Neutralizing antibodies against sars-cov-2 and methods of making, formulations, and uses thereof

Abstract

The application relates to the field of biotechnology and discloses a neutralizing antibody against a novel coronavirus as well as a preparation method, a preparation and an application thereof. The nanobody comprises complementarity determining regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2-4. The nanobody is clear in a recognition and binding mechanism with RBD, can be combined with SARS-CoV-2 efficiently and specifically, and the affinity can reach a picomolar level (Kd<1x10 ‑12 M); the nanobody recognizes and binds RBD in a different way from ACE2, still has good neutralizing biological activity (IC50: 16.37 nM), can obtain excellent effects in SARS-CoV-2 detection or diseases caused by SARS-CoV-2, and has a wide application prospect in the fields of biology and medicine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a neutralizing antibody against the novel coronavirus, its preparation method, formulation, and uses, and particularly to a nanobody against the novel coronavirus SARS-CoV-2, its preparation method, formulation, pharmaceutical composition, detection kit, and uses. Background Technology

[0002] 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 viruses is an effective means of preventing viral transmission and infection.

[0003] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogen of the novel coronavirus infection (COVID-19) discovered at the end of 2019 and causing global spread. Its infection relies 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. Due to its high infectivity, rapid spread, 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.

[0004] Nanobodies possess 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; however, there are currently no marketed nanobody drugs targeting SARS-CoV-2. Summary of the Invention

[0005] To overcome the aforementioned shortcomings of existing technologies, this invention provides a nanobody against the novel coronavirus SARS-CoV-2, its preparation method, and its uses. The nanobody exhibits a strong ability to specifically bind to the novel coronavirus SARS-CoV-2 (Kd < 1 x 10⁻⁶). -12 With clear binding epitope information, the application of the nanobody provides a wider range of means for the diagnosis and treatment of SARS-CoV-2.

[0006] This invention provides an amino acid sequence (SC4) of a nanobody that resists the RBD (Receptor-Binding 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:

[0007] The amino acid sequence of SC4:

[0008] QVQLQESGGGLVQAGGSLRLSCAASGSDFSSSTMGWYRQAPGKQREFVAISSEGSTSYAGSVKGRFTISRDNAKNTVYLQMNSLEPEDTAVYYCNVVDRWYDYWGQGTQVTVS (SEQ ID NO. 1).

[0009] In this invention, the nanobody comprises three complementarity-determining regions, CDR1, CDR2, and CDR3, whose amino acid sequences are as follows:

[0010] CDR1 sequence: GSDFSSST (SEQ ID NO.2)

[0011] CDR2 sequence: ISSEGST (SEQ ID NO.3)

[0012] CDR3 sequence: NVVDRWYDY (SEQ ID NO.4).

[0013] 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.

[0014] In one specific embodiment, the nanobody includes at least CDR1.

[0015] In one specific embodiment, the nanobody includes at least CDR2.

[0016] In one specific embodiment, the nanobody includes at least CDR3.

[0017] 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:

[0018] FR1 sequence: QVQLQESGGGLVQAGGSLRLSCAAS (SEQ ID NO.5);

[0019] FR2 sequence: MGWYRQAPGKQREFVA (SEQ ID NO. 6);

[0020] FR3 sequence: SYAGSVKGRFTISRDNAKNTVYLQMNSLEPEDTAVYYC (SEQ ID NO.7);

[0021] FR4 sequence: WGQGTQVTVS (SEQ ID NO.8).

[0022] The nanobody described in this invention mainly recognizes the receptor-binding motif region (RBM) that binds to the RBD.

[0023] This invention further provides biomaterials and derived antibodies related to the nanobody. One aspect of this invention provides an isolated polynucleotide encoding the antibody against the novel coronavirus SARS-CoV-2. The nucleic acid sequence is as follows:

[0024] CAGGTGCAGGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGGCTGGAGGAAGCCTGAGACTGTCCTGTGCCGCCAGCGGCAGCGACTTCAGCTCTCCACCATGGGCTGGTACAGACAGGCCCCGGCAAGCAGAGAGAGTTCGTGGCCATCAGCTCCGAGGGCAGCACCTCC TACGCCGGCTCCGTGAAGGGCAGGTTCACAATCAGCAGAGATAACGCCAAGAACACAGTGTACCTGCGATGAATAGCCTGGAGCCTGAGGACACCGCCGTGTACTACTGCAATGTGGTGGACAGATGGTACGACTACTGGGGCCAGGGCACCCAGGTGACAGTGAGC (SEQ ID NO.9).

