Nanobodies against SARS-CoV-2, their preparation methods and applications

Abstract

The application relates to the technical field of biology, and discloses a nanobody targeting a receptor binding domain of a novel coronavirus SARS-CoV-2 as well as a preparation method and application thereof. The application also discloses biological materials, derivative antibodies and detection reagents related to the nanobody. The nanobody disclosed by the application mainly recognizes a receptor binding motif region combined with RBD. The nanobody comprises complementarity determining regions CDR1, CDR2 and CDR3, and can also comprise a framework region FR. The nanobody disclosed by the application can be combined with SARS-CoV-2 in a high-efficiency and specific manner, and the affinity can reach a picomolar level. Meanwhile, the nanobody provided by the application has good neutralization biological activity, and the recognition and combination mechanism is clear, so that excellent effects can be obtained in SARS-CoV-2 detection or diseases caused by SARS-CoV-2. The application can be applied to the fields of biology and medicine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a nanobody against the novel coronavirus SARS-CoV-2, its preparation method, and its uses. Background Technology

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) belongs to the beta-coronavirus family and is the pathogen of COVID-19, a novel coronavirus pneumonia disease discovered at the end of 2019 and causing global spread. Due to its high infectivity, rapid transmission, and high mortality rate, and the lack of a completely effective treatment, it has become one of the most important infectious diseases threatening global public health security. Therefore, the development of drugs, preventive vaccines, and related antibodies against this virus is particularly urgent.

[0003] Currently, vaccination is the primary method for preventing and controlling infectious diseases. Several vaccines against SARS-CoV-2 are available globally, including inactivated vaccines, subunit vaccines, viral vector vaccines, and nucleic acid vaccines. These vaccines have long development cycles and high production costs. The current low vaccination rate and frequent viral mutations keep us highly vigilant.

[0004] Neutralizing antibodies are a class of antibodies that can bind to viral epitopes, reducing viral infectivity by inhibiting viral invasion. Neutralizing antibodies are widely available; they can be obtained from convalescent plasma or by immunizing against different viral antigenic epitopes to obtain antibodies that recognize multiple epitopes. Their preparation processes are relatively mature, and the research and development cycle is short. Therefore, developing high-titer neutralizing antibodies against SARS-CoV-2 is also an effective means of preventing viral transmission and infection.

[0005] SARS-CoV-2 virus infection of the host depends on the recognition and binding of the receptor-binding domain (RBD) of its spike protein S to angiotensin-converting enzyme 2 (ACE2) on host epithelial cells, followed by fusion with the cell membrane, thus completing the infection process. Therefore, blocking the binding of the S protein to ACE2 is an effective preventive or therapeutic measure to interrupt viral infection.

[0006] In nature, some camels and sharks possess antibodies that lack the light chain and contain only the heavy chain (heavy chain antibodies). These antibodies have a variable region of approximately 12-15 kDa, can recognize and bind antigens with extremely high affinity, and are the smallest active antigen-binding fragments; therefore, they are also called nanobodies. Nanobodies have advantages such as small molecular weight, strong penetrability, ease of expression, ease of genetic modification, and ease of binding to multiple epitopes. Therefore, these antibodies can serve as candidates for effective neutralizing antibodies, and currently, there are no marketed nanobody drugs targeting SARS-CoV-2. Summary of the Invention

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In view of the shortcomings of the existing technology, this invention uses recombinant SARS-CoV-2 RBD protein expressed in vitro to immunize alpacas four times, then isolates peripheral blood lymphocytes and extracts total RNA from the cells, which is subsequently reverse transcribed into cDNA. Using this cDNA as a template, nanobody sequences are amplified with specific primers to construct a phage library. After three rounds of biological elimination screening, FSEC interaction screening, pseudovirus neutralizing activity verification, and high-resolution complex crystal analysis, nanobodies against the SARS-CoV-2 S protein RBD domain (RBD: Receptor-Binding Domain) are isolated and obtained.

[0009] This invention discloses an amino acid sequence of the above-mentioned nanobody and a method for its preparation. Figure 1 The nanobody exhibits a strong ability to specifically bind to the novel coronavirus SARS-CoV-2 (Kd < 1x10⁻¹). -12 With M) and clear binding epitope information, the application of the nanobody provides a wider range of means for the diagnosis and treatment of SARS-CoV-2, and addresses the problems in the prior art.

