Neutralizing antibodies against sars-cov-2 and uses thereof

Abstract

This invention discloses neutralizing antibodies against SARS-CoV-2 or their antigen-binding portions and their applications. The neutralizing antibody against SARS-CoV-2 or its antigen-binding moiety contains a heavy chain variable region named VH and a light chain variable region named VL. Both VH and VL consist of a determinant complementary region and a framework region. The determinant complementary regions of both VH and VL consist of CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 of VH is shown in positions 26-33 of SEQ ID No. 3; the amino acid sequence of CDR2 of VH is shown in positions 51-57 of SEQ ID No. 3; the amino acid sequence of CDR3 of VH is shown in positions 96-106 of SEQ ID No. 3; the amino acid sequence of CDR1 of VL is shown in positions 27-32 of SEQ ID No. 4; the amino acid sequence of CDR2 of VL is shown in positions 50-52 of SEQ ID No. 4; and the amino acid sequence of CDR3 of VL is shown in positions 89-99 of SEQ ID No. 4. This neutralizing antibody has highly efficient neutralizing activity and can broadly neutralize SARS-CoV-2 variants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to neutralizing antibodies against SARS-CoV-2 or their antigen-binding portions and their applications. Background Technology

[0002] The isolation and identification of monoclonal antibodies provides new strategies for the treatment and prevention of infectious diseases, and also offers effective antigenic targets for vaccine design. Monoclonal antibodies can not only rapidly and effectively block viral infection via Fab, but also enhance the body's immune response to viruses through Fc effector function. Antibody-mediated immune enhancement can also better control viral replication and clear infected cells, thereby reducing viral load. Furthermore, Fc modification can significantly prolong antibody half-life, thus reducing the dosage and frequency of use. Various techniques can be used for the preparation of monoclonal antibodies, including hybridoma technology, EBV transformation, phage display, flow cytometry, 10X Genomics and beacon, and the combined use of these techniques; samples can be obtained from immunized animals, infected individuals, and volunteers immunized with vaccines.

[0003] SARS-CoV-2, belonging to the subgenus Sarbecovirus of the family Coronaviridae, was first reported in late 2019. SARS-CoV-2 infection in humans can present as mild or asymptomatic, but can also lead to severe and critical illness. Its high transmissibility has led to a global pandemic. Existing vaccines are effective in preventing SARS-CoV-2 infection, but with the ongoing global pandemic, new mutant strains are constantly emerging, posing a significant challenge to vaccine efficacy. Studies show that mutations in the receptor binding domain (RBD), such as K417N / E484K / N501Y, can significantly reduce the neutralizing effect of post-immunization plasma. In particular, the emergence of the Omicron variant has caused the vast majority of immune plasma and monoclonal antibodies to lose their neutralizing activity. Therefore, the isolation and identification of broadly neutralizing antibodies (bNAbs) against SARS-CoV-2 can provide new methods for COVID-19 prevention and control, as well as vaccine design.

[0004] Currently, several monoclonal antibodies against SARS-CoV-2 have been approved for emergency use, but they require combination therapy. More than twenty antibodies have entered the clinical evaluation stage. The efficacy evaluation of most antibodies is limited to wild-type SARS-CoV-2 strains or a few mutant strains. Mutations at key sites in these mutant strains can reduce the neutralizing activity of the antibodies, leading to partial or even complete drug resistance. Therefore, it is necessary to isolate and identify more anti-SARS-CoV-2 neutralizing antibodies, especially highly effective and broad-spectrum neutralizing antibodies, to provide more candidate antibodies for clinical applications. Furthermore, most existing anti-SARS-CoV-2 neutralizing antibodies are derived from infected individuals or immunized animals. Infected samples pose certain biosafety risks during handling, and animal-derived antibodies require humanization. Summary of the Invention

[0005] One of the objectives of this invention is to develop a highly efficient and broad-spectrum monoclonal antibody against SARS-CoV-2.

[0006] This invention provides a neutralizing antibody against SARS-CoV-2 or its antigen-binding portion thereof, wherein the neutralizing antibody or its antigen-binding portion contains a compound named V. H The heavy chain variable region and its name is V L The light chain variable region, the V H and V L Each is composed of a cluster complement region and a frame region; the V H and the V L The complementary regions of the determinant clusters are all composed of CDR1, CDR2, and CDR3;

[0007] The V H The amino acid sequence of CDR1 is shown in positions 26-33 of SEQ ID No. 3;

[0008] The V H The amino acid sequence of CDR2 is shown in positions 51-57 of SEQ ID No. 3;

[0009] The V H The amino acid sequence of CDR3 is shown in positions 96-106 of SEQ ID No. 3;

[0010] The V L The amino acid sequence of CDR1 is shown in positions 27-32 of SEQ ID No. 4;

[0011] The V L The amino acid sequence of CDR2 is shown in positions 50-52 of SEQ ID No. 4;

[0012] The V LThe amino acid sequence of CDR3 is shown in positions 89-99 of SEQ ID No. 4.

[0013] For example, V H The amino acid sequence is shown in SEQ ID No. 3. For example, V L The amino acid sequence is shown in SEQ ID No. 4.

[0014] Optionally, according to the above-described neutralizing antibody or its antigen-binding portion, the neutralizing antibody is any of the following:

[0015] a) From the above V H And the V mentioned above L The resulting single-chain antibody;

[0016] b) A fusion antibody containing the single-chain antibody described in a);

[0017] c) Contains the V as described in claim 1 H and the V as described in claim 1 L Fab;

[0018] d) Contains the V as described in claim 1 H and the V as described in claim 1 L Complete antibody.

[0019] The present invention also provides biomaterials related to the above-described neutralizing antibodies or their antigen-binding portions, wherein the biomaterials are any of the following:

[0020] B1) Nucleic acid molecules that encode the aforementioned antibodies or their antigen-binding portions;

[0021] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) A recombinant vector containing the nucleic acid molecules described in B1);

[0023] B4) A recombinant vector containing the expression cassette described in B2);

[0024] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0025] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0026] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0027] B8) Recombinant microorganisms containing the recombinant vector described in B4);

[0028] B9) Cell lines containing the nucleic acid molecules described in B1);

[0029] B10) Cell lines containing the expression cassette described in B2);

[0030] B11) Cell lines containing the recombinant vector described in B3);

[0031] B12) contains the recombinant vector described in B4) cell lines.

[0032] The cell lines are, for example, 293T cells and / or 293F cells.

[0033] The antigen-binding portion may be selected from at least one of the following: Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, sc-Fv, and dimer.

[0034] The "Fab fragment" consists of a light chain and a heavy chain with a CH1 and variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0035] A “Fab” fragment contains a light chain and a portion or segment of a heavy chain, the portion or segment containing a VH domain and a CH1 domain, as well as a region between the CH1 and CH2 domains.