[0025] Another aspect of the present invention provides a construct containing the isolated polynucleotide.

[0026] 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.

[0027] 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.

[0028] In some embodiments of the present invention, the host cell is selected from one or more combinations of E. coli MC1061 cells, Trichoplusia niHigh Five cells, BL21(DE3) cells, Expi293 cells and E. coli SS320 cells.

[0029] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and containing one or more nanobodies according to the above description.

[0030] The present invention also provides a detection reagent or detection kit containing one or more of the nanobodies as described above.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) that have 70%, 75%, 80%, 85%, 90%, 95%, 99%, 98%, or 99% or more, and possess the function of the nanobody. Specifically, this includes polypeptide fragments that have the function of the nanobody, 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.

[0039] 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.

[0040] The beneficial effects of this invention are as follows:

[0041] (1) In this invention, lemurs were immunized four times with SARS-CoV-2 RBD protein expressed in vitro. Peripheral blood lymphocytes were then isolated and total RNA was extracted from the cells, subsequently reverse transcribed into cDNA. Using this cDNA as a template, nanobody sequences were 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 that recognize and bind to the RBD domain of the SARS-CoV-2 S protein were isolated and obtained. Figure 1 ).

[0042] (2) The nanobody (SC4) isolated and identified by this invention can efficiently neutralize SARS-CoV-2. Its recognition and binding mode to RBD is different from that of ACE2, but it has a highly specific recognition and binding ability to the RBD of the SARS-CoV-2 virus S protein; the affinity can reach Kd<1x10 -12 M, and has virus-neutralizing activity, with an IC50 of approximately 16.37 nM.

[0043] (3) Structural analysis of the RBD-SC4 complex high-resolution crystal showed that the binding site of the nanobody to RBD is different from that of ACE2 RBD binding site, and the interaction mechanism is clear. It can be developed into a conjugate antibody with multi-epitope recognition, thereby enhancing the neutralizing activity against the virus and avoiding or reducing the escape phenomenon of antiviral.

[0044] (4) 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.

[0045] (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.

[0046] (6) The nanobody described in this invention can play an important role in the prevention, screening, diagnosis and / or treatment of SARS-CoV-2 virus and may have the following applications: 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.

[0047] Glossary:

[0048] Nanobody: The variable region VHH of a heavy chain antibody.

[0049] 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.

[0050] 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.

[0051] FSEC: Fluorescent molecular sieve, an experimental technique for qualitative analysis of interactions between biomolecules.

[0052] Kd: Dissociation constant, which reflects the strength of the affinity between substances; the smaller the value, the stronger the affinity. Attached Figure Description

[0053] Figure 1The 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).

[0054] Figure 2 The results show the purification results of the RBD antigen.

[0055] Figure 3 The image shows the detection of alpaca mRNA extracted samples and the nanobody gene sequence amplified by two rounds of PCR.

[0056] 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.

[0057] Figure 5 The results are shown as FSEC results for SC4-RBD. The x-axis represents the elution volume of the target protein (mL), and the y-axis represents the absorbance of A482 or A508 (nm). NC indicates FSEC results for fluorescently labeled RBD; SC4 indicates FSEC results for SC4-RBD.

[0058] Figure 6 The results are shown as SC4 affinity assay results. 2 μg / mL biotin-labeled RBD was immobilized onto the SC sensor, equilibrated, and then placed in SC4 solutions of different concentrations to detect the BLI signal.

[0059] Figure 7 The results of the neutralization experiment are shown for SC4 and the positive control group (other nanobodies with neutralizing activity). SARS-CoV-2 pseudovirus was incubated with different concentrations of nanobodies before infecting VeroE6-hACE2 cells, and the infection rate was detected by flow cytometry. The x-axis represents the logarithm of SC4 protein concentration, and the y-axis represents the percentage of virus neutralization efficiency.

[0060] Figure 8 The results of purification and crystallization of the SC4-RBD 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).