[0010] This invention provides a nanobody amino acid sequence (SA4) against the RBD domain of the SARS-CoV-2 S protein. The nanobody sequence includes three complementarity-determining regions, CDR1, CDR2, and CDR3, and its amino acid sequence is as follows:

[0011] 1) The amino acid sequence of SA4:

[0012] QVQLQESGGGLVQPGGSLRLSCAASGSFFEFGTVGWFRQAPGKQRELVSRITGNDHRYYADSVKGRFTISRDNDETTVYLQMDSLKPEDTAIYHCNILEGQRWSNYWGQGTQVTVS(SEQ ID NO:1)

[0013] 2) CDR1 sequence: GSFFEFGT (SEQ ID NO:2)

[0014] 3) CDR2 sequence: ITGNDHR (SEQ ID NO:3)

[0015] 4) CDR3 sequence: NILEGQRWSNY (SEQ ID NO:4)

[0016] The nanobody described in this invention may be an antibody containing one or more of the above-mentioned CDR1, CDR2 and CDR3, that is, the nanobody may contain CDR1, or contain CDR2, or contain CDR3, or contain CDR1 and CDR2, or contain CDR1 and CDR3, or contain CDR2 and CDR3, or contain CDR1, CDR2 and CDR3.

[0017] In one specific embodiment, the nanobody includes at least CDR1.

[0018] In one specific embodiment, the nanobody includes at least CDR2.

[0019] In one specific embodiment, the nanobody includes at least CDR3.

[0020] In this invention, the further included component is a frame region, which is any frame region capable of realizing the function of the nanobody described in this invention, including human or mouse-derived components. In this invention, the nanobody may include one or more of the following frame regions:

[0021] FR1 sequence: QVQLQESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 5);

[0022] FR2 sequence: VGWFRQAPGKQRELVSR (SEQ ID NO: 6);

[0023] FR3 sequence: YYADSVKGRFTISRDNDETTVYLQMDSLKPEDTAIYHC (SEQ ID NO: 7);

[0024] FR4 sequence: WGQGTQVTVS (SEQ ID NO:8).

[0025] In this invention, the nanobody can be in various antibody forms, including but not limited to complete antibodies, antibody fragments, human antibodies, humanized antibodies, and genetically modified antibodies, such as monoclonal antibodies, chimeric antibodies, or recombinant antibodies, as well as fragments of these antibodies, provided that they retain the properties described in this invention.

[0026] In another aspect, the present invention provides an isolated polynucleotide encoding the aforementioned antibody against the novel coronavirus SARS-CoV-2. The sequence of this polynucleotide is as follows:

[0027] CAGGGTGCAGCTGCAGGAGTCCGGCGGCGGACTGGTGCAGCCTGGAGGAAGCCTGAGACTGTCCTGCGCCGCCAGCGGCTCCTTCTTCGAGTTCGGCACAGTGGGCTGGTCAGGCAGGCCCCCGGCAAGCAGAGGGAGCTGGTTCCAGGATCACCGGCAACGATCACAGATACTA CGCCGATAGCGTGAAGGGCAGGTTCACCATCTCCAGGGACAACGACGAGACCACCGTGTACCTGCAGATGGACAGCCTGAAGCCTGAGGACACAGCCATCTACCACTGCAATATCCTGGAGGGCCAGAGGTGGTCCAATTACTGGGGCCAGGGCACCCAGGTGACAGTGTCC(SEQ ID NO:9).

[0028] In this invention, polypeptides with a certain degree of amino acid sequence identity with the nanobody are also within the scope of protection of this invention. This includes antibodies or antibody fragments (with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) that have 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more sequence identity with the nanobody and possess the function of the nanobody. Specifically, the nanobody is obtained by substituting, deleting, or adding one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3), or by adding one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) to the N-terminus and / or C-terminus, and is a polypeptide fragment having the function of the nanobody.

[0029] Another aspect of the present invention provides a construct containing the isolated polynucleotide.

[0030] In some embodiments of the present invention, the construct is constructed by inserting the isolated polynucleotide into the multiple cloning site of the expression vector. The expression vector in the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0031] Another aspect of the present invention provides an expression system for nanobodies, the expression system containing the exogenous polynucleotide integrated into the construct or genome. Any cell suitable for expression via the expression vector can serve as a host cell; for example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0032] In some embodiments of the present invention, the host cell is selected from one or more combinations of E. coli MC1061 cells, BL21(DE3) cells, Expi293 cells, Trichoplusia ni High Five cells and E. coli SS320 cells.

[0033] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and containing one or more nanobodies according to the above description.

[0034] The present invention also provides a detection reagent or detection kit containing one or more of the nanobodies as described above.

[0035] The present invention further provides biomaterials, derived antibodies, etc. related to the nanobody.

[0036] The nanobody described in this invention primarily recognizes and binds to the receptor-binding motif region (RBM) of the RBD.

[0037] Another aspect of the present invention provides a method for preparing the anti-SARS-CoV-2 nanobody, comprising the following steps: culturing the expression system of the anti-SARS-CoV-2 nanobody under suitable conditions for expressing the anti-SARS-CoV-2 nanobody, thereby expressing the anti-SARS-CoV-2 nanobody, and purifying and separating the anti-SARS-CoV-2 nanobody.

