Llama-derived nanobody s102 and applications thereof

Abstract

The present application relates to a llama-derived nanobody and its application, in particular to a llama-derived nanobody S102 or an antigen-binding fragment thereof binding to SARS-CoV-2 S2 and its application, wherein the antibody comprises a heavy chain variable region, and the heavy chain variable region comprises 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 S102 of the present application has great potential clinical application prospect in preventing, treating and detecting SARS-CoV-2 original strain and its variant strain and related coronavirus infection.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202111365268.1, filed on November 17, 2021, entitled “An Alpaca-Derived Nanobody S102 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 and its application, and more specifically, to an alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2S2, 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 application in the preparation of drugs for the prevention, treatment, or detection of SARS-CoV-2 and / or related coronavirus infections. Background Technology

[0004] The novel coronavirus (also known as SARS-CoV-2 or COVID-19) of the family Coronaviridae, as well as the severe acute respiratory syndrome coronavirus (SARS-CoV) of the same family, are also major pathogens affecting the human respiratory system. They are mainly transmitted through droplets, aerosols, and contact. Therefore, these viruses that cause respiratory diseases seriously endanger public health and safety, and pose a great threat to the health and safety of the people, as well as to national economic development and social stability.

[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 interacts with the host cell receptor protein ACE2 and triggers 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 consists of two subunits, S1 and S2. The S2 subunit is involved in the fusion of the viral membrane and cell membrane. Antibodies targeting the S2 subunit can exert antiviral effects through a neutralization mechanism. Especially against RNA viruses like SARS-CoV-2, which are prone to mutation and immune escape, single-specific antibodies are insufficient to meet long-term therapeutic needs. Therefore, the isolation and identification of neutralizing antibodies targeting different epitopes is urgently needed.

[0007] Nanobodies, also known as single-domain antibodies (VHHs), possess several unique advantages compared to traditional mAbs (~150 kDa): smaller molecular weight (~15 kDa), lower immunogenicity, better solubility and stability, and a longer CDR3 region. These properties allow nanobodies to be used as single domains or modular units to construct more complex molecules, such as multivalent antibodies targeting different antigens to broaden their spectrum of activity. Importantly, nanobodies can be easily nebulized and delivered directly to the lungs via inhalers, making them potential drugs for treating respiratory diseases. Therefore, isolating and identifying cross-reactive nanobodies is crucial for building a potential drug reserve to address the current COVID-19 pandemic and future coronavirus infections. 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 SARS-CoV-2S2, 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 affinity to the SARS-CoV-2S2 protein, effectively inhibiting infection by the original SARS-CoV-2 strain and its series of variant strains, 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. It can be administered via nebulization, reaching directly to the lungs, and has a faster onset of action, providing a potential treatment 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-2S2, 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., GFTFSSYA).

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

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

[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., QVQLQESGGGLVQPGGSLRLSCAAS), SEQ ID NO:5 (i.e., MAWYRQAPGKERELVAV), SEQ ID NO:6 (i.e., ADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCHA), 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] CAGGGTGCAGCTGCAGGAGAGCGGAGGAGGGCTGGTGCAGCCCGGAGGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGATTTACCTTTAGCAGCTATGCCATGGCATGGTATAGACAGGCCCCTGGAAAAGAGAGAGAGCTGGTGGCAGTGATTGGAAGCTTCGTGACCAACTACGC CGACAGCGTTAAGGGAAGGTTCACCATCAGCAGAGACAACGCAAAAAACATGGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCCACGCCAGAAGAGTGCAGGTGGAGAGAAGCGAGTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC.

[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-2S2 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] Preferably, the related coronavirus is SARS-CoV, GX / P2V / 2017, GD / 1 / 2019, or RaTG13.

[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 binds to SARS-CoV-2S2 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, GX / P2V / 2017, GD / 1 / 2019, or RaTG13.

[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-2S2 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, GX / P2V / 2017, GD / 1 / 2019, or RaTG13.