[0061] Figure 9 This diagram shows a structural comparison of the SC4-recognized RBD epitopes and the ACE2-recognized epitopes. The different epitopes of the SC4 and ACE2-recognized RBDs are marked with dashed ellipses. Detailed Implementation

[0062] 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.

[0063] 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.

[0064] Example 1. Expression and purification of the RBD domain of the SARS-CoV-2 S protein

[0065] RBD (uniport: P0DTC2, amino acids 330-541) was constructed into the pFastBac1 vector. The reading frame coding sequences from N-terminus to 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 (LEVLFQG P) (SEQ ID NO.11), Gly-Ser linker sequence, Avi tag (GLNDIFEAQKIEWHE) (SEQ ID NO.12), Gly-Ser linker sequence, and 10×His tag.

[0066] 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 TrisHCl, pH 8.0) containing 20 mM imidazole. The protein was then eluted with buffer A containing 300 mM imidazole.

[0067] 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 separated and purified using a molecular sieve (Superdex Increase 200 10 / 300 GL). 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.

[0068] 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 / 300 GL). The fractions containing the target protein were mixed together, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C for phage display screening.

[0069] Example 2. Nanobody Screening

[0070] 2.1 Alpaca Immunization

[0071] 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 centrifuged at 3000 g 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.

[0072] 2.2 Phage Library Construction

[0073] The PBLs in Case 2.1 were performed using RNAsio Plus (TaKaRa) lysis, 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 700 bp product was excised from the gel and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme) according to the manufacturer's instructions. The second round of PCR used this product as a template, employing the specific primer VHH-BspQI-F (5′-ATATGCTCTTCAAGTCAGGTGCAGCTGCAGGAGTCTGGRGGAGG-3′,

[0074] The encoding gene of the nanobody was amplified by 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 obtained fragments were digested with BspQI and ligated into the pDX-init vector. The constructed library was electroporated into E. coli SS320 strain to obtain the phage display library.

[0075] 2.3 Phage Display

[0076] 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 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 μL of MyOne Streptavidin C1 was first incubated with 50 nM biotinylated RBD, followed by incubation of the amplified and collected phage solution for 20 min. 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, and purified in vitro before being 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.

[0077] 2.4 ELISA Screening

[0078] Forty-seven clones containing the VHH gene were selected: A1~A12, B1~B12, C1~C12, and D1~D11 (corresponding to, respectively). Figure 4 (SA1~SA12, SB1~SB12, SC1~SC12 and SD1~SD11) and a positive control clone from a human library ( Figure 4The culture medium (PC) was transferred to 96-well plates and incubated at 37°C and 300 rpm for 5 h. Then, it was transferred at a ratio of 1:20 (v / v) to 1 mL of TB medium containing 25 μg / mL chloramphenicol. After 2 h of incubation, the temperature was lowered to 22°C and incubated for another 1.5 h. Then, 0.02% (w / v) arabinose was added, and the mixture was induced for 17 h. Centrifuge at 3,220 g for 30 min to collect cells, discard the supernatant, add 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), resuspend the cells, and vortex at room temperature for 30 min. Add 0.9 mL of TBS buffer containing 1 mM MgCl2 (150 mM NaCl, 20 mM Tris-HCl pH 7.4), mix well, and centrifuge at 3,220 g at 4°C for 30 min. The supernatant containing VHH protein can be used directly for ELISA or FSEC detection.

[0079] The day before the ELISA, Maxi-Sorp 96-well plates (Cat. 442404, ThermoFisher) were coated with Protein A and incubated at 4°C for 16 h. 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 min, 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 min 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 min. 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 4 Of the 47 clones, 45 were positive (excluding B5 and D8), indicating that the nanobodies expressed by these 45 positive clones can bind to RBD. The remaining two clones (B5 and D8) with a ratio of less than 1.5 may have bound to RBD but with low affinity or did not express nanobodies.

[0080] 2.5 Fluorescent Molecular Screening - FSEC

[0081] 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 (SC4, 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 fluorescent 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, SC4 can shift the peak of the fluorescently labeled RBD in molecular sieves.

[0082] Example 3: Purification of Nanobodies

[0083] 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 containing 25 mg / L chloramphenicol (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. 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-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 150 mM NaCl and 2 mM MgCl2 to a final concentration. 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 Mimidazole. 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.