[0038] The present invention further provides a method for inhibiting the activity of the novel coronavirus SARS-CoV-2, comprising administering an effective amount of the nanobody or related biomaterial as described above, or a pharmaceutical composition containing the nanobody, to an individual in need of treatment.

[0039] The present invention further provides a method for preventing and / or treating disease caused by the novel coronavirus SARS-CoV-2, comprising administering an effective amount of the nanobody or related biomaterial as described above, or a pharmaceutical composition containing the nanobody, to an individual in need of treatment.

[0040] The present invention further provides a method for immunological testing and analysis for non-disease diagnosis and treatment purposes, comprising administering an effective amount of the nanobody or related biomaterial as described above, or a pharmaceutical composition containing the nanobody, to an individual in need of treatment.

[0041] The present invention further provides a method for antagonizing the SARS-CoV-2 RBD receptor, comprising administering an effective amount of the nanobody or related biomaterial as described above, or a pharmaceutical composition comprising the nanobody, to an individual in need of treatment.

[0042] The present invention further provides a method for using the nanobody in combination with other reagents or drugs, including administering an effective amount of the nanobody or its related biomaterial as described above, or a pharmaceutical composition containing the nanobody, to an individual in need of treatment.

[0043] The host cells used in this invention are all existing technologies and can be obtained directly through commercial channels. The culture media used in the culture are also various conventional culture media. Those skilled in the art can select suitable culture media based on experience and culture the cells under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using appropriate methods (such as temperature change or chemical induction), and the cells are cultured for a further period of time. The anti-SARS-CoV-2 antibodies in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified using various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0044] The beneficial effects of this invention are as follows:

[0045] (1) This invention isolates and identifies a nanobody (SA4) capable of efficiently and specifically neutralizing SARS-CoV-2, exhibiting highly specific recognition and binding ability to the SARS-CoV-2 viral S protein RBD, primarily recognizing and binding to the receptor-binding motif (RBM) of the RBD; the affinity can reach Kd < 1x10 -12 M, and has good virus neutralizing activity, with an IC50 of approximately 12.68 nM.

[0046] (2) Structural analysis of the SA4-RBD complex in high-resolution crystals showed that the binding site of the nanobody to RBD overlapped with the main binding site of ACE2 RBD, thereby competitively inhibiting the binding of RBD protein to ACE2, and the interaction mechanism was clear.

[0047] (3) The nanoantibodies described in this invention are not easy to aggregate, can withstand high temperature, strong acid and strong alkali, and have good structural and physicochemical stability.

[0048] (5) The nanobody described in this invention can be expressed efficiently in Escherichia coli with high expression efficiency, is easy to separate and purify, and has low cost.

[0049] Therefore, the nanobody recognition and binding mechanism described in this invention is clear, and it can achieve excellent results in the detection, screening, and diagnosis of SARS-CoV-2, or play an important role in the effective immune protection in the prevention and / or treatment of SARS-CoV-2 virus. It may have the following applications in the biological and medical fields: I) application in the preparation of SARS-CoV-2 inhibitors; II) application in the preparation of drugs for the prevention and / or treatment of diseases caused by SARS-CoV-2; III) application in immunological testing and analysis for non-disease diagnosis and treatment purposes or in the preparation of SARS-CoV-2 detection reagents or kits; IV) application in the preparation of SARS-CoV-2 RBD receptor antagonists; V) application in combination with other reagents or drugs.

[0050] Glossary:

[0051] Nanobody: The variable region VHH of a heavy chain antibody.

[0052] A "complementarity-determining region" or "CDR" refers to one of three hypervariable regions within the heavy chain or light chain variable region of an antibody molecule, forming an N-terminal antigen-binding facet complementary to the three-dimensional structure of the bound antigen. Starting from the N-terminus of the heavy or light chain, these complementarity-determining regions are designated as "CDR1," "CDR2," and "CDR3," respectively. CDRs are involved in antigen-antibody binding, and CDR3 contains a unique region specific for antigen-antibody binding. Therefore, an antigen-binding site may include six CDRs, comprising CDR regions from each of the V regions of the heavy and light chains.

[0053] The amino acid sequence complementarity-determining region CDR1 (complementary region CDR1) is the first variable region sequence of the nanobody; the amino acid sequence complementarity-determining region CDR2 (complementary region CDR2) is the second variable region sequence of the nanobody; and the amino acid sequence complementarity-determining region CDR3 (complementary region CDR3) is the third variable region sequence of the nanobody.

[0054] IC50: Half-inhibitory concentration, the concentration of a drug or inhibitor required to inhibit a specified biological process (or a component of that process, such as an enzyme, receptor, or cell) by half.

