Llama-derived nanobody r67 and applications thereof

Abstract

The present application relates to a llama-derived nanobody and its application, in particular to a llama-derived nanobody R67 or an antigen-binding fragment thereof capable of binding to SARS-CoV-2 RBD 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 R67 of the present application can bind to SARS-CoV-2 RBD protein with high affinity, and can neutralize SARS-CoV-2 original strain and a series of variant strains with high neutralization activity, thereby inhibiting the infection. The nanobody R67 of the present application has great potential application value in the clinical treatment, prevention and / or detection of SARS-CoV-2 original strain and its variant strains and related coronavirus infection.

Description

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 202111363955.X, filed on November 17, 2021, entitled “An Alpaca-Derived Nanobody R67 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 R67 and its applications, and more specifically, to an alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2RBD, 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] 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.

[0005] 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.

[0006] 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

[0007] Invention objectives

[0008] The present application aims to provide a llama-derived nanobody or antigen-binding fragment thereof binding to SARS-CoV-2 RBD, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a preparation method thereof, a transformed cell, and a pharmaceutical composition comprising the same, and their use in the preparation of a drug for preventing or treating COVID-19. The llama-derived nanobody or antigen-binding fragment thereof of the present application is a high-titer nanobody with strong binding ability to the RBD protein of the original strain and its variant strains of SARS-CoV-2 and related coronaviruses, and can effectively inhibit the infection of the original strain and its series of variant strains of SARS-CoV-2 and related coronaviruses. The nanobody has the advantages of small molecular weight (~ 15 kDa), low immunogenicity, better solubility and stability, and longer CDR3 region, and can be administered by nebulization to reach the lungs directly and act faster, thus providing a potential treatment strategy for COVID-19 or other coronavirus infections.

[0009] Solution

[0010] To achieve the above-mentioned object, the present application provides the following technical solutions.

[0011] In a first aspect, the present application provides a llama-derived nanobody or antigen-binding fragment thereof binding to SARS-CoV-2 RBD, wherein the antibody comprises a heavy chain variable region comprising CDRs as follows:

[0012] a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 (i.e., GFTLDYYA),

[0013] a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2 (i.e., ISPSGTST),

[0014] and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 3 (i.e., AAASPSYYYCSHHEVEYDY).

[0015] In specific embodiments, the heavy chain variable region further comprises four framework regions FR1-4, which are arranged in order interlaced with the CDR1, CDR2 and CDR3.

[0016] In a preferred embodiment, the amino acid sequences of FR1-4 are as set forth in SEQ ID NO: 4 (i.e., QVQLQESGGGLVRPGGSLRLSCAAS), SEQ ID NO: 5 (i.e., IGWFRQAPGKEREGVSC), SEQ ID NO: 6 (i.e., NYADSVKGRFTISRDNAKNTVYLQIDSLKPEDTAIYYC), and SEQ ID NO: 7 (i.e., WGQGTQVTVSS), respectively.

[0017] In a preferred embodiment, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 8:

[0018] wherein the underlined portions are FR1-4, respectively, and the blacked portions are CDR1, CDR2, and CDR3 of the heavy chain variable region, respectively.

[0019] In a second aspect, the present application provides a polynucleotide encoding the llama-derived nanobody or antigen-binding fragment thereof according to the first aspect described above.

[0020] Further, the polynucleotide is DNA or mRNA.

[0021] Further, the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 9:

[0022] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCGGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTCCTAGTGGTACGAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATAGACAGCCTGAAACCTGAGGACACAGCCATTTATTACTGTGCAGCAGCTTCTCCCTCATATTACTACTGTTCACATCATGAGGTTGAGTATGACTACTGGGGCCAGGGGACCCAGGTGACCGTGAGCTCT.

[0023] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide according to the second aspect described above.

[0024] 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.

[0025] Fourthly, the present invention provides an expression vector comprising the nucleic acid construct as described in the third aspect above.

[0026] 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.

[0027] 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-2RBD 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.

[0028] Preferably, the pharmaceutical composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation.