[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 relates to the development of nanobody drugs targeting the novel coronavirus. Through immunizing alpacas with SARS-CoV-2S protein, constructing an antibody library, and screening for specific nanobodies using phage display technology, a nanobody binding to SARS-CoV-2S2 was identified and named nanobody S102. Surface plasmon resonance (SPR) assays confirmed that nanobody S102 of this invention binds to SARS-CoV-2S2 with high affinity. Furthermore, antibody neutralization assays demonstrated that nanobody S102 of this invention can neutralize the original SARS-CoV-2 strain and its variants, as well as related coronaviruses, with high neutralizing activity.

[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 S-his protein described in Example 1 of this invention;

[0053] Figure 2 This is a schematic diagram of the molecular sieve chromatography and Western blot identification results of the SARS-CoV-2 WT S2-his protein described in Example 1 of the present invention;

[0054] Figure 3 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the nanobody S102 described in Example 4 of the present invention;

[0055] Figure 4 This is a graph showing the affinity identification results between the nanobody S102 and the SARS-CoV-2 WT S2 protein described in Example 5 of this invention;

[0056] Figure 5 This is a schematic diagram illustrating the effect of the nanobody S102 on SARS-CoV-2WT pseudovirus infection as measured in Example 7 of the present invention.

[0057] Figure 6 This is a schematic diagram illustrating the effect of the nanobody S102 on the pseudovirus infection of related coronaviruses SARS-CoV, GX / P2V / 2017, GD / 1 / 2019 and RaTG13, as measured in Example 7 of the present invention. Detailed Implementation

[0058] 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.

[0059] 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.

[0060] The present invention will now be described in detail.

[0061] definition

[0062] "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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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 phage was purchased from StrataGene, and plasmid pMES4 was purchased from Addgene; Vero cells were purchased from ATCC CCL81.

[0067] 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: 14-19) in this invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0068] Example 1: Expression and purification of SARS-CoV-2 original strain (WT)S-his and SARS-CoV-2 WT S2-his proteins

[0069] A signal peptide coding sequence (as shown in SEQ ID NO:11) was appended to the 5' end of the SARS-CoV-2 WT S protein coding sequence (as shown in SEQ ID NO:10), and a six-histidine tag (hexa-His-tag) coding sequence and a translation stop codon (TGA) were appended to the 3' end. The protein was then constructed into the pCAGGS vector via restriction endonuclease sites EcoRI and XhoI, and transfected into 293F cells for SARS-CoV-2 WT S-his protein expression. Cell culture medium containing the target protein was subjected to nickel ion affinity chromatography (HisTrap). TM Excel (GE) and gel filtration chromatography (Superose) TM After purification with 6% Increase 10 / 300GL (GE), a relatively pure target protein, SARS-CoV-2 WT S-his, was obtained. The SDS-PAGE analysis of the SARS-CoV-2 WT S-his protein revealed a size of approximately 200 kDa, as shown in the results. Figure 1 As shown.

[0070] Similarly, a six-histidine tag (hexa-His-tag) and a translation stop codon (TGA) were added to the 3' end of the SARS-CoV-2 WT S2 protein coding sequence (as shown in SEQ ID NO:12). Using restriction endonuclease sites EcoRI and XhoI, this sequence was constructed into the pFastBac1 vector (sequence shown in SEQ ID NO:13). The site-specific Tn7 transposon was used to transpose the foreign gene constructed on the pFastBac plasmid into the bacmid in *E. coli* DH10Bac, generating a recombinant bacmid. The bacmid was transfected into sf9 cells, producing a recombinant baculovirus capable of expressing the target gene. Viral titer was confirmed using a plaque assay. The confirmed viral titer was used to infect sf9 cells for large-scale viral amplification. The P3 virus could be directly used for protein expression; the virus was used to infect Hi5 cells for SARS-CoV-2 WT S2-his protein expression. Cell culture medium containing the target protein was purified by nickel affinity chromatography (HisTrap™ excel (GE Healthcare)) and gel filtration chromatography (Superdex™ 200Increase 10 / 300GL column (GE Healthcare)) to obtain a relatively pure target protein. The Western blot analysis of the SARS-CoV-2 WT S2-his protein showed a size of approximately 60 kDa, as shown in the results. Figure 2 .