[0055] FSEC: Fluorescent molecular sieve, an experimental method for qualitative analysis of interactions between biomolecules.

[0056] Kd: dissociation constant, which reflects the affinity between substances; the smaller the value, the stronger the affinity. Attached Figure Description

[0057] Figure 1 The diagram shows the nanobody screening technology process; in the FSEC detection graph, the horizontal axis represents volume (mL) and the vertical axis represents FL 482 / 508 (mV).

[0058] Figure 2 The results show the purification results of the RBD antigen.

[0059] Figure 3 The results show the detection of alpaca mRNA extracted samples and the results of two rounds of PCR amplification of nanobody gene sequences.

[0060] Figure 4 This image shows nanobodies bound to RBD identified by ELISA. The letters on the horizontal axis (SA1, SA2, etc.) represent clone numbers; the vertical axis represents the ratio of the absorbance values ​​of the experimental group to those of the negative control group.

[0061] Figure 5 The results are shown as FSEC results for SA4-RBD. The horizontal axis represents the elution volume of the target protein (mL), and the vertical axis represents the absorbance of A482 or A508 (nm). NC indicates the FSEC results for fluorescently labeled RBD; SA4 indicates the FSEC results for SA4-RBD.

[0062] Figure 6 The results are shown as SA4 affinity assay results. 2 μg / mL biotin-labeled RBD was immobilized onto the SA sensor, equilibrated, and then placed in SA4 solutions of different concentrations to detect the BLI signal.

[0063] Figure 7 The results of the neutralization experiment are shown for SA4 and the positive control group (other nanobodies with neutralizing activity). SARS-CoV-2 pseudovirus was incubated with different concentrations of SA4 nanobodies, and then used to infect VeroE6-hACE2 cells. The infection rate of the cells was detected by flow cytometry. The horizontal axis represents the logarithm of the SA4 protein concentration, and the vertical axis represents the percentage of virus neutralization efficiency.

[0064] Figure 8The results of purification and crystallization of the RBD-SA4 complex are shown. The horizontal axis represents the volume (mL) of the target protein eluted, and the vertical axis represents the A280 absorbance value (mAU).

[0065] Figure 9 This diagram shows a structural comparison of the SA4 and ACE2 recognition epitopes. The main recognition epitopes of SA4 and ACE2 overlap, and are marked with dashed ellipses. Detailed Implementation

[0066] The present invention is further illustrated below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The processes, conditions, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0067] The present invention does not have any special restrictions on the source of the coding sequence; any gene synthesis method well known in the art can be used.

[0068] Example 1. Expression and purification of the RBD domain of the SARS-CoV-2 S protein

[0069] RBD (uniport: P0DTC2, amino acids 330-541) was constructed into the pFastBac1 vector. The reading frame coding sequences from the N-terminus to the C-terminus are: Honeybee Melittin secretion signal peptide (KFLVNVALVFMVVYISYIYAA) (SEQ ID NO:10), Gly-Ser linker sequence, RBD target protein sequence, Gly-Ser linker sequence, 3C protease cleavage site (LEVLFQGP) (SEQ ID NO:11), Gly-Ser linker sequence, Avi tag (GLNDIFEAQKIEWHE) (SEQ ID NO:12), Gly-Ser linker sequence, and 10×His tag.

[0070] RBD expression was performed in Trichoplusia ni High Five suspension cells via secretory expression. The supernatant was collected, filtered through a 0.22 μm filter, and then mixed with 20 mM imidazole and 3 mL of Ni-Smart beads (SA035100) packing material. The mixture was incubated at 4°C with stirring for 3 hours. The supernatant-packing material mixture was then added to a gravity column, the packing material was collected, and the mixture was washed with 10 column volumes of buffer A (150 mM NaCl, 20 mM Tris HCl, pH 8.0) containing 20 mM imidazole. The protein was then eluted with buffer A containing 300 mM imidazole.

[0071] RBD antigen sample preparation: The affinity-purified sample was added to 3C protease at a ratio of 100:1 and digested at 4°C for 16 hours. The protein was then further purified by molecular sieve separation (Superdex Increase 200 10 / 300GL). Figure 2 The portions containing the target protein are mixed together, concentrated to 2 mg / mL, and then aliquoted. The samples are used directly for immunization or flash-frozen in liquid nitrogen and stored at -80°C.

[0072] Biotinylation labeling of RBD: 0.8 mg / mL RBD was mixed with 22 μg / mL purified BirA (biotin ligase), 5 mM ATP, 10 mM magnesium acetate, and 44 μM biotin were added, and the mixture was incubated at 4 °C for 16 hours. The protein was then further purified using a molecular sieve (Superdex Increase 200 10 / 300GL). The fractions containing the target protein were mixed, aliquoted, flash-frozen in liquid nitrogen, and stored at -80 °C for phage display screening.

