Neutralizing antibody d7 against novel coronavirus and application thereof

Abstract

The application discloses an anti-SARS-CoV-2 neutralizing antibody d7 and application thereof. The application provides the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region of the antibody d7 against SARS-CoV-2 in sequence as shown in SEQ ID No. 1, 2 and 3; and the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region in sequence as shown in SEQ ID No. 4, 5 and 6. The application prepares an anti-SARS-CoV-2 neutralizing antibody d7. Experiments prove that the antibody d7 can specifically combine with the S protein RBD of SARS-CoV-2 and can neutralize various SARS-CoV-2 epidemic mutant strains. The antibody provided by the application can be used for the prevention and treatment of coronavirus infection and has important biological and medical significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a neutralizing antibody d7 against novel coronavirus and application thereof. BACKGROUND

[0002] SARS-CoV-2 is an enveloped, linear single-stranded positive-sense RNA virus, whose genome encodes four structural proteins, namely spike (S), envelope (E), membrane (M) and nucleocapsid (N), and eight accessory proteins. SARS-CoV-2 virus is very similar to SARS-CoV at the amino acid level, but there are some significant differences. Like other zoonotic coronaviruses, SARS-CoV-2 has a large number of spike (S) proteins on its surface, which are composed of S1 and S2 subunits, forming a homotrimeric viral spike, which interacts with specific protein receptors on the surface of host cells. After binding to the cell receptor, the S protein is cleaved by the protease of the host cell, activating the fusion of the viral and cell membranes. This interaction is mediated by the S1 receptor-binding domain (RBD), which binds to the peptidase domain (PD) of the host cell receptor angiotensin-converting enzyme-2 (hACE2). The S1 contains the receptor-binding domain (RBD) that directly binds to ACE2 and the N-terminal domain (NTD). Structural studies show that the S protein has different conformations. In the pre-fusion stage, the RBD switches between a closed and an open conformation for hACE2 interaction. These two states are referred to as the "down" and "up" conformations, where down corresponds to the receptor-unbound state and up corresponds to the receptor-bound state, the latter being considered less stable. In the post-fusion stage, receptor binding causes the pre-fusion trimer to misalign, causing the S1 subunit to fall off and the S2 subunit to undergo a conformational change, thus triggering the fusion of the host membrane.

[0003] Neutralizing antibodies are key components of the host immune response to viral pathogens, and antibody therapies for treating COVID-19 are being developed. The S protein of SARS-CoV-2 is the main target of neutralizing antibodies, and some monoclonal antibody monotherapy and combination therapy have been granted emergency use authorization for COVID-19 treatment, and more therapies are being developed. Although a variety of targeted antibodies have been developed so far, the coronavirus is still spreading, and new genetic mutations can occur at any time and change the transmissibility and immune escape ability of the coronavirus. Several representative mutant strains of SARS-CoV-2 have been reported so far: Alpha, Beta, Gamma, Delta, and Omicron, and some studies have shown that some mutations occur in the key positions of the virus binding to neutralizing antibodies, thereby causing the virus to escape some monoclonal antibodies. In terms of mutations, the virus is developing in a direction that may eventually lead to the escape of our current therapeutic and preventive interventions against the S protein of the virus. If the virus continues to spread, more key mutations will continue to accumulate. Therefore, it is still necessary to develop new therapeutic and preventive methods, and the development of new coronavirus antibodies with stronger neutralizing activity and higher coverage is still very important. SUMMARY

[0004] The purpose of the present application is to provide a neutralizing antibody against the novel coronavirus and its application.

[0005] In a first aspect, the present application claims an antibody against SARS-CoV-2.

[0006] The antibody against SARS-CoV-2 claimed in the present application, named d7, has the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively; and the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively.

[0007] H-CDR1, H-CDR2 and H-CDR3 are three complementarity determining regions in the heavy chain variable region, and L-CDR1, L-CDR2 and L-HCDR3 are three complementarity determining regions in the light chain variable region.

[0008] Further, the amino acid sequence of the heavy chain variable region is SEQ ID No. 7 from the 1st to the 120th amino acid from the N-terminal, or has at least 90% identity with SEQ ID No. 7 from the 1st to the 120th amino acid from the N-terminal (the inconsistent part can be in the framework region (FR)). The amino acid sequence of the light chain variable region is SEQ ID No. 8 from the 1st to the 110th amino acid from the N-terminal, or has at least 90% identity with SEQ ID No. 8 from the 1st to the 110th amino acid from the N-terminal (the inconsistent part can be in the framework region (FR)).