[0084] Example 4: Combining Affinity Detection

[0085] The binding kinetics of SC4 and RBD were detected using biomembrane interferometry (BLI) with an Octet RED96 instrument. To determine the binding affinity between RBD and SC4, 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 SC4 for 300 s. The sensor was then 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 Kt of SC4 binding to RBD... D Value less than 1 pM ( Figure 6 ).

[0086] Example 5: SARS-CoV-2 virus neutralization experiment

[0087] 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 h, the culture supernatant was filtered through a 0.45 µm filter to obtain pseudoviruses, which could be used for viral infection.

[0088] 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, SC4 and neutralizing positive controls (PC1, PC2, and PC3) were pre-incubated with the pseudovirus at 37°C for 1 h. After 6 h of infection, the medium was replaced with pseudovirus-free medium (Dulbecco's modified Eagle's medium-2% fetal calfserum). 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, SC4 is active against SARS-CoV-2 pseudovirus, IC 50 =16.37nM.

[0089] Example 6: Preparation, crystallization and structural analysis of SC4-RBD complex

[0090] To obtain the SC4-RBD complex, the prepared RBD and nanobody SC4 (amino acid sequence shown in SEQ ID NO. 1) were mixed together at a molar ratio of 1:1.5 and incubated at 4°C for 2 h. The complex was then separated using a molecular sieve (Superdex Increase 200 10 / 300 GL column). The separated RBD-SC4 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 SC4 and RBD form a stable and homogeneous complex, with an elution volume of about 15 mL.

[0091] 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 0.2 M Potassium phosphate dibasic solution and 20% w / v Polyethylene glycol 3,350. Glycerol was added to the crystal growth conditions to a final concentration of approximately 25%, and after approximately 120 seconds, the crystals were rapidly transferred and frozen in liquid nitrogen.

[0092] 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 0.97915 Å. The 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 modeled using 2F in Coot. o -F c The graph was manually adjusted and refined using Phenix, and finally the structure was displayed using PyMol.

[0093] The crystal structure of the SC4-RBD complex was resolved to a resolution of 3.0 Å (Table 1). The crystal results show that SC4 and RBD bind in a 1:1 stoichiometric ratio. Structural alignment reveals that the epitope recognized by SC4 does not overlap with the epitope recognized by ACE2. Figure 9 ).

[0094] Table 1 SC4-RBD Diffraction Data and Structural Parameters

[0095]

[0096] a. Highest shell parameter

[0097] 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> Neutralizing antibodies against the novel coronavirus, their preparation methods, formulations, and uses <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 113 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asp Phe Ser Ser Ser 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Ile Ser Ser Glu Gly Ser Thr Ser Tyr Ala Gly Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val 85 90 95 Val Asp Arg Trp Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Gly Ser Asp Phe Ser Ser Ser Thr 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <400> 3 Ile Ser Ser Glu Gly Ser Thr 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 Asn Val Val Asp Arg Trp Tyr Asp Tyr 1 5 <210> 5 <211> 25 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <400> 6 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val Ala 1 5 10 15 <210> 7 <211> 38 <212> PRT <213> Artificial Sequence <400> 7 Ser Tyr Ala Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr 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> 339 <212> DNA <213> Artificial Sequence <400> 9 caggtgcagc tgcaggagag cggcggcgga ctggtgcagg ctggaggaag cctgagactg 60 tcctgtgccg ccagcggcag cgacttcagc tcctccacca tgggctggta cagacaggcc 120 cccggcaagc agagagagtt cgtggccatc agctccgagg gcagcacctc ctacgccggc 180 tccgtgaagg gcaggttcac aatcagcaga gataacgcca agaacacagt gtacctgcag 240 atgaatagcc tggagcctga ggacaccgcc gtgtactact gcaatgtggt ggacagatgg 300 tacgactact ggggccaggg cacccaggtg acagtgagc 339 <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 <2十一> 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 It should be noted that there seems to be an error in "2十一" in line 31, which is likely a misrepresentation and should probably be "211". The above translation is based on the best understanding of the text with this correction.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 one or more of 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 the construct or genome according to claim 6.

8. A pharmaceutical composition, characterized in that, It comprises one or more of the nanobodies 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 one or more of the 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 medicines 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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