[0073] Example 2. Nanobody Screening

[0074] 2.1 Alpaca Immunization

[0075] Before immunization, the immunogen (2 mg / mL, 500 μL) prepared in Example 1 was mixed with GERBU adjuvant (GERBUAJUVANT P#3111) at a volume ratio of 1:1 to form an emulsion. The antigen-adjuvant emulsion was then subcutaneously injected at 10 points near the arch lymph nodes on the alpaca's neck. Booster immunizations were performed every two weeks, for a total of four immunization experiments. Before the first immunization, 3 mL of blood was collected and allowed to clot at room temperature for 2 hours. The blood was then centrifuged at 3000g for 5 minutes at room temperature, and the supernatant was collected as pre-immunization serum. Serum from the first to fourth immunizations was collected using the same method, and serum antibody titers were determined by ELISA. After the fourth immunization, serum antibody titers were higher than 10. 6 Collect 80 ml of blood in an EDTA-coated blood collection tube. Immediately invert the tube twice to inhibit clotting. Separate peripheral blood lymphocytes (PBLs) using Ficoll Plus 1.077 according to the manufacturer's instructions.

[0076] 2.2 Phage Library Construction

[0077] The PBLs from Example 2 were lysed using RNAsio Plus (TaKaRa) and RNA was extracted. Figure 3A). Then, cDNA was prepared using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). The VHH fragment was further obtained through two rounds of PCR. The primers for the first PCR step were CALL001 (5′-GTCCTGGCTGCTCTTCTACAAGG-3′) (SEQ ID NO.13) and CALL002 (5′-GGTACGTGCTGTTGAACTGTTCC-3′) (SEQ ID NO.14), amplifying the heavy chain variable regions of all immunoglobulins from the cDNA, including the VH (1000bp) variable region of ordinary antibodies and the VHH (700bp) variable region of heavy chain antibodies. Figure 3 B). Then, VH and VHH were separated by agarose gel electrophoresis. A 700bp product was excised from the gel and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme) according to the manufacturer's instructions. A second round of PCR was performed using this product as a template, amplifying the encoding gene of the nanobody using specific primers VHH-BspQI-F (5′-ATATGCTCTTCAAGTCAGGTGCAGCTGCAGGAGTCTGGRGGAGG-3′) (SEQ ID NO.15) and VHH-BspQI-R (5′-TATAGCTCTTCCTGCCGAGGAGACGGTGACCTGGGT-3′) (SEQ ID NO.16). Figure 3 C); These two primers bind to the FR1 and FR4 positions encoding the nanobody, respectively. The resulting fragments are digested with BspQI and ligated into the pDX-init vector. The constructed library is then electroporated into E. coli SS320 strain to obtain a phage display library.

[0078] 2.3 Phage Display

[0079] This invention involved three rounds of phage display. The first round used 96-well plates coated with 60 nM neutravidin protein. Purified phage particles were first incubated with 50 nM biotinylated RBD (from Example 1), then aliquoted at 100 μL / well into 96-well plates. After binding at room temperature, non-specifically bound phages were washed away, and the remaining phages were released by digestion with 0.25 mg / mL trypsin. The selected phages were used to infect E. coli SS320, amplified in vivo, purified in vitro, and then used for the second round of phage display. In the second round of demonstration, this invention used magnetic beads as the experimental medium. 12 μM MyOne Streptavidin C1 was first incubated with 50 nM biotinylated RBD, followed by incubation for 20 min with the amplified and collected phage solution. After washing, 5 μM non-biotinylated RBD was added for competitive binding, removing some phages with low affinity or rapid dissociation. Finally, the selected phages were digested with trypsin to release them. The selected phages were then used to infect E. coli SS320, amplified in vivo, purified in vitro, and then used for the third round of phage display (referring to the second round experimental procedure). After three rounds of screening, the selected VHH gene was cloned into the expression vector pSb_init. The target protein contains the pelb secretion signal peptide (MSKYLLPTAAAGLLLLAAQPAMA; SEQ ID NO.17) at the N-terminus and the Myc tag and 6xHis tag at the C-terminus. The gene plasmid was transformed into E. coli MC1061 (commercially available) for further screening of single clones.