[0029] More preferably, the nasal spray is selected from aerosols, sprays, and powders.

[0030] More preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0031] More preferably, the tablet is a sublingual tablet;

[0032] More preferably, the granules are fine granules;

[0033] More preferably, the powder is a granule;

[0034] More preferably, the pills are small pills.

[0035] 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.

[0036] 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-2RBD 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.

[0037] Preferably, the novel coronavirus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

[0038] 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.

[0039] Preferably, the relevant coronavirus is SARS-CoV, RaTG13, RshSTT182, RsYN04, GX / P2V / 2017, or GD / 1 / 2019.

[0040] 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 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.

[0041] Preferably, the relevant coronavirus is SARS-CoV, RaTG13, RshSTT182, RsYN04, GX / P2V / 2017, or GD / 1 / 2019.

[0042] 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-2RBD as described in the first aspect above.

[0043] Preferably, the novel coronavirus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

[0044] 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.

[0045] Preferably, the relevant coronavirus is SARS-CoV, RaTG13, RshSTT182, RsYN04, GX / P2V / 2017, or GD / 1 / 2019.

[0046] 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.

[0047] Beneficial effects

[0048] This invention relates to the development of nanobody drugs targeting the novel coronavirus. Through immunizing alpacas with SARS-CoV-2 RBD and NTD proteins, constructing an antibody library, and screening for specific nanobodies using phage display technology, a nanobody with high affinity and specific binding to the SARS-CoV-2 RBD was identified and named nanobody R67. The inventors confirmed through surface plasmon resonance (SPR) testing that nanobody R67 can bind with high affinity to the original SARS-CoV-2 strain, its variants, and related coronaviruses' RBDs. 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 nanobody R67 is a high-affinity, high-neutralizing alpaca-derived nanobody targeting the original SARS-CoV-2 strain, its variants, and related coronaviruses.

[0049] 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

[0050] 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.

[0051] Figure 1This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2WT RBD-his protein described in Example 1 of this invention;

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

[0053] Figure 3 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2WT RBD-hFc protein described in Example 1 of this invention;

[0054] Figure 4 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the nanobody R67 described in Example 4 of the present invention;

[0055] Figure 5 This is a schematic diagram illustrating the effect of nanobody R67 in neutralizing the pseudovirus infection of VSV-SARS-CoV-2, as measured in Example 7 of this invention. In this diagram, A represents the effect of nanobody R67 in neutralizing the pseudovirus infection of the original SARS-CoV-2 WT strain; B represents the effect of nanobody R67 in neutralizing the pseudovirus infection of the SARS-CoV-2 variant strain Alpha (B.1.1.7); C represents the effect of nanobody R67 in neutralizing the pseudovirus infection of the SARS-CoV-2 variant strain Beta (B.1.351); D represents the effect of nanobody R67 in neutralizing the pseudovirus infection of the SARS-CoV-2 variant strain Gamma (P.1); E represents the effect of nanobody R67 in neutralizing the pseudovirus infection of the SARS-CoV-2 variant strain Kappa (B.1.617.1); and F represents the effect of nanobody R67 in neutralizing the pseudovirus infection of the SARS-CoV-2 variant strain Delta (B.1.617.2). 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-2WT NTD-his and SARS-CoV-2WT and their 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-2WT 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-2WT 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-2WT 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 for expression of SARS-CoV-2WT 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-2WT 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-2WT 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 200Increase 10 / 300GL column (GE Healthcare), a relatively pure target protein can be obtained. SDS-PAGE analysis of the SARS-CoV-2RBD-his protein revealed a size of approximately 30 kDa, as shown in the results. Figure 1 The SDS-PAGE analysis of the SARS-CoV-2 NTD-his protein revealed a size of approximately 60 kDa, as shown in the results. Figure 2 The SDS-PAGE analysis of the SARS-CoV-2 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 the SARS-CoV-2 WTRBD-hFc protein, RBD-hFc proteins of SARS-CoV-2 variant strains Alpha, Beta, Gamma, Kappa, and Delta, as well as related coronaviruses SARS-CoV, RaTG13, RshSTT182, RsYN04, GX / P2V / 2017, and GD / 1 / 2019, were prepared for surface plasmon resonance analysis. The 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 protein and SARS-CoV-2 WTNTD, each with a 6-histidine tag, were prepared and diluted to a final volume of 1 mL with PBS. After emulsification with 1 mL of complete Freund's adjuvant for 5 min, the mixture was 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-2WT 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-2WT 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 R67 is obtained through sequencing.