[0036] The “F(ab')2 segment” contains two light chains and two heavy chains, the heavy chains containing a portion of a constant region between the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains. The F(ab')2 segment is thus composed of two Fab' segments, which are linked together by disulfide bonds between the two heavy chains.

[0037] The “Fv segment” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0038] "Single-chain Fv" or "scFv" refers to an antibody fragment containing both the VH and VL domains of the antibody, where these domains exist as a single polypeptide chain. Typically, the Fv polypeptide also includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0039] "Diasome" refers to a small antibody fragment with two antigen-binding sites, wherein the fragment contains a heavy chain variable domain (VH) and a light chain variable domain (VL) linked to it within the same polypeptide chain (VH-VL or VL-VH). By using a linker too short to allow pairing between the two domains on the same chain, each domain is forced to pair with a complementary domain of the other chain, thereby creating two antigen-binding sites.

[0040] Optionally, based on the above-described biological material, the nucleic acid molecule described in B1) is a gene encoding the above-described neutralizing antibody or its antigen-binding portion.

[0041] Optionally, based on the above-described biological material, the gene is a DNA molecule as described in either A) or B):

[0042] A) The V H The encoded sequence of CDR1 is shown in bits 76-99 of SEQ ID No. 1, wherein V H The encoded sequence of CDR2 is shown in bits 151-171 of SEQ ID No. 1, wherein V H The encoded sequence of CDR3 is shown in bits 286-318 of SEQ ID No. 1; the V L The encoded sequence of CDR1 is shown in bits 79-96 of SEQ ID No. 2, wherein V L The CDR2 encoded sequence is shown in bits 148-156 of SEQ ID No. 2, wherein the V L The encoded sequence of CDR3 is shown in bits 265-297 of SEQ ID No. 2;

[0043] B) has more than 90% identity with the DNA molecule defined in A) and encodes the DNA of the neutralizing antibody or its antigen-binding portion.

[0044] For example, V H The encoded sequence is shown in SEQ ID No. 1. For example, V L The encoded sequence is shown in SEQ ID No. 2.

[0045] In this article, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0046] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0047] The present invention also provides products for treating or preventing SARS-CoV-2 infection, said products comprising the above-described neutralizing antibody or its antigen-binding portion or the above-described biological material.

[0048] The following applications of the above-mentioned neutralizing antibodies or their antigen-binding portions, the above-mentioned biological materials, or the above-mentioned products also fall within the scope of protection of this invention:

[0049] X1. Application in the preparation of drugs for treating SARS-CoV-2 infection;

[0050] X2. Application in the treatment of SARS-CoV-2 infection;

[0051] X3. Application in the preparation of drugs for the prevention of SARS-CoV-2 infection;

[0052] X4. Application in the prevention of SARS-CoV-2 infection.

[0053] This invention also provides a method for screening SARS-CoV-2 specific memory B cells, including...

[0054] (1) Using pseudoviruses of wild-type SARS-CoV-2 and SARS-CoV-2 mutant strains and biological plasma after immunization with inactivated COVID-19 vaccine, a virus neutralization test was conducted to screen for candidate samples for neutralizing antibody sorting. The SARS-CoV-2 mutant strains include Alpha, Beta v1, Beta v2, Beta v3, Gamma, Delta, Lambda, Kappa, Epsilon and Zeta. The candidate samples for neutralizing antibody sorting are the biological plasma that neutralized more than half of the pseudoviruses in the virus neutralization test and had a geometric mean of neutralization titer >30.

[0055] (2) Using the RBD and RBDsa proteins of SARS-CoV-2 as sorting antigens, SARS-CoV-2-specific single memory B cells were sorted from peripheral blood mononuclear cells of the candidate samples.

[0056] This invention also provides a method for preparing neutralizing antibodies against SARS-CoV-2, including...

[0057] (1) Using pseudoviruses of wild-type SARS-CoV-2 and SARS-CoV-2 mutant strains and biological plasma after immunization with inactivated COVID-19 vaccine, a virus neutralization test was conducted to screen for candidate samples for neutralizing antibody sorting. The SARS-CoV-2 mutant strains include Alpha, Beta v1, Beta v2, Beta v3, Gamma, Delta, Lambda, Kappa, Epsilon and Zeta. The candidate samples for neutralizing antibody sorting are the biological plasma that neutralized more than half of the pseudoviruses in the virus neutralization test and had a geometric mean of neutralization titer >30.

[0058] (2) Using the RBD and RBDsa proteins of SARS-CoV-2 as sorting antigens, SARS-CoV-2-specific single memory B cells were sorted from peripheral blood mononuclear cells of the candidate samples;

[0059] (3) Obtain the gene sequences of the heavy chain variable region and light chain variable region of the antibody through the single memory B cell;

[0060] (4) The neutralizing antibody is prepared by means of the gene sequences of the heavy chain variable region and the light chain variable region of the antibody.

[0061] Step (3) can specifically involve using the SARS-CoV-2-specific single memory B cell cDNA as a template, amplifying the heavy chain and light chain variable region genes of the antibody using a nested PCR system to obtain amplification products; screening the amplification products, with the screening criteria being that the lengths of the amplification products of the antibody's heavy chain variable region gene and the antibody's light chain variable region gene are both 300-400 bp; and sequencing the screened amplification products to obtain the gene sequences of the antibody's heavy chain variable region and light chain variable region.

[0062] Step (4) may specifically include: performing sequence analysis on the gene sequences of the heavy chain variable region and the light chain variable region of the antibody, antibody expression, antibody purification, antibody binding activity screening test and / or antibody neutralization test to prepare the neutralizing antibody.

[0063] The antibody expression, for example, involves linking the genes of the heavy and light chain variable regions of the antibody to an expression vector via homologous recombination and expressing the antibody in 293T and / or 293F cells.

[0064] The antibody purification process, for example, involves incubating the expression supernatant of 293T and / or 293F cells with 1 / 1000 of rProtein G microspheres at room temperature for 2 hours, followed by adding the mixture to a chromatography column for antibody purification.

[0065] The antibody binding activity screening assay, for example, uses wild-type RBD protein of SARS-CoV-2, wild-type spike protein, S1 protein of Beta mutant strain and / or S1 protein of Delta mutant strain to screen neutralizing antibodies with binding activity from purified antibodies.

[0066] The antibody neutralization test, for example, uses pseudoviruses of SARS-CoV-2 wild type, Alpha, Beta, and Delta to screen neutralizing antibodies with neutralizing activity from those with binding activity; these neutralizing antibodies are the neutralizing antibodies mentioned above.

[0067] Optionally, according to the screening method or the preparation method described above, the inactivated COVID-19 vaccine is BBIBP-CorV.