[0080] 2.4 ELISA Screening

[0081] Forty-seven clones containing the VHH gene were selected. Figure 4 (SA1-SA12, SB1-SB12, SC1-SC12 and SD1-SD11) and a positive control clone from a human library ( Figure 4Cells were transferred from PC to 96-well plates and cultured at 37°C and 300 rpm for 5 h. Then, they were transferred at a 1:20 (v / v) ratio to 1 mL of TB medium containing 25 μg / mL chloramphenicol. After 2 h of culture, the temperature was lowered to 22°C and cultured for another 1.5 h. Then, 0.02% (w / v) arabinose was added, and the cells were induced for 17 h. Cells were collected by centrifugation at 3,220 g for 30 min, the supernatant was discarded, and 0.1 mL of TES buffer (20% (w / v) sucrose, 0.5 mM EDTA, 0.5 μg / mL lysozyme, 50 mM Tis-HCl, pH 8.0) was added to resuspend the cells. The cells were then vortexed at room temperature for 30 min, and 0.9 mL of TBS buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 1 mM MgCl2 was added. After mixing, the cells were centrifuged at 3,220 g for 30 min at 4°C. The supernatant containing VHH protein can be used directly for ELISA or FSEC detection.

[0082] The day before the ELISA, Maxi-Sorp 96-well plates (Cat. 442404, Thermo Fisher) were coated with Protein A and incubated at 4°C for 16 hours. The next day, the Protein A solution was discarded, and the 96-well plates were blocked with 0.5% (w / v) bovine serum albumin (BSA) dissolved in TBS. After incubation at room temperature for 30 minutes, the BSA solution was discarded, and the plates were washed three times with TBS. Then, 0.1 mL of anti-myc antibody diluted 1:2,000 was added, and the plates were incubated at room temperature for 20 minutes to allow the antibody to bind to Protein A. The anti-myc antibody solution was discarded, and the plates were washed three times with TBST (TBS supplemented with 0.05% (v / v) Tween 20). The prepared VHH protein with the Myc tag was added to the 96-well plates and incubated at room temperature for 20 minutes. The protein solution was discarded, and the plates were washed three times with TBST. Add 0.1 mL of 50 nM biotinylated RBD or MBP (the maltose-binding protein, as a negative control), incubate at room temperature for 20 min, discard the solution, and wash the 96-well plate three times with TBST. Add streptavidin conjugated with horseradish peroxidase (HRP) to each well (1:5,000, Cat S2438, Sigma). Incubate at room temperature for 20 min, and wash three more times with TBST. Add 0.1 mL of developing solution (51 mM Na2HPO4, 24 mM citric acid, 0.006% (v / v) H2O2, 0.1 mg / mL 3,3',5,5'-tetramethylbenzidine), incubate at room temperature, and measure the absorbance at 650 nm after color development. A positive clone with affinity is considered to have a ratio of absorbance between the experimental group and the negative control group higher than 1.5. Figure 4Of the 47 clones, 45 were positive (excluding B5 and D8), indicating that the nanobodies expressed by these 45 positive clones could bind to RBD. The remaining two clones (SB5 and SD8) with a ratio of less than 1.5 may have bound to RBD but with low affinity or did not express nanobodies.

[0083] 2.5 Fluorescent Molecular Screening - FSEC

[0084] Biotinylated RBDs were mixed with fluorescein-labeled streptavidin (Cat 16955, AAT Bioquest) at a 1:1 ratio to prepare fluorescently labeled RBDs, which were then stored on ice. The periplasmic extract (SA4, amino acid sequence as shown in SEQ ID NO.1) was mixed with the fluorescently labeled RBDs at a volume ratio of 1:1.5, and then subjected to fluorescence molecular sieve (FSEC) analysis (the fluorescently labeled RBDs served as a control (NC)). The final concentration of RBDs in the FSEC was 500 nM, and fluorescence at 482 / 508 nm was detected. Results Figure 5 As shown, SA4 can shift the peak of the fluorescently labeled RBD in molecular sieves.

[0085] Example 3: Purification of Nanobody

[0086] The gene encoding the nanobody was constructed into the pSb-init vector, which also has a Myc tag and a 6xHis tag at its C-terminus. The constructed plasmid was transformed into E. coli MC1061 for expression. The general procedure was as follows: Cells were cultured in TB medium (0.17M KH2PO4 and 0.72M K2HPO4, 1.2% (w / v) peptone, 2.4% (w / v) yeast extract, and 0.5% (v / v) glycerol) at 37°C and 220 rpm, containing 25 mg / L chloramphenicol. When the bacteria grew to an OD of approximately 0.5, the temperature was lowered to 22°C, and the cells were cultured for another 1.5. Then, 0.02% (w / v) arabinose was added to induce expression for 16 h. Collect cultured cells by centrifugation, resuspend each liter of cells in 20 mL of TES-high Buffer (0.5 M sucrose, 0.5 mM EDTA, and 0.2 M Tris, Tris-HCl pH 8.0), and incubate at 4 °C for 30 min. Then add 40 mL of ice water and stir at 4 °C for 1 h. Centrifuge at 10000 g at 4 °C for 30 min, collect the supernatant, and add NaCl and MgCl2 to a final concentration of 150 mM. Incubate the treated supernatant with 3 mL of Ni-Smart beads (SA035100) for 30 min to bind, then wash with buffer A containing 5 mM imidazole. Elute the target protein with buffer A containing 250 mM imidazole. The eluted target protein can be used directly for the next step of RBD-Nanobody complex purification, crystallization, or other physicochemical tests, or flash-frozen in liquid nitrogen and stored at -80 °C.