[0080] Table 2. Reaction Primers

[0081]

[0082] Example 4: Expression and purification of nanobody R67

[0083] To make the heavy chain variable region of R67 more complete, the coding sequence of QVQLQ (CAGGTGCAGCTGCAG, SED ID NO:28) was added to the 5' end of the core coding sequence of R67 obtained in Example 3, and the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT, SED ID NO:29) 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 R67 of this application. Then, a signal peptide (SED ID NO:21) was added before it, followed by the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA. The sequence was constructed into the pCAGGS vector through 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 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 4 The purified nanobody R67 was obtained.

[0084] Example 5: Detection of antibody binding ability to SARS-CoV-2 RBD 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 to immobilize the RBD-hFc proteins of the original SARS-CoV-2 strain and its variants (Alpha, Beta, Gamma, Kappa, and Delta) and related coronaviruses (SARS-CoV, RaTG13, RshSTT182, RsYN04, GX / P2V / 2017, and GD / 1 / 2019) obtained in Example 1 onto the chip, with an immobilization volume of approximately 100 RU. The R67 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. Kinetic curves of the R67 nanobody binding to the SARS-CoV-2 RBD protein were obtained. The binding constant (ka), dissociation constant (kd), and equilibrium dissociation constant (K) of nanobody R67 binding to the original SARS-CoV-2 strain and its variants (Alpha, Beta, Gamma, Kappa, and Delta) and related coronaviruses (SARS-CoV, RaTG13, RshSTT182, RsYN04, GX / P2V / 2017, and GD / 1 / 2019) of RBD. 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 R67 can bind with high affinity to the RBD proteins 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, RsYN04, GX / P2V / 2017, and GD / 1 / 2019.

[0087] Table 3. Binding constant (ka), dissociation constant (kd), and equilibrium dissociation constant (K) of antibody R67 with the original SARS-CoV-2 strain, its variants, and related coronaviruses' RBD protein. D )

[0088]

[0089]

[0090] Example 6: Packaging of original SARS-CoV-2 strains and variant pseudoviruses

[0091] 1) The last 18 amino acids of the S protein encoding the original SARS-CoV-2 strain (WT) and the variant strains (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), and Delta (B.1.617.2)) were removed, and the remaining S protein sequences were synthesized (synthesis services were provided by Genewiz Suzhou) to obtain the nucleotide sequences of the genes SARS-CoV-2-WT-S-del18, B.1.1.7-S-del18, B.1.351-S-del18, P.1-S-del18, B.1.617.1-S-del18, and B.1.617.2-S-del18, as shown in SEQ ID NO:22~27, respectively.

[0092] 2) The protein gene obtained in 1) was cloned into the pCAGGS vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18, pCAGGS-B.1.1.7-S-del18, pCAGGS-B.1.351-S-del18, pCAGGS-P.1-S-del18, pCAGGS-B.1.617.1-S-del18, and pCAGGS-B.1.617.2-S-del18.

[0093] The packaging steps for the original SARS-CoV-2 strain and its variant pseudoviruses are as follows:

[0094] 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.

[0095] b. Transfection: Take the 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.

[0096] 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.

[0097] 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℃.

[0098] Pseudoviruses were obtained from the original SARS-CoV-2 strain (SARS-CoV-2WT) and its variant strains (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), and Delta (B.1.617.2)).