[0068] This invention utilizes RBD and RBDsa (K417T, E484K, N501Y) proteins to specifically sort individual memory B cells from volunteers immunized with an inactivated COVID-19 vaccine, obtaining paired antibody heavy and light chain genes, and screening to obtain monoclonal antibodies against SARS-CoV-2. These monoclonal antibodies exhibit highly efficient neutralizing activity and can broadly neutralize SARS-CoV-2 variants. The discovery of this monoclonal antibody not only provides an alternative drug for the prevention and treatment of SARS-CoV-2 infection but also offers a technical reference for the development of monoclonal antibody drugs against viral infections. Attached Figure Description

[0069] Figure 1A This is a schematic diagram of sample addition to a 96-well plate in Example 9.

[0070] Figure 1B The neutralization titer (ID) of the sample in Example 1 50 ) Detection statistics chart.

[0071] Figure 2 This is a sorting diagram for Example 1.

[0072] Figure 3 This is an electrophoresis diagram of the PCR products from nested PCR in Example 1.

[0073] Figure 4 The results of sequence analysis of the variable regions of the heavy and light chains in Example 1 are shown.

[0074] Figure 5 This is the Western Blot result of the antibody expression supernatant from Example 2.

[0075] Figure 6 This is the initial screening result of the binding activity of the antibody expression supernatant in Example 2.

[0076] Figure 7 This is the initial screening result of the neutralizing activity of the antibody expression supernatant in Example 2.

[0077] Figure 8 The SDS-PAGE results are for the purified proteins of the antibody in Examples 28-9D.

[0078] Figure 9 The results show the affinity of the antibody 38-9D in Examples 38-9D to the wild-type RBD protein.

[0079] Figure 10A The pseudovirus neutralizing activity (IC50) of the antibody in Example 3, Table 7.8-9D. 50 (μg / ml).

[0080] Figure 10B This is a statistical chart of the neutralizing activity of the antibody pseudovirus in Examples 38-9D.

[0081] Figure 11 The results of the live virus neutralization test for antibody in Example 48-9D are shown. Detailed Implementation

[0082] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0084] SARS-CoV-2 wild-type and mutant strains (Alpha, Beta v1, Beta v2, Beta v3, Gamma, Delta, Lambda, Kappa, Epsilon, Zeta, Eta, Iota v1, Iota v2, Mu, Omicron BA.1, and Omicron BA.2) pseudoviruses: self-made, references from [source missing].

[0085] MMWR Morb Mortal Wkly Rep.2021 Jan 22;70(3):95-99.

[0086] Cell.2021 Apr 29;184(9):2372-2383.e9.

[0087] Cell.2021 Aug 5;184(16):4220-4236.e13.

[0088] Microbiol Spectr.2021 Oct 31;9(2):e0078921.

[0089] Infect Genet Evol. 2021 Nov;95:105038.

[0090] Nature.2022 Feb;602(7898):664-670.

[0091] Nature. 2022 Mar 3. doi: 10.1038 / s41586-022-04594-4.

[0092] Live SARS-CoV-2 virus: wild type (IME-BJ01 strain, Genbank No. MT291831); Beta (CSTR: 16698.06.NPRC2.062100001); Delta (CSTR.16698.06.NPRC6.CCPM-BV-049-2105-6), Omicron (SARS-CoV-2strainOmicronCoV / human / CHN_CVRI-01 / 2022).

[0093] GM: DEME (Thermo, C11995500BT) + 10% FBS (Hyclone, SH30084) + 1% Bismuth Substance Antibody (GIBCO, 15140122).

[0094] HeLa-hACE2: A gift from Professor Ding Qiang of Tsinghua University, recorded in PLoS Pathog. 2021 Nov 8; 17(11): e1010053.).

[0095] Vero cells: (ATCC: CCL81)

[0096] Bright-Lite TM Test reagents: vazyme, DD1204.

[0097] RPMI 1640 medium: Thermo, C22400500BT (+10% FBS).

[0098] Cell staining buffer: PBS (biosharp) + 2% FBS.

[0099] Biotinylated SARS-CoV-2 RBD: acrobiosystems, SPD-C82E9.

[0100] Biotinylated RBDsa protein: acrobiosystems, SPD-C82E7.

[0101] Wild-type RBD protein of SARS-CoV-2: acrobiosystems, SPD-C52H1.

[0102] Wild-type spike protein of SARS-CoV-2: acrobiosystems, SPN-C52H9.

[0103] S1 protein of the SARS-CoV-2 Beta mutant strain: Sino Biological, 40591-V08H15.

[0104] The S1 protein of the Delta mutant strain of SARS-CoV-2: acrobiosystems, S1N-C52Hu.

[0105] Fluorescein-labeled antibodies: CD3-Pacific Blue (Biolegend, 300431), CD8a-Pacific Blue (Biolegend, 301023), CD14-Pacific Blue (Biolegend, 325616), CD19-FITC (Biolegend, 302206), CD27-PerCP-Cy5.5 (Biolegend, 356408), PE Streptavidin (Biolegend, 405203), APC Streptavidin (Biolegend, 405207)

[0106] Lysis buffer: 0.5×PBS, 10mM DTT (Invitrogen, 18064022), 10U RNase Inhibitor (NEB, M0307L).

[0107] The expression vectors CMV for the heavy and light chains were provided by Professor Zhang Linqi's laboratory at Tsinghua University and documented in Nature. 2020 Aug; 584(7819): 115-119.

[0108] Example 1: Preparation of anti-SARS-CoV-2 monoclonal antibody

[0109] (I) Plasma neutralization activity detection

[0110] 1. Sample: Plasma from volunteers who received two immunizations with the inactivated COVID-19 vaccine (BBIBP-CorV).

[0111] 2. Neutralization Activity Assay: The neutralizing titers of 28 samples against SARS-CoV-2 wild-type and mutant strains (Alpha, Betav1, Beta v2, Beta v3, Gamma, Delta, Lambda, Kappa, Epsilon, Zeta) pseudoviruses were determined. The method is as follows:

[0112] (1) Plasma samples (named No. 1-28 plasma) were heat-inactivated at 56℃ for 30 min and then briefly centrifuged to remove insoluble matter.

[0113] (2) In a 96-well cell culture plate, add 150 μl of GM (DEME + 10% FBS + 1% double antibody) to the cell control (column 1, i.e. C in Figure 1), add 100 μl of GM to the remaining wells, and add 30 μl of GM to wells 3A-3H and 8A-8H (as shown in Figure 1, 1-12 are columns 1-12, AH is row AH, for example, 3A is the treatment well in row A of column 3 in the 96-well plate).

[0114] (3) Take 22.5 μl of plasma No. 1 and add it to 3A-3B, plasma No. 2 and add it to 3C-3D, in sequence, that is, the initial plasma dilution is 1:10.

[0115] (4) Mix the plasma sample thoroughly, take 50 μl and transfer it into the next row of processing wells, and serially dilute it 3 times until the 7th or 12th row.

[0116] (5) Thaw the SARS-CoV-2 pseudovirus and dilute it to 4000 TCID with GM. 50 / ml, take 50μl (200TCID) 50 Add it to columns 2-12.