[0009] Further, the amino acid sequence of the heavy chain of the antibody is SEQ ID No. 7, or has at least 90% identity with SEQ ID No. 7 (the inconsistent part can be in the framework region (FR)). The amino acid sequence of the light chain of the antibody is SEQ ID No. 8, or has at least 90% identity with SEQ ID No. 8 (the inconsistent part can be in the framework region (FR)).

[0010] In a second aspect, the present application claims to protect an antigen binding fragment derived from the antibody of the first aspect.

[0011] The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, human antibody, chimeric antibody, bispecific antibody or multispecific antibody.

[0012] dAb consists of a VH or VL domain, is some of the smallest functional antibody fragments, retaining full antigen-binding specificity. dAb is about one-tenth the mass of a normal antibody molecule. Although dAb contains only three of the six complementarity determining regions from an affinity antibody, they do exhibit antigen-binding specificity and affinity. dAb can maintain significant stability under harsh temperature pressure and chemical denaturation conditions.

[0013] Bispecific antibody (BsAb, simply bispecific antibody) refers to an artificial antibody that can specifically bind to two antigens or antigen epitopes at the same time.

[0014] In a third aspect, the present application claims to protect a nucleic acid molecule.

[0015] The nucleic acid molecule claimed by the present application encodes the antibody of the first aspect or the antigen binding fragment of the second aspect.

[0016] Further, in the nucleic acid molecule, the nucleotide sequences encoding H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are shown as SEQ ID No. 9 from 5' end at positions 79-105, 148-177 and 292-324, respectively. In the nucleic acid molecule, the nucleotide sequences encoding L-CDR1, L-CDR2 and L-HCDR3 in the light chain variable region are shown as SEQ ID No. 10 from 5' end at positions 67-99, 157-186 and 271-300, respectively.

[0017] Further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region is SEQ ID No. 9 from 5' end at positions 1-360 or has at least 90% identity to SEQ ID No. 9 from 5' end at positions 1-360; the nucleotide sequence encoding the light chain variable region is SEQ ID No. 10 from 5' end at positions 1-330 or has at least 90% identity to SEQ ID No. 10 from 5' end at positions 1-330.

[0018] More specifically, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain is SEQ ID No. 9 or has at least 90% identity to SEQ ID No. 9; the nucleotide sequence encoding the light chain is SEQ ID No. 10 or has at least 90% identity to SEQ ID No. 10.

[0019] In a fourth aspect, the present application claims an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacteria comprising the nucleic acid molecule of the first aspect.

[0020] In the detailed description of the present application, the recombinant expression vector expressing the heavy chain of the antibody is obtained by cloning SEQ ID No. 9 (the coding gene of the antibody heavy chain) into the restriction sites NheI and XbaI of pcDNA3.1 vector; the recombinant expression vector expressing the light chain of the antibody is obtained by cloning SEQ ID No. 10 (the coding gene of the antibody light chain) into the restriction sites NheI and XbaI of pcDNA3.1 vector. The recombinant cell is obtained by co-transfecting 293F cells with the two recombinant expression vectors expressing the heavy chain and the light chain of the antibody, respectively.

[0021] In a fifth aspect, the present application claims a pharmaceutical composition.

[0022] The pharmaceutical composition claimed by the present application comprises:

[0023] (A1) the antibody of the first aspect or the antigen binding fragment of the second aspect; and

[0024] (A2) a pharmaceutically acceptable excipient, diluent or carrier.

[0025] In a sixth aspect, the present application seeks protection for the use of any one of:

[0026] (B1) the nucleic acid molecule of the third aspect above or the expression cassette of the fourth aspect above or the recombinant vector or the recombinant cell or the recombinant bacterium of the fifth aspect above in the manufacture of the antibody of the first aspect above or the antigen binding fragment of the second aspect above or the pharmaceutical composition of the fifth aspect above;

[0027] (B2) the antibody of the first aspect above or the antigen binding fragment of the second aspect above in the manufacture of the pharmaceutical composition of the fifth aspect above;