[0071] Example 2: Construction of an alpaca immune and antibody library

[0072] 200 μg of the SARS-CoV-2 WT S protein with a 6-histidine tag prepared in Example 1 was 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. Subsequent immunizations were performed every two weeks, with the S protein emulsified using MF59 water-soluble adjuvant. On day 12 after the fifth 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... 12-18cDNA 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.

[0073] 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.

[0074] Table 1. Reaction primers

[0075]

[0076] Example 3: Screening for specific nanobodies using phage display technology

[0077] 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.

[0078] 2×10 11The 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 S-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 S-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. VCSM13 helper phage was then added and cultured overnight. The culture was centrifuged the next day, and the phage supernatant was used for ELISA experiments (using SARS-CoV-2 WT S-his protein as the coating antigen). When OD... 450nM When 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 S102 is obtained through sequencing.

[0079] Table 2. Reaction Primers

[0080]

[0081] Example 4: Expression and purification of nanobody S102

[0082] To make the heavy chain variable region of S102 more complete, the coding sequence of QVQLQ (CAGGTGCAGCTGCAG, SEQ ID NO:22) was added to the 5' end of the core coding sequence of S102 obtained in Example 3, and the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT, SEQ ID NO:23) 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 S102 of this application. Then, a signal peptide (SED ID NO:20) 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) TMAfter purification using 75% increase 10 / 300 GL column (GE Healthcare), a relatively pure target protein was obtained. The target peak was determined by SDS-PAGE, and the results are as follows: Figure 3 The purified nanobody S102 was obtained.

[0083] Example 5: Detection of antibody binding ability to SARS-CoV-2 WT S2 using surface plasmon resonance technology

[0084] Surface plasmon resonance analysis was performed using a Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0085] SA chips (purchased from GE Healthcare) were used. The SARS-CoV-2 S2-biotin protein obtained in Example 1 was immobilized on the chip using the affinity between the SA chip and biotin. The S102 antibody 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), loading the protein one concentration at a time from low to high. Representative kinetic curves of the S102 nanobody binding to the SARS-CoV-2 S2 protein are shown below. Figure 4 As shown. The kinetics of the binding constant (ka), dissociation constant (kd), and equilibrium dissociation constant (K) of the nanobody S102 to SARS-CoV-2S2 are shown. D As shown in Table 3, these parameters were calculated using BIAevaluation software 8K (Biacore, Inc.). The results in Table 3 demonstrate that the nanobody S102 can bind to SARS-CoV-2 S2 with high affinity.

[0086] Table 3. Binding constant (ka), dissociation constant (kd), and equilibrium dissociation constant (K) of antibodies binding to SARS-CoV-2S2 protein D )

[0087]

[0088] Example 6: Packaging of the original SARS-CoV-2 strain and its variants, and related coronavirus pseudoviruses

[0089] 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 services were provided by Suzhou Genewiz), resulting in the SARS-CoV-2-WT-S-del18 gene, whose nucleotide sequence is shown in SEQ ID NO:21.

[0090] Similarly, the last 18 amino acids of the S protein of the SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa, and Delta, as well as related coronaviruses SARS-CoV, GX / P2V / 2017, GD / 1 / 2019, and RaTG13, were removed, and the remaining S protein sequences were synthesized to obtain the S-del18 genes of these strains; the gene sequences of the S protein of the above strains can be obtained from the public database NCBI.

[0091] 2) The S-del18 genes obtained in 1) were cloned into the pCAGGS vector to obtain the expression plasmid pCAGGS-S-del18.

[0092] The packaging steps for the pseudoviruses of the above strains are as follows:

[0093] 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.

[0094] b. Transfection: Take the expression plasmid of S protein with the last 18 amino acids removed from step 2) above, and transfect it with PEI at a ratio of 30 μg plasmid / 10 cm cell culture dish. 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.

[0095] 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.

[0096] 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 clean bench to remove cell debris, aliquot, and freeze at -80℃.