[0117] (6) Incubate at 37℃ for 1 hour.

[0118] (7) Digest HeLa-hACE2 cells, resuspend them in GM, and the cell concentration is 1.3 × 10⁻⁶. 5 per ml.

[0119] (8) Take 100 μl of the HeLa-hACE2 cell suspension obtained in step (7) (i.e., 1.3 × 10⁻⁶ cells per 100 μl solution). 4 (1 cell) is added to each processing well of the cell plate.

[0120] (9) Place the cell plate in an incubator at 37°C and 5% CO2 for 48 hours.

[0121] (10) Reading: After 48 hours, discard the cell culture medium, wash once with 200 μl PBS, and pat dry; add 100 μl Bright-Lite to each well. TMAfter the test reagents have been left at room temperature for 2 minutes, the plate is read using an ELISA reader.

[0122] (11) Calculate the neutralizing titer (ID) of the plasma. 50 The viral inhibition percentage (%) of plasma at each dilution = (VT) / (VC), where T is the ELISA reader reading of the plasma sample and C is the cell control. Figure 1A The first column of the image shows the ELISA reader reading, where V represents the virus control. Figure 1A The second column of the image shows the microplate reader reading; the ID is calculated using GraphPad Prism's log(inhibitor) vs. response--Variable slope. 50 .

[0123] 3. Results Analysis: See Figure 1B After activity screening, the neutralizing titer (ID) was determined. 50 The geometric mean (GMT) of the samples was greater than 30, and the samples with more than half of the strains (>5) were neutralized (the titer was greater than the detection limit (10)). These were plasma samples 1, 2, 3, 4, 11, 17, and 25. These samples can be used as candidate samples for neutralizing antibody sorting. 50 The 50% inhibition dilution is represented by GMT, which is the geometric mean titer.

[0124] (II) Sorting of SARS-CoV-2-specific single memory B cells

[0125] (1) PBMC resuscitation: The frozen PBMCs obtained in step (I) above (approximately 1×10⁻⁶) of the candidate sample were revived. 7 The samples were rapidly revived in a 37°C water bath and resuspended in 10 ml of RPMI 1640 medium (+10% FBS) preheated to 37°C, and centrifuged at 400×g for 5 min.

[0126] (2) Labeling: Discard the culture medium, resuspend the cells in 10 ml of pre-chilled cell staining buffer, centrifuge at 400×g for 5 min, discard the supernatant, and resuspend the cells in 100 μl of cell staining buffer; add 0.2 μg of SARS-CoV-2 RBD or RBDsa protein to the cells, mix thoroughly, and incubate at 4℃ for 1 h; wash twice with 2 ml of pre-chilled cell staining buffer, and resuspend the cells in 95 μl of cell staining buffer; mix the fluorescently labeled antibodies (CD3-Pacifc Blue, CD8a-Pacific Blue, CD14-Pacific Blue, CD19-FITC, CD27-PerCP-Cy5.5, PE Streptavidin, APCStreptavidin) into the cell suspension, and incubate at 4℃ for 30 min; wash twice with 2 ml of pre-chilled cell staining buffer, and resuspend in 400 μl of cell staining buffer to obtain labeled PBMCs.

[0127] (3) Sorting: The labeled PBMCs were analyzed and sorted using a Sony MA900 Cell Sorter flow cytometer to separate CD3+ cells. - CD8 - CD14 - CD19 + CD27 + RBD + / RBDsa + B cells (see) Figure 2 These are antigen-specific memory B cells, with 3-15 cells obtained from each sample. The antigen-specific memory B cells were individually aliquoted into 96-well PCR plates containing 4 μl of lysis buffer (0.5×PBS, 10 mM DTT, 10 U RNase inhibitor).

[0128] (4) Freezing: Quickly place in dry ice and use directly for reverse transcription or move to -80°C freezer for freezing.

[0129] (II) Single B-cell gene cloning

[0130] 1. Reverse transcription

[0131] After thawing the aforementioned frozen antigen-specific memory B cells, reverse transcription was performed by adding reagents according to the instructions of the High Capacity cDNA Reverse Transcription Kit (Thermo, 4368813). The steps are as follows:

[0132] (1) Add to each well

[0133]

[0134] (2) Heat at 65℃ for 5 minutes, then place on ice for at least 2 minutes.

[0135] (3) Add 2 μl of 10X RT Buffer, 0.25 μl of RNase Inhibitor (40 U / μl), and 1 μl of MultiScribe. TM Reverse Transcriptase.

[0136] (4) Reaction conditions: 25℃ for 10 min, 37℃ for 120 min, then inactivation at 85℃ for 5 min.

[0137] 2. Nested PCR

[0138] The first round of PCR template uses the cDNA obtained by reverse transcription in step 1 above, and the second round of PCR template uses the product of the first round of PCR.

[0139] (1) Nested PCR of heavy chain variable region

[0140] The primers are shown in Table 1.

[0141] Table 1. Nested PCR primers for heavy chain variable regions

[0142]

[0143]

[0144] The first round of nested PCR reaction system for the antibody heavy chain gene was as follows: PrimerSTAR Max Premix (2×) 10 μl; Primer mix: 5′ primer 0.6 μl; 3′ Cγ CH1 0.6 μl; Template 5 μl; sterile distilled water 3.8 μl; total volume 20 μl.

[0145] PCR reaction conditions: pre-denaturation 98℃ for 2 min; 98℃ for 10 s, 56℃ for 5 s, 72℃ for 10 s, 35 cycles; extension 72℃ for 5 min.

[0146] The second round of nested PCR reaction system for the antibody heavy chain gene was as follows: PrimerSTAR Max Premix (2×) 10 μl; Primer mix: 5′ primer 0.6 μl; Primer mix: 3′ primer 0.6 μl; Template 2 μl; Sterile distilled water 6.8 μl; Total volume 20 μl.

[0147] PCR reaction conditions: pre-denaturation 98℃ for 2 min; 98℃ for 10 s, 58℃ for 5 s, 72℃ for 10 s, 35 cycles; extension 72℃ for 5 min.

[0148] (2) Nested PCR of the variable region of the light chain (kappa)

[0149] The primers are shown in Table 2.

[0150] Table 2. Nested PCR primers for the light chain (kappa) variable region

[0151]

[0152]

[0153] The first round of nested PCR reaction system for the antibody light chain (kappa) gene was as follows: PrimerSTAR Max Premix (2×) 10 μl; Primer mix: 5′ primer 0.6 μl; 3′ Cκ 543 0.6 μl; Template 5 μl; sterile distilled water 3.8 μl; total volume 20 μl.

[0154] PCR reaction conditions: pre-denaturation 98℃ for 2 min; 98℃ for 10 s, 56℃ for 5 s, 72℃ for 10 s, 35 cycles; final extension 72℃ for 5 min.