[0028] (B3) the antibody of the first aspect above or the antigen binding fragment of the second aspect above or the nucleic acid molecule of the third aspect above or the expression cassette of the fourth aspect above or the recombinant vector or the recombinant cell or the recombinant bacterium of the fifth aspect above or the pharmaceutical composition of the fifth aspect above in the manufacture of a product for the prophylaxis and / or treatment of a disease caused by SARS-CoV-2 infection;

[0029] (B4) the antibody of the first aspect above or the antigen binding fragment of the second aspect above or the nucleic acid molecule of the third aspect above or the expression cassette of the fourth aspect above or the recombinant vector or the recombinant cell or the recombinant bacterium of the fifth aspect above or the pharmaceutical composition of the fifth aspect above in the manufacture of a product for the inhibition of SARS-CoV-2 infection;

[0030] (B5) the antibody of the first aspect above or the antigen binding fragment of the second aspect above or the nucleic acid molecule of the third aspect above or the expression cassette of the fourth aspect above or the recombinant vector or the recombinant cell or the recombinant bacterium of the fifth aspect above or the pharmaceutical composition of the fifth aspect above in the manufacture of a product for the detection of SARS-CoV-2;

[0031] (B6) the antibody of the first aspect above or the antigen binding fragment of the second aspect above or the nucleic acid molecule of the third aspect above or the expression cassette of the fourth aspect above or the recombinant vector or the recombinant cell or the recombinant bacterium of the fifth aspect above or the pharmaceutical composition of the fifth aspect above in the manufacture of a product for the neutralization of SARS-CoV-2;

[0032] (B7) the antibody of the first aspect above or the antigen binding fragment of the second aspect above or the nucleic acid molecule of the third aspect above or the expression cassette of the fourth aspect above or the recombinant vector or the recombinant cell or the recombinant bacterium of the fifth aspect above or the pharmaceutical composition of the fifth aspect above in the manufacture of a product for the detection of the S protein or the RBD of the S protein of SARS-CoV-2;

[0033] (B8) Use of the antibody of the first aspect or the antigen binding fragment of the second aspect or the nucleic acid molecule of the third aspect or the expression cassette of the fourth aspect or the recombinant vector of the fifth aspect or the recombinant cell of the sixth aspect or the recombinant bacteria of the seventh aspect or the pharmaceutical composition of the eighth aspect in the preparation of a product for binding to the S protein or the RBD of the S protein of SARS-CoV-2.

[0034] The present application prepares a neutralizing antibody d7 against SARS-CoV-2. Experiments show that the antibody d7 can specifically bind to the RBD of the S protein of SARS-CoV-2 and can neutralize a plurality of SARS-CoV-2 epidemic mutant strains. The antibody provided by the present application can be used for the prevention and treatment of coronavirus infection, and has important biological and medical significance. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The backbone sequence of pCDNA3.1 (+).

[0036] Figure 2 The SDS-PAGE result chart of eluted and purified antibody d7 after Protein G purification.

[0037] Figure 3 The neutralization experiment result chart of antibody d7 on each epidemic strain of SARS-CoV-2.

[0038] Figure 4 The SPR experiment result chart of antibody d7 on S-RBD of SARS-CoV-2 and several epidemic strains. DETAILED DESCRIPTION

[0039] The present application will be further described in detail below in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0040] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0041] Example 1, Preparation of neutralizing antibody d7 against SARS-CoV-2

[0042] I. Construction of recombinant expression vector for expressing antibody d7

[0043] The heavy chain amino acid sequence of the anti-SARS-CoV-2 neutralizing antibody d7 (heavy chain constant region is IgG1, light chain is Kappa) involved in this invention is shown in SEQ ID No. 7, and the light chain amino acid sequence is shown in SEQ ID No. 8. Specifically, positions 1-120 of SEQ ID No. 7 are the heavy chain variable region (positions 27-35, 50-59, and 98-108 are three CDRs); positions 1-110 of SEQ ID No. 8 are the light chain variable region (positions 23-33, 53-62, and 91-100 are three CDRs).