[0099] Example 7: Detection of pseudovirus infection by neutralizing the original SARS-CoV-2 strain and its series of variant strains using nanobody R67

[0100] The purified nanobody R67 obtained in Example 4 was serially diluted 5-fold from 50 μg / mL to the 9th gradient (120 pg / mL). This diluted nanobody R67 was then mixed with 1.6 x 10⁻⁶ ppm of the solution. 4 TCID 50 The original SARS-CoV-2 strain and a series of variant 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 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 original SARS-CoV-2 strain and the series of variant pseudoviruses was calculated based on the number of cells exhibiting GFP fluorescence. The results are shown below. Figure 5 As shown in A to 5F, the statistical results are presented in Table 4.

[0101] Table 4. Neutralizing effect of nanobody R67 against the original SARS-CoV-2 virus and a series of variant pseudoviruses.

[0102]

[0103] *Among them, IC 50 (μg / mL) a This represents the half-maximal inhibitory concentration (WMC) of nanobody R67.

[0104] As shown in Table 4, the nanobody R67 can neutralize the original SARS-CoV-2 strain and a series of variant pseudoviruses with high neutralizing activity.

[0105] In summary, nanobody R67 can serve as a high- and neutralizing alpaca-derived nanobody targeting the original strain and variants of the novel coronavirus (SARS-CoV-2).

[0106] Example 8: Detection of live virus infection by neutralizing the original SARS-CoV-2 strain and its variants using nanobody R67

[0107] In this embodiment, the neutralizing effect of nanobody R67 on the original SARS-CoV-2 virus and its variants Alpha, Beta, and Delta live viruses was determined by a live virus neutralization assay based on the cytopathic effect (CPE). The specific steps are as follows:

[0108] The R67 nanobody was serially diluted 2-fold to the 11th gradient, with four replicates per gradient and 50 μL per well. Each dilution was then mixed with an equal volume of 100 TCID50. 50 The original SARS-CoV-2 strain and its variants Alpha, Beta, and Delta were 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 against live SARS-CoV-2 infection 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.

[0109] The results showed that the nanobody R67 had an IC50 value against the original SARS-CoV-2 virus and its variants Alpha, Beta, and Delta live viruses. 50 The values ​​were 0.10 μg / ml, 0.46 μg / ml, 0.11 μg / ml, and 0.11 μg / ml, respectively, indicating that the nanobody R67 has a good inhibitory effect on both the original and variant live strains of the novel coronavirus.

[0110] In summary, the broad-spectrum nanobody R67 can serve as a candidate antibody drug for the prevention, treatment, and detection of the original strain of SARS-CoV-2 and its variants, as well as related coronavirus infections.

[0111] 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 2, characterized in that, The amino acid sequences of FR1-4 are shown in 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 as described in 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; The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated formulations, and ointments; The parenteral preparations include transdermal preparations, ointments, plasters, topical liquids, and injectable preparations.

15. The pharmaceutical composition according to claim 14, characterized in that, The tablets are sublingual tablets; The granules are fine granules; The powder is a granule; The pills are small pills; The injectable formulation is a push-in formulation.

16. The use of an alpaca-derived nanobody or antigen-binding fragment thereof conjugated to SARS-CoV-2 RBD as described in any one of claims 1 to 4, the nucleotide sequence as described in any one of claims 5 to 7, the nucleic acid construct as described in claim 8 or 9, the expression vector as described in claim 10, the transformed cells as described in claim 11, or the pharmaceutical composition as described in any one of claims 12 to 15 in the preparation of a medicament for the prevention, treatment, or detection of SARS-CoV-2 and / or related coronavirus infections; wherein, The relevant coronaviruses are SARS-CoV, RaTG13, RshSTT182, RsYN04, GX / P2V / 2017, or GD / 1 / 2019.

17. The application according to claim 16, characterized in that, The SARS-CoV-2 mentioned refers to the original SARS-CoV-2 strain and / or a variant of SARS-CoV-2.

18. The application according to claim 17, characterized in that, The SARS-CoV-2 variant strains mentioned are the Alpha, Beta, Gamma, Kappa and / or Delta variant strains of SARS-CoV-2.

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