[0155] The second round of nested PCR reaction system for the light chain (kappa) gene of the antibody was as follows: PrimerSTAR Max Premix (2×) 10 μl; Primer mix: 5′ primer 0.6 μl; Primer mix: 3′ primer 0.6 μl; Template 2 μl; sterile distilled water 6.8 μl; total volume 20 μl.

[0156] PCR reaction conditions: pre-denaturation 98℃ for 2 min; 98℃ for 10 s, 58℃ for 5 s, 72℃ for 10 s, 35 cycles; extension 72℃ for 5 min.

[0157] (3) Lambda variable region nested PCR

[0158] The primers are shown in Figure 3.

[0159] Table 3. Lambda Variable Region Nested PCR Primers

[0160]

[0161]

[0162] The first-round nested PCR reaction system for the light chain Lambda gene of the antibody was as follows: PrimerSTARMax Premix (2×) 10 μl; Primer mix: 5′ primer 0.6 μl; 3′ Cλ 0.6 μl; Template 5 μl; sterile distilled water 3.8 μl; total volume 20 μl.

[0163] PCR reaction conditions: pre-denaturation 98℃ for 2 min; 98℃ for 10 s, 58℃ for 5 s, 72℃ for 10 s, 35 cycles; final extension 72℃ for 5 min.

[0164] The second round of nested PCR reaction system for the light chain (Lambda) gene of the antibody was as follows: PrimerSTAR Max Premix (2×) 10 μl; Primer mix: 5′ primer 0.6 μl; 3′ Cl 0.6 μl; Template 2 μl; sterile distilled water 6.8 μl; total volume 20 μl.

[0165] PCR reaction conditions: pre-denaturation 98℃ for 2 min; 98℃ for 10 s, 60℃ for 5 s, 72℃ for 10 s, 35 cycles; extension 72℃ for 5 min.

[0166] Results analysis: The PCR products from the last round were detected using 1% agarose gel electrophoresis (see electrophoresis results below). Figure 3 Each column of electrophoresis results represents the electrophoresis results of PCR products from nested heavy chain variable region PCR and nested light chain variable region PCR performed on the same memory B cell. PCR products with positive fragments at 300bp-400bp and with paired heavy and light chains (i.e., both nested heavy chain variable region PCR products and nested light chain variable region PCR products show positive fragments at 300bp-400bp) are then gel recovered.

[0167] 3. Gel recovery and purification of PCR products

[0168] PCR products were purified by gel extraction using a standard agarose gel DNA recovery kit (Tiangen, DP209).

[0169] The specific method is as follows.

[0170] (1) Column equilibration step: Add 500 μl of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0171] (2) Cut a single target DNA band from the agarose gel and place it in a clean centrifuge tube, and weigh it.

[0172] (3) Add an equal volume of PC solution to the gel block (if the gel weight is 0.1g, its volume can be regarded as 100μl, then add 100μl of PC solution), place in a 50℃ water bath for about 10min, and gently turn the centrifuge tube up and down continuously to ensure that the gel block is fully dissolved.

[0173] (4) Add the solution obtained in the previous step to an adsorption column CB2 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column CB2 into the collection tube.

[0174] (5) Add 600 μl of washing solution PW (with anhydrous ethanol added) to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube; repeat the operation steps.

[0175] (6) Place the adsorption column CB2 into the collection tube and centrifuge at 12,000 rpm for 2 min to remove as much of the washing solution as possible. Place the adsorption column at room temperature for a few minutes to dry completely.

[0176] (7) Place the adsorption column CB2 into a clean centrifuge tube, add an appropriate amount of preheated pure water dropwise to the middle of the adsorption membrane, and let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min and collect the DNA solution; add the collected DNA solution dropwise onto the adsorption membrane, and let it stand at room temperature for 2 min; centrifuge at 12,000 rpm for 2 min and collect the DNA solution again to improve the DNA recovery.

[0177] The concentration of purified DNA fragments was determined using Nanodrop.

[0178] (III) Variable Region Gene Linking Vectors

[0179] 1. The expression vectors CMV for the heavy and light chains were digested with restriction endonucleases to obtain the digestion products. The reaction system is as follows:

[0180]

[0181]

[0182] Reaction conditions: 37℃ for 20 min.

[0183] After enzyme digestion, agarose gel electrophoresis and gel recovery purification were performed, using the same method as above.

[0184] 2. The PCR gel-recovered products of the heavy and light chains were subjected to homologous recombination with the double-digested expression vector to obtain ligation products. The reaction system is as follows:

[0185]

[0186] Mix the above reaction solutions thoroughly and perform the ligation reaction at 50°C for 20 min to obtain CMV-H, CMV-k and CMV-λ ligation products.

[0187] 3. The ligation product transforms competent cells DH5α

[0188] (1) Melt competent cells DH5α (100 μl) on an ice bath, add CMV-H, CMV-k or CMV-λ ligation products (10 μl) respectively, mix gently, and let stand on ice for 30 min.

[0189] (2) Place it in a water bath at 42°C for 45 seconds to heat shock, then quickly transfer it to an ice bath and let it stand for 2 minutes.

[0190] (3) Add 500 μl of antibiotic-free sterile culture medium LB to the centrifuge tube, mix well, and then revive in a shaker at 37°C and 180 rpm for 1 hour.

[0191] (4) Spread the revived bacterial solution evenly onto LB medium containing ampicillin. After the bacterial solution is absorbed, invert the plate and place it in a 37°C incubator overnight.

[0192] 4. Vaccination

[0193] Pick a single colony growing on the LB plate and add it to 3 ml of ampicillin-resistant liquid LB medium. Incubate at 37°C and 220 rpm for 6-7 hours on a shaker. Pick 2 colonies from each plate.

[0194] 5. Identification of positive clones

[0195] The bacterial culture was sent to a biotechnology company for sequencing identification. The obtained sequences were then subjected to Ig BLAST alignment analysis to screen for successfully paired antibody sequences. The specific criteria for successful pairing were: 1. The antibody sequence contains a CDR region; 2. The antibody sequence does not contain a terminator; 3. The antibody sequence is within the reading frame.

[0196] The results are as follows Figure 4As shown, 18 pairs of antibody sequences were obtained. Figure A shows the percentage of germline genes in the heavy chain variable region, the light chain kappa chain variable region, and the light chain lambda chain variable region, respectively. Figure B shows the lengths of the heavy chain complementarity-determining region (CDR) and the light chain CDR, respectively. Figure C shows the mutation statistics for the heavy chain and light chain variable regions. The heavy chain variable regions originated from 9 different germline genes, with IGHV3-53 and IGHV3-30 having the highest percentages. Compared to germline genes, the mutation frequency of the heavy chain variable regions ranged from 0% to 5.4%. Analysis of the CDR3 loop length showed that the heavy chain variable regions ranged from 11 aa to 21 aa. The light chains are predominantly kappa chains (13 in total) and lambda chains (5 in total). The V gene sequences are mainly IGKV4-1 and IGLV3-21. Compared with germline genes, the mutation frequency of the variable regions of the light chains is between 0.4% and 4.9%. Analysis of the length of the complementary determining region (CDR) CDR3 loop shows that the variable regions of the light chains are 8aa-12aa long.