[0044] The protein sequence of antibody d7 was converted into a DNA sequence (the heavy chain encoding gene is shown in SEQ ID No. 9, and the light chain encoding gene is shown in SEQ ID No. 10). NheI (GCTAGC) and XbaI (TCTAGA) double restriction sites and homologous arms were added to the ends of SEQ ID No. 9 and SEQ ID No. 10 respectively by overlapping PCR. The DNA was then ligated into the double-enzyme-digested (NheI and XbaI) pCDNA3.1(+) vector using Gibson Assembly. The ligation product was transformed into DH5α cells for amplification and plasmid extraction to obtain the expression vector. Figure 1 The backbone sequence of pCDNA3.1(+) was shown. The recombinant plasmid ligated into SEQ ID No. 9, which was finally verified by sequencing, was named pCDNA3.1-H, and the recombinant plasmid ligated into SEQ ID No. 10 was named pCDNA3.1-L.

[0045] pCDNA3.1-H structural description: The recombinant plasmid obtained by cloning the DNA fragment shown in SEQ ID No. 9 between NheI and XbaI in the pCDNA3.1(+) vector.

[0046] pCDNA3.1-L structural description: The recombinant plasmid obtained by cloning the DNA fragment shown in SEQ ID No. 10 between NheI and XbaI in the pCDNA3.1(+) vector.

[0047] II. Large-scale culture / Transfection in 1L shake flasks

[0048] According to the standard operating procedure (SOP), to culture suspension 293F cells (ThermoFisher), generally start with a 250ml cell culture shake flask and culture to a volume of 30ml-100ml. Follow the 0.5×10⁻⁶ ratio. 6 Inoculate cells at a rate of 1 cells / ml into 300 ml of culture medium in a 1 L shake flask and incubate at 37°C, 120 rpm, 5% CO2 in a shaker until the cell density reaches 1 × 10⁻⁶ cells / ml.6 cells / ml. 300 μg of DNA (i.e. 150 μg of each of the recombinant expression vectors pCDNA3.1-H and pCDNA3.1-L obtained in step one) was added to 30 ml of PBS and 1.2 ml of sterile filtered PEI solution (0.5 mg / ml) was added to the PBS / DNA mixture, which was left to stand for 20 min before the cells were added. After transfection, the cells were incubated in a shaking incubator for 48 h. The cell culture medium supernatant was separated from the cell pellet by centrifugation at 3000 g for 5 min and the supernatant was harvested.

[0049] III. Purification of the protein complex extracted from the cell culture medium supernatant

[0050] The supernatant from the cell culture medium after 48 h of incubation after transfection was filtered through a 0.45 μm filter. The filtered supernatant was incubated with 1.25 ml of Protein G column material (Cytiva) per litre of filtered supernatant, eluted with 10 mM citric acid / 200 mM NaCl buffer and then separated on a Q-sepharose ion exchange column (Cytiva) using 10 mM phosphate buffer / 200 mM NaCl as the loading buffer. The eluate was then purified on a Sephacryal 200 gel chromatography column (Cytiva) using 10 mM phosphate buffer / 150 mM NaCl as the elution buffer. The eluate was the d7 antibody stock solution.

[0051] A sample of 10 μl of the concentrated protein was taken and 2x protein loading buffer was added for electrophoresis. The protein was filtered through a 0.22 μm filter. Figure 2 The d7 antibody heavy chain and light chain proteins of interest expressed in the cell supernatant from the 2 L (8 x 250 ml) of 293F culture medium after transient transfection are shown. The proteins of interest were extracted from the cell supernatant and purified. Figure 2 As can be seen from the SDS-PAGE electrophoresis pattern, the proteins of interest were expressed in the cell supernatant from the d7 antibody heavy chain and light chain bacterial expression at a molecular weight of approximately 48-63 KDa and 25 KDa, respectively, which is close to the theoretical value (heavy chain 50 KD and light chain 25 KD) (the actual molecular weight is slightly larger than the theoretical value due to the glycosylation process of the protein), and there is essentially no impurity band after purification. The constructed expression strain was stored in a -80 °C freezer for future use. It is estimated that the yield of purified protein from 1 L of culture medium is close to 1 mg.

[0052] Example 2, Antibody d7 and SARS-CoV-2 pseudovirus neutralization assay

[0053] The pseudovirus neutralization assay uses vesicular stomatitis virus (VSV) as its basic framework, replacing its receptor-binding protein G with the Spike protein of SARS-CoV-2 to simulate its entry into cells and its inhibition by drugs. Compared with in vitro experiments, the pseudovirus simulates the process of viral infection of cells, resulting in more realistic and reliable experimental results. Antibody d7 performed exceptionally well in in vitro experiments, therefore further experiments are needed to confirm its therapeutic efficacy.