[0097] We obtained the original SARS-CoV-2 strain (SARS-CoV-2 WT) and its variants Alpha, Beta, Gamma, Kappa and Delta, as well as pseudoviruses of related coronaviruses SARS-CoV, GX / P2V / 2017, GD / 1 / 2019 and RaTG13.

[0098] Example 7: Detection of SARS-CoV-2 original strains and their variants, as well as related coronavirus pseudovirus infections, by nanobody S102.

[0099] The purified nanobody S102 obtained in Example 4 was serially diluted 5-fold to the 9th gradient. The diluted nanobody S102 was then mixed with 1.6 × 10⁻⁶ ppm of the nanobody. 4 TCID 50 The original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Kappa, and Delta obtained in Example 6, along with pseudoviruses of related coronaviruses SARS-CoV, GX / P2V / 2017, GD / 1 / 2019, and RaTG1, were mixed and incubated at 37°C for 1 hour. The mixture was then added to 96-well plates pre-seeded with Vero cells (ATCC CCL81). After incubation for 18–20 hours, the results were detected using a CQ1 Confocal Quantitative Image Cytometer (Yokogawa). The neutralizing capacity of the antibody against the aforementioned pseudoviruses, i.e., the half-maximal inhibitory concentration (IC50), was calculated based on the number of cells exhibiting GFP fluorescence. 50 ).

[0100] The results showed that the IC50 of the nanobody S102 against the original SARS-CoV-2 strain was [missing information]. 50 The concentration was 1.45 μg / ml, and the representative inhibition curve was as follows: Figure 5 As shown; IC50 values ​​for SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa, and Delta. 50 The results are shown in Table 4; IC50 values ​​for related coronaviruses SARS-CoV, GX / P2V / 2017, GD / 1 / 2019, and RaTG13. 50 The results are shown in Table 5, and the representative inhibition curves are shown below. Figure 6 As shown.

[0101] Table 4. Neutralizing effect of nanobody S102 on pseudoviruses of SARS-CoV-2 variant strains

[0102]

[0103] Table 5. Neutralizing effect of nanobody S102 on pseudoviruses of related coronaviruses

[0104]

[0105] Example 8: Detection of SARS-CoV-2 variant Delta live virus infection neutralized by nanobody S102

[0106] In this embodiment, the neutralizing effect of nanobody S102 on the Delta variant of SARS-CoV-2 was determined by a live virus neutralization assay based on the cytopathic effect (CPE). The specific steps are as follows:

[0107] The nanobody S102 was serially diluted 2-fold to the 11th gradient, with four replicate wells per gradient and 50 μL per well. Each dilution was then mixed with an equal volume of 100 TCID50. 50 The SARS-CoV-2 variant Delta was incubated at 37°C; after 1 hour, the mixture was added to suspended Vero cells and incubated at 37°C for another 3 days; cytopathic effects were observed and recorded; the IC50 of this nanobody in inhibiting live infection of the SARS-CoV-2 variant Delta was calculated using GraphPad Prism 7.0. 50 The experiment was conducted in a biosafety level 3 (BSL3) laboratory at the Chinese Center for Disease Control and Prevention.

[0108] The results showed that the S102 nanobody had an IC50 against the Delta live virus variant of the SARS-CoV-2 virus. 50 The value was 26.3 μg / ml, indicating that the nanobody S102 has a good inhibitory effect on the live virus of the SARS-CoV-2 variant strain.

[0109] In summary, the nanobody S102 could serve as a candidate antibody drug with high and neutral activity against the original strain of SARS-CoV-2 and its variants (e.g., Alpha, Beta, Gamma, Kappa, or Delta) and related coronaviruses (including SARS-CoV, GX / P2V / 2017, GD / 1 / 2019, and RaTG13) for the prevention, treatment, and / or detection of these viruses.

[0110] 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 S2, 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-2S2 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-2S2 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 S2 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 S2 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 S2 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 15, characterized in that, The parenteral preparations include transdermal preparations, ointments, plasters, topical liquids, and injectable preparations.

21. The pharmaceutical composition according to claim 15, 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 S2 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, GX / P2V / 2017, GD / 1 / 2019, or RaTG13.

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