[0197] The heavy chain variable region coding gene sequence of monoclonal antibody 8-9D is shown in SEQ ID No. 1, where positions 76-99 are the CDR1 coding sequence, positions 151-171 are the CDR2 coding sequence, and positions 286-318 are the CDR3 coding sequence. The amino acid sequence of the expressed heavy chain variable region is shown in SEQ ID No. 3, where positions 26-33 are the CDR1 sequence, positions 51-57 are the CDR2 sequence, and positions 96-106 are the CDR3 sequence.

[0198] The gene sequence encoding the light chain variable region of monoclonal antibody 8-9D is shown in SEQ ID No. 2, wherein positions 79-96 are the coding sequence for CDR1, positions 148-156 are the coding sequence for CDR2, and positions 265-297 are the coding sequence for CDR3. The amino acid sequence of the expressed light chain variable region is shown in SEQ ID No. 4, wherein positions 27-32 are the CDR1 sequence, positions 50-52 are the CDR2 sequence, and positions 89-99 are the CDR3 sequence.

[0199] The DNA fragments of the antibody sequences described above can also be prepared artificially.

[0200] Example 2: Preparation of Monoclonal Antibodies

[0201] (I) Initial screening of antibody activity

[0202] Antibody expression in the 1.293T cell system

[0203] Transfect 293T cells with the successfully paired antibody heavy chain and light chain gene expression vectors:

[0204] (1) 24 hours before transfection, 5×10⁵ plants were seeded in each well of a 12-well plate. 5 293 T cells.

[0205] (2) On the day of transfection, observe the degree of cell confluence, which should be 70%-80%.

[0206] (3) Take 1 μg of plasmid DNA extracted by the plasmid mini-prep kit (Tiangen, DP103) and dilute it with 60 μl of Opti-MEM medium.

[0207] (4) Take 4 μl 2000 Reagent was diluted with 60 μl of Opti-MEM medium.

[0208] (5) Mix the diluted DNA and Mix 2000 at a volume ratio of 1:1 to obtain a mixed solution and incubate at room temperature for 5 minutes.

[0209] (6) Take 120 μl of the mixture and gently add it to 293T cells, and incubate at 37°C with 5% CO2.

[0210] (7) After culturing for 48 h, collect cells and supernatant, centrifuge at 12,000 rpm for 2 min, and collect cell culture supernatant and cell pellet. Store cell culture supernatant at -20℃. Add 1 ml of pre-chilled PBS to a centrifuge tube containing cell pellet, mix the cell pellet by pipetting, centrifuge at 12,000 rpm for 2 min, discard the supernatant, wash twice, add 100 μl of cell lysis buffer (Promega, E1531), incubate on ice for 10 min, centrifuge at 12,000 rpm for 5 min, and collect the supernatant. Perform Western blotting (using horseradish enzyme-labeled goat anti-human IgG (H+L), ZSGB-BIO, ZB-2304) on the cell culture supernatant and the supernatant collected after lysis pelleting.

[0211] Some results are as follows Figure 5 As shown, most antibodies can be expressed and secreted into the cell supernatant.

[0212] 2. Initial screening of antibody binding activity

[0213] (1) Coating: The wild-type RBD protein, wild-type spike protein, S1 protein of Beta mutant and S1 protein of Delta mutant of SARS-CoV-2 were diluted with PBS to 0.5 μg / ml, and 100 μl / well was added to each well of a 96-well ELISA plate and coated overnight at 4°C.

[0214] (2) Blocking: Discard the coating solution, add 200 μl of blocking solution (PBST + 3% BSA) to each well, and block at 37°C for 1 h.

[0215] (3) Washing: Discard the blocking solution and wash 3 times with PBST.

[0216] (4) Samples: Dilute the supernatant of the aforementioned antibody expression 2-fold and 10-fold, and add 100 μl / well to the 96-well ELISA plate that has completed step (3). In addition, add an equal amount of negative control (supernatant of untransfected 293T cells) to the 96-well ELISA plate that has completed step (3). Incubate at 37°C for 2 h.

[0217] (5) Washing: Discard the sample and wash with PBST 5 times.

[0218] (6) Secondary antibody: Dilute goat anti-human IgG antibody with diluent (PBST+1% BSA) at a ratio of 1:50000, add 100 μl to each well, and incubate at 37°C for 30 min.

[0219] (7) Washing: Discard the sample and wash 5 times with PBST.

[0220] (8) Color development: Add 100 μl of color development solution to each well and incubate at room temperature in the dark for 15 min.

[0221] (9) Termination: Add 50 μl of termination solution to each well.

[0222] (10) Reading: Use an ELISA reader to read the absorbance at 450nm and 630nm. The result is the 450nm reading minus the 630nm reading.

[0223] (11) Results Analysis: A value at a 2-fold dilution greater than 2.5 times that of the negative control and greater than 0.1 is considered to have binding activity. Samples that can bind to one or more of the four antigens are considered to have binding activity (see [link to relevant documentation]). Figure 6 The results showed that antibody 8-9D had strong binding activity against all four antigens.

[0224] 3. Initial screening of antibody neutralizing activity

[0225] (1) Screening antibodies with binding activity for initial neutralization activity.

[0226] (2) In a 96-well cell culture plate, as shown in Figure 1, add 150 μl of GM (DMEM + 10% FBS + 1% double antibody) to the cell control (column 1), add 130 μl of GM to the sample initial dilution wells (columns 3 and 8), and add 100 μl of GM to the remaining wells.

[0227] (3) Add 22.5 μl of antibody expression supernatant to the initial dilution well of the sample, mix thoroughly, and transfer 50 μl to the next row of processing wells. The initial dilution is 10, and the samples are serially diluted 3 times.

[0228] (4) Thaw the SARS-CoV-2 pseudoviruses (wild-type, Alpha, Beta, Delta pseudoviruses) and dilute them with GM to 4000 TCID. 50 / ml, take 50μl (200TCID) 50 Add to columns 2-12.

[0229] (5) Incubate at 37℃ for 1 hour.

[0230] (6) Digest HeLa-hACE2 cells, resuspend them in GM, and the cell concentration is 1.3 × 10⁻⁶. 5 per ml.

[0231] (7) Take 100 μl of cell suspension (i.e., per 1.3 × 10⁻⁶ cells). 4 Add one cell to the cell plate and incubate at 37°C with 5% CO2 for 48 hours.

[0232] (8) Reading: After 48 hours, discard the cell culture medium, wash once with 200 μl PBS, and pat dry; add 100 μl Bright-Lite to each well. TM After the test reagents have been left at room temperature for 2 minutes, the plate is read using an ELISA reader.