[0054] I. Preparation of pseudoviruses

[0055] Based on our previous research, we constructed pseudoviruses and mutant strains of SARS-CoV-2. Specifically, one day before transfection, 293T cells were digested and adjusted to a concentration of 5 × 10⁻⁶ cells / cells. 5 –7×10 5 The concentration was set at 15 ml of cells / mL. Then, the cells in 15 ml of culture medium were transferred to T75 cell culture medium and incubated overnight at 37°C with 5% CO2. When the cells reached 70%-90% coverage, the supernatant was discarded, and a solution of 7 × 10⁻⁶ cells / mL was used. 4 TCID 50 The virus was infected with 15 ml of VSV-ΔG-luciferase plasmid expression vector system (Kerafast: EH1008) at a concentration of / mL. Simultaneously, 30 μg of S protein expression plasmids from different prevalent strains (at a concentration of / mL) were used. Figure 1 Using pCDNA3.1(+) as a vector, recombinant plasmids were obtained by inserting all the S protein encoding genes between the NheI and XbaI restriction sites of the pCDNA3.1(+) vector and transducing them into cells. Cells were cultured at 37°C in a 5% CO2 incubator. After 6-8 hours, the cell supernatant was discarded, and the cells were gently washed twice with PBS + 1% FBS. 15 mL of fresh DMEM was added to the T75 cell culture flask. After culturing at 37°C in a 5% CO2 incubator for 24 hours, the culture supernatant containing the pseudovirus was collected, filtered, and aliquoted to obtain VSV pseudoviruses with S-WT, S-alpha, S-beta, S-gamma, S-delta, S-lambda, or S-Mu (representing the S proteins of wild-type SARS-CoV-2, the alpha, beta, gamma, delta, lambda, and Mu epidemic strains of SARS-CoV-2, respectively). These were frozen at -80°C for later use.

[0056] Wherein, the S proteins of 7 SARS-CoV-2 are from wild type SARS-CoV-2, alpha, beta, gamma, delta, lambda and Mu prevalent strains of SARS-CoV-2 respectively. The amino acid sequence of S protein from wild type SARS-CoV-2 (i.e. Wuhan-Hu-1 strain) is the same as NCBI Reference Sequence: YP_009724390.1, and the corresponding coding gene sequence is NCBI Reference Sequence: NC_045512.2 (21563..25384), NCBI Gene ID is 43740568; the amino acid sequence of S protein from alpha prevalent strain is changed from YP_009724390.1 as follows: deletion of 69-70th amino acid residues, deletion of 144th amino acid residue, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, and the corresponding coding gene is changed from NC_045512.2 (21563..25384) as follows: deletion of 205-210th nucleotides, deletion of 430-432th nucleotides, mutation of 1501-1503th nucleotides to tat, mutation of 1708-1710th nucleotides to gat, mutation of 1840-1842th nucleotides to ggt, mutation of 2041-2043th nucleotides to cat, mutation of 2146-2148th nucleotides to atc, mutation of 2944-2946th nucleotides to gcc, mutation of 3352-3354th nucleotides to cac; the amino acid sequence of S protein from beta prevalent strain is changed from YP_009724390.1 as follows: L18F, D80A, D215G, deletion of 242-244th amino acid residues, K417N, E484K, N501Y, D614G, A701V, and the corresponding coding gene is changed from NC_045512.2 (21563..25384) as follows: mutation of 52-54th nucleotides to ttc, mutation of 238-240th nucleotides to gcc, mutation of 643-645th nucleotides to ggc, deletion of 724-732th nucleotides, mutation of 1249-1251th nucleotides to aat, mutation of 1450-1452th nucleotides to aag, mutation of 1501-1503th nucleotides to tat, mutation of 1840-1842th nucleotides to ggt, mutation of 2101-2103th nucleotides to gta; the amino acid sequence of S protein from gamma prevalent strain is changed from YP_009724390.1 as follows: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F, and the corresponding coding gene is changed from NC_045512.2 (21563..25384) the following changes occur: mutations ttcttc at positions 52-54, aac at positions 58-60, tct at positions 76-78, tac at positions 412-414, agc at positions 568-570, agc at positions 1249-1251, aag at positions 1450-1452, tat at positions 1501-1503, ggt at positions 1840-1842, tat at positions 1963-1965, atc at positions 3079-3081, ttcttc at positions 3526-3528; the amino acid sequence of the S protein from the delta variant compared to YP_009724390.1 has the following changes: T19R, G142D, deletion of amino acid residues 156-157, R158G, L452R, T478K, D614G, P681R, D950N, the corresponding coding gene compared to NC_045512.2 (21563..25384) has the following changes: mutations aga at positions 55-57, gag at positions 424-426, deletion of nucleotides 466-471, mutations ggc at positions 472-474, aga at positions 1354-1356, aag at positions 1432-1434, mutations ggt at positions 1840-1842, aga at positions 2041-2043, mutations aac at positions 2848-2850; the amino acid sequence of the S protein from the lambda variant compared to YP_009724390.1 has the following changes: G75V, T76I, deletion of amino acid residues 246-252, L452Q, F490S, D614G, T859N, the corresponding coding gene compared to NC_045512.2 (21563..25384) has the following changes: mutations gtt at positions 223-225, att at positions 226-228, deletion of nucleotides 736-756, mutations cag at positions 1354-1356, tct at positions 1468-1470, mutations ggt at positions 1840-1842, mutations aat at positions 2575-2577; the amino acid sequence of the S protein from the Mu variant compared to YP_009724390.1 has the following changes: T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N, the corresponding coding gene compared to NC_045512.2 (21563..25384) the following changes were made: mutations at positions 283-285 to att, at positions 430-432 to tct, at positions 433-435 to aac, at positions 1036-1038 to aaa, at positions 1450-1452 to aaa, at positions 1501-1503 to tat, at positions 1840-1842 to ggt, at positions 2041-2043 to cat, and at positions 2848-2850 to aac.

