, Delta, and related coronaviruses SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, and GD / 1 / 2019 obtained in Example 1 were immobilized on the chip, with an immobilization volume of approximately 100 RU. The R218 protein was serially diluted with PBST buffer (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.05% Tween) from low to high concentrations. The kinetic curves of the binding of the nanobody R218 of this invention to the RBD proteins of the above viral strains were obtained, and the results were evaluated using BIAevaluation software. Using 8K (Biacore, Inc.) software, the binding constant (ka), dissociation constant (kd), and equilibrium dissociation constant (K) of the nanobody R218 binding to the RBD protein of these viral strains were calculated. D As shown in Table 3, the results indicate that the nanobody R218 can bind with high affinity to the RBD of the original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Kappa, and Delta, as well as related coronaviruses SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, and GD / 1 / 2019, demonstrating good broad-spectrum binding.

[0087] Table 3. Equilibrium dissociation constants (K218) of nanobody R218 and antigen RBD D )result

[0088]

[0089]

[0090] Example 6: Packaging of pseudoviruses from the original SARS-CoV-2 strain

[0091] 1) The gene encoding the last 18 amino acids of the S protein of the original SARS-CoV-2 strain (WT) was removed, and the remaining sequence of the S protein was synthesized (synthesis service provided by Suzhou Genewiz), to obtain the nucleotide sequence of the SARS-CoV-2-WT-S-del18 gene, as shown in SEQ ID NO:22.

[0092] Similarly, the last 18 amino acids of the S protein in the SARS-CoV-2 mutant strains Beta and Delta were removed, and the remaining S protein sequence was synthesized to obtain the S-del18 gene of the mutant strains Beta and Delta; the gene sequences of the S protein of the mutant strains Beta and Delta can be obtained from the public database NCBI.

[0093] 2) The S-del18 genes obtained in 1) were cloned into the pCAGGS vector to obtain the expression plasmid pCAGGS-S-del18.

[0094] The packaging steps for the original SARS-CoV-2 strain and its variant pseudoviruses are as follows:

[0095] a. Cell preparation: Seed HEK293T cells in a 10cm cell culture dish and allow the cell confluence to reach approximately 80% by the second day. The culture medium is DMEM containing 10% FBS.

[0096] b. Transfection: Take the expression plasmids of each S protein from step 2) above, remove the last 18 amino acids, 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.

[0097] 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.

[0098] 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℃.

[0099] The original SARS-CoV-2 strain (SARS-CoV-2 WT) and its variant strains Beta and Delta were obtained as pseudoviruses.

[0100] Example 7: Detection of SARS-CoV-2 original strain and its variant pseudovirus infection by nanobody R218

[0101] The purified nanobody R218 obtained in Example 4 was serially diluted 5-fold to the 9th gradient. This diluted nanobody R218 was then mixed with 1.6 × 10⁻⁶ ppm... 4 TCID 50The SARS-CoV-2 original strain and its variants Beta and Delta pseudoviruses obtained in Example 6 were mixed and incubated at 37°C for 1 hour, then added to 96-well plates pre-inoculated with Vero cells (purchased from ATCCCCL81). After incubation for 18–20 hours, the results were detected using a CQ1 Confocal Quantitative Image Cytometer (Yokogawa). Based on the number of cells displaying GFP fluorescence, the neutralizing capacity of the antibody against the aforementioned SARS-CoV-2 original strain and its variants Beta and Delta pseudoviruses, i.e., the half-maximal inhibitory concentration (IC50), was calculated. 50 ).

[0102] The results showed that the IC50 of the nanobody R218 against the original SARS-CoV-2 strain was [missing information]. 50 The concentration was 0.84 μg / ml, and its representative inhibition curve is shown below. Figure 4 As shown; IC50 of SARS-CoV-2 variants Beta and Delta 50 The concentrations were 3.48 μg / ml and 1.12 μg / ml, respectively.

[0103] In summary, the nanobody R218 could serve as a candidate antibody drug against the original strain of the novel coronavirus (SARS-CoV-2) and its variants (e.g., Alpha, Beta, Gamma, Kappa, or Delta) as well as related coronaviruses (including SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, and GD / 1 / 2019) for the prevention, treatment, and / or detection of these viruses.

[0104] 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.

Claims

1. An alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD, comprising a heavy chain variable region. The heavy chain variable region comprises 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.

2. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 RBD according to claim 1, characterized in that, The heavy chain variable region also includes four frame regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

3. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 RBD according to claim 1, characterized in that, The FR1-4 are shown as SEQ ID NO:4, 5, 6, and 7, respectively.

4. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 RBD according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

8.

5. A polynucleotide encoding an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD as described in any one of claims 1 to 4.

6. The polynucleotide according to claim 5, characterized in that, The polynucleotide is DNA or mRNA.

7. The polynucleotide according to claim 5, characterized in that, The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

9.

8. A nucleic acid construct comprising the polynucleotide of any one of claims 5 to 7.

9. The nucleic acid construct according to claim 8, characterized in that, It also includes at least one expression regulatory element operatively linked to the polynucleotide.

10. An expression vector comprising the nucleic acid construct of claim 8 or 9.

11. A transformed cell comprising the polynucleotide of any one of claims 5 to 7, the nucleic acid construct of claim 8 or 9, or the expression vector of claim 10.

12. A pharmaceutical composition comprising an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD as described in any one of claims 1 to 4, a polynucleotide as described in any one of claims 5 to 7, a nucleic acid construct as described in claim 8 or 9, an expression vector as described in claim 10 or a transformed cell as described in claim 11, and a pharmaceutically acceptable carrier and / or excipient.

13. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is in the form of a nasal spray, oral preparation, suppository, or parenteral preparation.

14. The pharmaceutical composition according to claim 13, characterized in that, The nasal spray is selected from aerosols, sprays, and powders.

15. The pharmaceutical composition according to claim 13, characterized in that, The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated formulations, and ointments.

16. The pharmaceutical composition according to claim 15, characterized in that, The tablets mentioned are sublingual tablets.

17. The pharmaceutical composition according to claim 15, characterized in that, The granules are fine granules.

18. The pharmaceutical composition according to claim 15, characterized in that, The powder is a granule.

19. The pharmaceutical composition according to claim 15, characterized in that, The pills mentioned are small pills.

20. The pharmaceutical composition according to claim 13, characterized in that, The parenteral preparations include transdermal preparations, ointments, plasters, topical liquids, and injectable preparations.

21. The pharmaceutical composition according to claim 20, characterized in that, The injectable formulation is a push-in formulation.

22. The use of an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 RBD as described in any one of claims 1 to 4, a nucleotide sequence as described in any one of claims 5 to 7, a nucleic acid construct as described in claim 8 or 9, an expression vector as described in claim 10, a transformed cell as described in claim 11, or a pharmaceutical composition as described in any one of claims 12 to 21 in the preparation of a medicament for the prevention, treatment, or detection of SARS-CoV-2 and / or related coronavirus infections; The novel coronavirus is the original SARS-CoV-2 strain and / or a variant of SARS-CoV-2; The SARS-CoV-2 variant strains mentioned are Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and / or Delta (B.1.617.2) variant strains of SARS-CoV-2; The relevant coronaviruses are SARS-CoV, RaTG13, RshSTT182, RacCS203, Rco319, RsYN04, GX / P2V / 2017, or GD / 1 / 2019.

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