[0233] (9) Calculate the 50% inhibitory titer (ID) of the antibody. 50 Inhibition rate (i.e., neutralization percentage) = (VT) / (VC) × 100%, where T is the ELISA reader reading for the antibody to be tested, C is the ELISA reader reading for the cell control, and V is the ELISA reader reading for the virus control; ID is calculated using GraphPad Prism's log(inhibitor) vs. response -- Variable slope. 50 .

[0234] (10) Results analysis: Among them, 8-9D can neutralize 4 test pseudostrains (see Figure 7 At the initial dilution, an inhibition rate of more than 50% against the virus is considered to indicate neutralization.

[0235] (II) Antibody Mass Expression and Purification

[0236] Antibody expression in the 1.293F cell system

[0237] (1) One day before transfection, 293F cells in the logarithmic growth phase with a viability greater than 90% were transfected at 1.5 × 10⁻⁶ mg / L. 6Inoculate the culture medium at a density of 125 ml per 1 ml and incubate in a constant temperature shaker at 37°C, 5% CO2, and 150 rpm (125 ml shake flask).

[0238] (2) On the day of transfection, samples were taken to count cell density and viability. Cell density was 2 × 10⁻⁶. 6 -3×10 6 Cells / ml, viability greater than 90%. Adjust cell density to 2×10⁶. 6 Cells / ml, with each vial containing 30ml of cell solution.

[0239] (3) Preparation of transfection solution: Dilute 60 μg of DNA extracted using the endotoxin-free plasmid large-scale extraction kit (Tiangen, DP117-T) with 150 mM NaCl solution (heavy chain to light chain molar ratio 1:1) to a total volume of 0.75 ml and mix gently; dilute 120 μl of Sinofection transfection reagent with 150 mM NaCl to a total volume of 0.75 ml and mix gently; let the diluted DNA and transfection reagent stand separately for about 5 minutes, then mix gently to a total volume of 1.5 ml, and then let stand at room temperature for 10 minutes to obtain the transfection solution.

[0240] (4) Add the transfection solution dropwise to the cell solution obtained in step (2), and gently shake the culture flask while adding the solution. After shaking well, put it back on the shaker to continue culturing.

[0241] (5) Add 2% (v / v) SMS 293-SUPI feed solution (SinoBiological, M293-SUPI) 24 hours after transfection, and then add feed solution (v / v) every 48 hours thereafter. Collect samples 5 days after transfection.

[0242] 2. Affinity chromatography purification of antibodies

[0243] (1) Buffer preparation: The water and buffer were filtered through a 0.45 μm filter membrane, and the binding / washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0), elution buffer (0.1 M glycine, pH 3.0), and neutralization solution (1 M Tris-HCl, pH 8.5) were prepared.

[0244] (2) Sample preparation: Cell supernatant was filtered with a 0.45μm filter membrane to reduce impurities, improve protein purification efficiency and prevent column clogging.

[0245] (3) Sample purification

[0246] 1) Mix rProtein G Beads (Solarbio, R8300) with the cell supernatant obtained in “(2) Sample Preparation” and incubate on a shaker for 2 hours.

[0247] 2) Load an appropriate amount of rProtein G Beads into the chromatography column and equilibrate the column with a binding buffer of 5 column volumes.

[0248] 3) Add the incubated cell supernatant to the equilibrated rProtein G Beads and collect the eluent; add the eluent back to the chromatography column and collect the eluent.

[0249] 4) Wash with 10 column volumes of washing buffer to remove non-specifically adsorbed proteins.

[0250] 5) Use 10 column volumes of elution buffer to collect the eluent, which is the target protein component.

[0251] 6) Use 3 column volumes of binding buffer and 5 column volumes of deionized water to equilibrate the packing material in sequence, and finally equilibrate with 5 column volumes of 20% ethanol. Then store it in an equal volume of 20% ethanol at 4°C.

[0252] 7) Protein quantification of the purified antibody was performed using Nanodrop. The quantification results showed that the concentration of the purified antibody was 1 mg / ml-2 mg / ml. The concentration of antibody 8-9D was 1.46 mg / ml.

[0253] 8) The purified antibody was detected by SDS-PAGE electrophoresis. Results for days 8-9 are shown in the attached table. Figure 8 As shown in the figure, SDS-PAGE results indicate that the protein purity after affinity chromatography is high, suggesting that the target antibody can be used for in vitro activity analysis.

[0254] Example 3: Analysis of antibody 8-9D affinity and neutralizing activity

[0255] (I) Affinity analysis of monoclonal antibodies

[0256] 1. Detection of the binding ability of monoclonal antibody 8-9D to SARS-CoV-2 RBD using BLI.

[0257] (1) Ligand coupling: Biotin-labeled 10 μg / ml RBD protein (acrobiosystems, SPD-C82E9) was coupled to SA sensor (SARTORIUS, 18-0009) using Octet RED 384 high-throughput intermolecular interaction instrument.

[0258] (2) Dilute antibody 8-9D serially with PBS at 340nM for 6 gradients, starting with 2-fold.

[0259] (3) Set up the injection analysis from low concentration to high concentration as shown in Table 4 below.

[0260] Table 4. Injection settings from low to high concentration

[0261]

[0262] (4) Data analysis: Make the settings as shown in Table 5 in the analysis software and perform curve fitting.

[0263] Table 5 Analysis Software Settings

[0264]

[0265]

[0266] 2. Results Analysis: The affinity KD between 8-9D and wild-type RBD was 4.27 nM (see...). Figure 9 ). Figure 9 The binding and dissociation curves of 8-9D with wild-type RBD protein at different concentrations are shown.

[0267] (II) Neutralization Activity Analysis of Monoclonal Antibodies

[0268] 1. Neutralization assay to detect the neutralizing activity of monoclonal antibodies.

[0269] (1) In a 96-well cell culture plate, as shown in Figure 1, add 150 μl of GM (DMEM + 10% FBS + 1% double antibody) to the cell control (column 1), and add 100 μl of GM to the remaining wells.

[0270] (2) Add antibodies to the treatment wells 3A-3H of the 96-well cell culture plate (i.e., add antibody ① to 3A-3B, antibody ② to 3C-3D, and so on). The initial antibody concentration is 10 μg / ml. Add GM to make the total volume 150 μl.

[0271] (3) Mix the antibody thoroughly, take 50 μl and transfer it into the next row of processing wells, and serially dilute it 3-fold.

[0272] (4) Thaw the SARS-CoV-2 pseudovirus and dilute it with GM to 4000 TCID. 50 / ml, take 50μl (200TCID) 50 Add to columns 2-12 of a 96-well cell culture plate.

[0273] (5) Incubate at 37℃ for 1 hour.