[0057] II. Pseudovirus infection inhibition test

[0058] After RT-PCR quantitative analysis of the pseudovirus collected in step one, each pseudovirus was diluted to a titer of 2 x 10 5 TCID 50 / mL, and 100 μL was added to a 96-well cell culture plate. REGN10933, REGN10987, LY-cov555, JS016, AZD1061, S309, VHH-72 (Wuhan Costar Biological Technology Co., Ltd.: CSD00700, CSD00701, CSD00702, CSD00717, CSD00704, CSD00715, CSDVV00316) and d7 samples were gradient diluted (3-fold dilution starting from 100 nM, a total of 8 gradients) in 96-well plates and added to the corresponding wells with virus solution. Eight control groups with only virus solution and eight control groups with only cells were set up on the plate. After incubation at 37°C for 1 hour, wild-type ACE2 overexpressing cells (Yisen Biological Technology (Shanghai) Co., Ltd.: 41107ES03) were trypsinized and added to each well of the 96-well plate at a concentration of 2 x 10 4 / 100 μL cells. After 24 hours of incubation in a 37°C incubator with 5% carbon dioxide, the change in luciferase gene expression was detected to evaluate the neutralization effect of wild-type d7 antibody and the seven commercial antibodies (Table 1) on pseudovirus infection of ACE2 overexpressing cells, respectively. 100 μL of luciferase substrate (Perkinlemer) was added to each well, incubated at room temperature for 2 minutes, then transferred to a detection white plate, and measured using a luminometer (Perkinlemer). Each group contained two repeated experiments. The EC 50 value of each sample was calculated using the Reed-Muench method.

[0059] Table 1, Information of 7 commercial antibodies

[0060] Casirivimab (REGN10933) Wuhan CStone Biotech Co., Ltd. CSD00700 Imdevimab (REGN10987) Wuhan CStone Biotech Co., Ltd. CSD00701 Bamlanivimab (LY-cov555) Wuhan CStone Biotech Co., Ltd. CSD00702 Etesevimab (JS016) Wuhan CStone Biotech Co., Ltd. CSD00717 Cilgavimab (AZD1061) Wuhan CStone Biotech Co., Ltd. CSD00704 Sotrovimab (S309) Wuhan CStone Biotech Co., Ltd. CSD00715 VHH-72 Wuhan CStone Biotech Co., Ltd. CSDVV00316

[0061] The results are as follows Figure 3It can be seen that the antibody d7 has very strong inhibition effect on all mutant pseudoviruses in the experiment, and from the calculation results of EC50, it can be seen that the inhibition effect is obviously better than other antibodies, and compared with Cilgavimab and Imdevimab which are the best for various mutant viruses, the inhibition effect of antibody d7 on pseudovirus is increased by one order of magnitude.