[0087] Example 4: Combining Affinity Detection

[0088] The binding kinetics of SA4 and RBD were detected using biomembrane interferometry (BLI) on an Octet RED96 instrument. To determine the binding affinity between RBD and SA4, biotinylated RBD was diluted to a final concentration of 2 μg / mL with PBS-T (1xPBS, 0.005% (v / v) Tween 20). The diluted protein was immobilized onto a streptavidin tag at 30°C, equilibrated in PBS-T for 120 s (baseline), and then associated with different concentrations of SA4 for 300 s. The sensor was subsequently immersed in PBS-T solution for dissociation. Data processing was performed using Data Analysis 10.0 software, fitted according to 1:1 stoichiometry. The biomembrane interferometry (BLI) results showed that the Kb of SA4 binding to RBD... D Value less than 1pM ( Figure 6 ).

[0089] Example 5: SARS-CoV-2 virus neutralization experiment

[0090] The pseudovirus neutralization experiment was conducted as follows: HEK293T cells were transfected with a vector encoding the viral envelope glycoprotein, the mouse leukemia virus core / packaging component (MLV Gag-pol), and a retroviral transfer vector containing green fluorescent protein (GFP). After 48 hours, the culture supernatant was filtered through a 0.45 μm filter to obtain pseudoviruses, which could be used for viral infection.

[0091] 50 mL of VeroE6 cells expressing human ACE2 protein (VeroE6-hACE2 cells) were seeded into 48-well plates and cultured until the cell density reached 102. 4 100 μL of the collected pseudovirus was added to each well. Before infection, SA4 and a neutralizing positive control (PC) were pre-incubated with the pseudovirus at 37°C for 1 h. Six h after infection, the medium was replaced with pseudovirus-free medium (Dulbecco's modified Eagle's medium-2% fetal calf serum). GFP expression levels in cells were detected by activated fluorometry as an indicator of infection efficiency. The results of the neutralization experiment are shown below. Figure 7 As shown, SA4 is active against SARS-CoV-2 pseudovirus, IC 50 =12.68nM.

[0092] Example 6: Preparation, crystallization, and structural analysis of the SA4-RBD complex.

[0093] To obtain the SA4-RBD complex, the prepared RBD and the nanobody SA4 (amino acid sequence shown in SEQ ID NO. 1) were mixed together at a molar ratio of 1:2 and incubated at 4°C for 2 hours. The complex was then separated using a molecular sieve (Superdex Increase 20010 / 300 GL column). The separated SA4-RBD complex was concentrated to approximately 10 mg / mL for crystallization experiments, and the results are shown below. Figure 8 As shown, by Figure 8 It can be seen that SA4 and RBD form a stable and homogeneous complex, with an elution volume of about 15 mL.

[0094] This invention employs a seated drop method for initial crystal screening, with crystals cultured at 16°C. Crystals were observed after 36 hours. Crystals used for diffraction were grown in a solution of 0.2 M ammonium sulfate, 0.1 M sodium acetate trihydrate, pH 4.6, and 25% w / v polyethylene glycol 4,000. Approximately 25% glycerol was added to the crystal growth conditions, and after about 120 seconds, the crystals were rapidly transferred and frozen in liquid nitrogen.

[0095] The diffraction data were collected using the Pilatus 6M detector at the BL18U1 beamline of the Shanghai Synchrotron Radiation Facility, with an X-ray beam of 50 x 50 μm and a wavelength of [wavelength missing]. Data were integrated using XDS software and expanded and merged using Aimless. Structural analysis was performed using the corresponding RBD and VHH regions of 6M0J and 6ZXN as search models, with molecular substitution performed using Phaser. The model was implemented using 2F in Coot. o -F c The graph was manually adjusted and refined using Phenix, and finally the structure was displayed using PyMol.

[0096] The crystal structure of the SA4-RBD complex was resolved to a resolution of [resolution value missing]. (Table 1). Crystallographic results show that SA4 binds to RBD in a 1:1 stoichiometric ratio. Structural alignment shows that the epitope recognized by SA coincides with the epitope recognized by ACE2. Figure 9 ).