[0274] (6) Digest HeLa-hACE2 cells, resuspend them in GM, and the cell concentration is 1.3 × 10⁻⁶. 5 per ml.

[0275] (7) Take 100 μl of cell suspension (i.e., per 1.3 × 10⁻⁶ cells).4 Add one cell to the cell plate and incubate at 37°C with 5% CO2 for 48 hours.

[0276] (8) Reading: After 48 hours, discard the cell culture medium, wash once with 200 μl PBS, and pat dry; add 100 μl Bright-Lite to each well. TM After the test reagents have been left at room temperature for 2 minutes, the plate is read using an ELISA reader.

[0277] (9) Calculate the 50% inhibitory concentration (IC50) of the antibody. 50 Inhibition rate (i.e., neutralization percentage) = (VT) / (VC) × 100%, where T is the ELISA reader reading for the antibody being tested, C is the ELISA reader reading for the cell control, and V is the ELISA reader reading for the virus control; IC50 is calculated using GraphPad Prism's log(inhibitor) vs. response -- Variable slope. 50 .

[0278] If the antibody has neutralizing activity (IC50) against at least one pseudovirus 50 If the concentration of the antibody is less than 10 μg / ml, then the antibody is a neutralizing antibody against SARS-CoV-2.

[0279] 2. Results Analysis: The results are as follows Figure 10A and Figure 10B As shown, 8-9D is highly effective at neutralizing wild-type SARS-CoV-2 strains and 13 mutant strains, and also has good neutralizing activity against Omicron mutant strains.

[0280] Example 4: Monoclonal antibody 8-9D used to inhibit SARS-CoV-2 infection.

[0281] 1. Virus inhibition experiment

[0282] (1) Cell preparation: The day before the experiment, Vero cells were prepared at 5 × 10⁻⁶ cells per cell line. 3 / wells were inoculated into 96-well plates.

[0283] (2) Add 50 μl (100 TCID) 50SARS-CoV-2 wild-type (IME-BJ01 strain, Genbank No. MT291831), Beta (CSTR: 16698.06.NPRC2.062100001), Delta (CSTR.16698.06.NPRC6.CCPM-BV-049-2105-6), or Omicron (SARS-CoV-2strainOmicronCoV / human / CHN_CVRI-01 / 2022) live virus were incubated with equal volumes of different concentrations (2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.03125 μg / ml, 0.015625 μg / ml) of the monoclonal antibody 8-9D from Example 2 at 37°C for 1 h. Three replicates were set up for each dilution. The cell control was culture medium, and the virus control was 100 TCID. 50 Live SARS-CoV-2 virus.

[0284] (3) Add the mixture of virus and antibody from step (2) to the Vero cells from step (1) and incubate at 37°C for 3 days.

[0285] (4) Observe the cytopathic effect under a microscope and score the degree of cytopathic effect (- to +++) (for specific scoring criteria, see Nature. 2020 Aug; 584(7821): 450-456).

[0286] (5) Convert the lesion score into an inhibition percentage and calculate the IC using GraphPad Prism's log(inhibitor) vs. response--Variable slope. 50 .

[0287] like Figure 11 As shown in the results, monoclonal antibody 8-9D also exhibits good neutralizing activity against live SARS-CoV-2 strains, with an IC50 value against WT, Beta, Delta, and Omicron BA.1. 50 The values ​​were 0.057 μg / ml, 0.120 μg / ml, 0.283 μg / ml and 0.141 μg / ml, respectively.

[0288] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A neutralizing antibody against SARS-CoV-2 or its antigen-binding moiety, characterized in that: The neutralizing antibody or its antigen-binding moiety contains a substance named V. H The heavy chain variable region and its name is V L The light chain variable region, the V H and V L Each is composed of a cluster complement region and a frame region; the V H and the V L The complementary regions of the determinant clusters are all composed of CDR1, CDR2, and CDR3; The V H The amino acid sequence of CDR1 is shown in positions 26-33 of SEQ ID No. 3; The V H The amino acid sequence of CDR2 is shown in positions 51-57 of SEQ ID No. 3; The V H The amino acid sequence of CDR3 is shown in positions 96-106 of SEQ ID No. 3; The V L The amino acid sequence of CDR1 is shown in positions 27-32 of SEQ ID No. 4; The V L The amino acid sequence of CDR2 is shown in positions 50-52 of SEQ ID No. 4; The V L The amino acid sequence of CDR3 is shown in positions 89-99 of SEQ ID No.

4.

2. The neutralizing antibody or its antigen-binding portion according to claim 1, characterized in that: The neutralizing antibody is any one of the following: a) V as described in claim 1 H and the V as described in claim 1 L The resulting single-chain antibody; b) Contains the V as described in claim 1 H and the V as described in claim 1 L Fab; c) Contains the V as described in claim 1 H and the V as described in claim 1 L Complete antibody.

3. A biomaterial relating to the neutralizing antibody or its antigen-binding portion as described in claim 1 or 2, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the neutralizing antibody or its antigen-binding portion as described in claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecules described in B1); B4) A recombinant vector containing the expression cassette described in B2); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1); B6) Recombinant microorganisms containing the expression cassette described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) Recombinant microorganisms containing the recombinant vector described in B4); B9) Cell lines containing the nucleic acid molecules described in B1); B10) Cell lines containing the expression cassette described in B2); B11) Cell lines containing the recombinant vector described in B3); B12) contains the recombinant vector described in B4) cell lines.

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule is a gene encoding the neutralizing antibody or its antigen-binding portion as described in any one of claims 1 or 2.

5. The biomaterial according to claim 4, characterized in that: The gene is a DNA molecule as described in either A) or B): A) The V H The encoded sequence of CDR1 is shown in bits 76-99 of SEQ ID No. 1, wherein V H The encoded sequence of CDR2 is shown in bits 151-171 of SEQ ID No. 1, wherein V H The encoded sequence of CDR3 is shown in bits 286-318 of SEQ ID No. 1; the V L The encoded sequence of CDR1 is shown in bits 79-96 of SEQ ID No. 2, wherein V L The CDR2 encoded sequence is shown in bits 148-156 of SEQ ID No. 2, wherein the V L The encoded sequence of CDR3 is shown in bits 265-297 of SEQ ID No. 2; B) has more than 90% identity with the DNA molecule defined in A) and encodes the DNA of the neutralizing antibody or its antigen-binding portion.

6. A product for treating or preventing SARS-CoV-2 infection, characterized in that: The product comprises the neutralizing antibody or its antigen-binding portion as described in any one of claims 1 or 2, or the biological material as described in any one of claims 3-5.

7. Any of the following applications of the neutralizing antibody or its antigen-binding portion as described in any one of claims 1 or 2, the biomaterial as described in any one of claims 3-5, or the product as described in claim 6: X1. Application in the preparation of drugs for treating SARS-CoV-2 infection; X2. Application in the preparation of drugs for the prevention of SARS-CoV-2 infection.

Images