[0097] Table 1. SA4-RBD Diffraction Data and Structural Parameters

[0098]

[0099]

[0100] a. Highest shell parameter

[0101] 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 preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features; without departing from the spirit and scope of the inventive concept, all changes and advantages that those skilled in the art can conceive of should be covered within the scope of the technical solutions claimed in the present invention. sequence list <110> Nanjing Jingzhun Biotechnology Co., Ltd. <120> Nanobodies against SARS-CoV-2, their preparation methods and applications <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 116 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Phe Phe Glu Phe Gly 20 25 30 Thr Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ser Arg Ile Thr Gly Asn Asp His Arg Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Glu Thr Thr Val Tyr Leu 65 70 75 80 Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr His Cys Asn 85 90 95 Ile Leu Glu Gly Gln Arg Trp Ser Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser 115 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Gly Ser Phe Phe Glu Phe Gly Thr 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <400> 3 Ile Thr Gly Asn Asp His Arg 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <400> 4 Asn Ile Leu Glu Gly Gln Arg Trp Ser Asn Tyr 1 5 10 <210> 5 <211> 25 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <400> 6 Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ser 1 5 10 15 Arg <210> 7 <211> 38 <212> PRT <213> Artificial Sequence <400> 7 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Asp Glu Thr Thr Val Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Ile Tyr His Cys 35 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <400> 8 Trp Gly Gln Gly Thr Gln Val Thr Val Ser 1 5 10 <210> 9 <211> 348 <212> DNA <213> Artificial Sequence <400> 9 caggtgcagc tgcaggagtc cggcggcgga ctggtgcagc ctggaggaag cctgagactg 60 tcctgcgccg ccagcggctc cttcttcgag ttcggcacag tgggctggtt caggcaggcc 120 cccggcaagc agagggagct ggtgtccagg atcaccggca acgatcacag atactacgcc 180 gatagcgtga agggcaggtt caccatctcc agggacaacg acgagaccac cgtgtacctg 240 cagatggaca gcctgaagcc tgaggacaca gccatctacc actgcaatat cctggagggc 300 cagaggtggt ccaattactg gggccagggc acccaggtga cagtgtcc 348 <210> 10 <211> 21 <212> PRT <213> Artificial Sequence <400> 10 Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile Ser 1 5 10 15 Tyr Ile Tyr Ala Ala 20 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <400> 11 Leu Glu Val Leu Phe Gln Gly Pro 1 5 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <400> 12 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 13 <211> twenty three <212> DNA <213> Artificial Sequence <400> 13 gtcctggctg ctcttctaca agg 23 <210> 14 <211> twenty three <212> DNA <213> Artificial Sequence <400> 14 ggtacgtgct gttgaactgt tcc 23 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <400> 15 atatgctctt caagtcaggt gcagctgcag gagtctggrg gagg 44 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <400> 16 tatagctctt cctgccgagg agacggtgac ctgggt 36 <210> 17 <211> twenty three <212> PRT <213> Artificial Sequence <400> 17 Met Ser Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu 1 5 10 15 Ala Ala Gln Pro Ala Met Ala 20

Claims

1. A nanobody against the novel coronavirus SARS-CoV-2, characterized in that, The amino acid sequence of the nanobody includes the following complementarity-determining regions CDR1, CDR2 and CDR3: CDR1 as shown in SEQ ID NO:2; CDR2 as shown in SEQ ID NO:3; and CDR3 as shown in SEQ ID NO:

4.

2. The nanobody according to claim 1, characterized in that, The nanobody includes the following framework regions FR1, FR2, FR3 and FR4: FR1 as shown in SEQ ID NO:5; FR2 as shown in SEQ ID NO:6; FR3 as shown in SEQ ID NO:7; and FR4 as shown in SEQ ID NO:

8.

3. The nanobody according to claim 2, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

1.

4. A polynucleotide encoding the isolated nanobody according to claim 1.

5. The polynucleotide according to claim 4, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO:

9.

6. A construct, characterized in that, It contains the polynucleotide as described in claim 4.

7. An expression system for nanobodies, said expression system comprising an exogenous polynucleotide as described in claim 4 or 5 integrated into a construct or genome according to claim 6.

8. A pharmaceutical composition, characterized in that, It comprises the nanobody according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

9. A detection reagent or detection kit, characterized in that, It contains nanobodies according to any one of claims 1 to 3.

10. The following uses of the nanobody according to any one of claims 1 to 3, or the polynucleotide according to claim 4 or 5, or the construct according to claim 6, or the expression system according to claim 7, or the pharmaceutical composition according to claim 8: I) Use in the preparation of drugs for the prevention and / or treatment of disease caused by the novel coronavirus SARS-CoV-2; II) Application in immunological testing and analysis for non-disease diagnosis and treatment purposes, or in the preparation of detection reagents or kits for the novel coronavirus SARS-CoV-2.

11. A method for preparing nanobodies according to any one of claims 1 to 3, characterized in that, The process includes the following steps: culturing the nanobody expression system under suitable conditions for expressing the nanobody, thereby expressing the nanobody, and purifying and separating the nanobody.

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