[0062] Example 3, Affinity determination of d7 antibody and SARS-CoV-2 S protein RBD

[0063] For a typical affinity determination, a Biacore 2000 instrument was used in the experiment, which is based on surface plasmon resonance technology (SPR) and can sensitively reflect the change of refractive index of the chip surface. In order to study the interaction between molecules, one of the molecules is fixed on the chip surface, and the other molecule flows through the surface in the form of solution. The response value of SPR is proportional to the mass concentration change near the chip.

[0064] In a typical test, a CM5 chip was selected to determine the protein-protein interaction between d7 antibody and SARS-CoV-2 Spike-RBD. The CM5 chip is based on the strategy of covalent coupling, with medium capacity and general characteristics. The running buffer is HBS-EP (Cytiva). SARS-CoV-2 RBD or beta-RBD or delta-S1 (Yi Qiao God: 40592-V08H, 40592-V08H85-B, 40591-V49H2-B) (20 ng / μl) is dissolved in sodium acetate buffer (pH 4.5) and immobilized on the chip as a ligand, with a flow rate of 5 μl / min during immobilization, and stopped after about 30 s, with an immobilization amount of about 60 RU. The analyte is d7 antibody, with a concentration from 100 to 1.56 nM (2-fold dilution between each gradient), and the diluent is running buffer. The flow rate is set to 45 μl / min, the binding time is set to 180 seconds, and the dissociation time is 1800 seconds. After the end of each cycle, a glycine solution with pH 1.5 is used for regeneration for 30 seconds at a flow rate of 30 μl / min.

[0065] The signal curve during the reaction is shown in Figure 4 For the binding of the two proteins, in order to analyze its kinetic characteristics, it can be assumed that they conform to a simple interaction model, and it is assumed that [d7] (the concentration of the analyte) is constant, so the rate equation of the binding process is:

[0066]

[0067] And in the dissociation process, the rate equation is:

[0068]

[0069] We fit the binding curves for all concentration gradients and obtain the data in Table 2.

[0070] Table 2, Molecular interaction parameters of d7 antibody with several Spike-RBDs

[0071] Spike ka (1 / Ms) kd (1 / s) KD (nM) WT-RBD 1.30 x 10 6 ]]> 1.92 x 10 -4 ]]> 0.15 Beta-RBD 1.73 x 10 6 ]]> 1.05 x 10 -4 ]]> 0.06 Delta-S1 5.10 x 10 5 ]] 1.11 x 10 -4 ]] 0.23

[0072] The d7 antibody shows very strong RBD affinity for SARS-CoV-2 RBD or beta-RBD or delta-S1 in the tests, and it can be seen that the protein has a fast binding rate, and the dissociation curve shows that the binding of the protein is extremely stable. In addition, considering the binding and dissociation processes, we can calculate the dissociation constant KD, and the value is less than nM order of magnitude.

[0073] This example embodies the strong affinity of the d7 antibody for SARS-CoV-2 RBD or beta-RBD or delta-S1 protein.

[0074] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in this application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. An antibody against SARS-CoV-2, characterized in that: the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region of the antibody are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 in turn; and the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region of the antibody are shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 in turn.

2. The antibody according to claim 1, characterized in that: the amino acid sequence of the heavy chain variable region is SEQ ID No. 7 from the 1st to the 120th from the N terminus.

3. The antibody of claim 1, wherein: the amino acid sequence of the light chain variable region is SEQ ID No. 8 from the 1st to the 110th from the N terminus.

4. The antibody according to claim 1 or 2, characterized in that: the amino acid sequence of the heavy chain of the antibody is SEQ ID No.

7.

5. The antibody of claim 1 or 3, characterized in that: the amino acid sequence of the light chain of the antibody is SEQ ID No.

8.

6. An antigen binding fragment derived from the antibody of any one of claims 1-5; the antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv, single chain antibody, chimeric antibody, bispecific antibody or multispecific antibody; the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the antigen binding fragment are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 in turn; and the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the antigen binding fragment are shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 in turn.

7. A nucleic acid molecule, characterized in that: the nucleic acid molecule encodes the antibody of any one of claims 1-5 or the antigen binding fragment of claim 6.

8. The nucleic acid molecule of claim 7, wherein: in the nucleic acid molecule, the nucleotide sequences encoding H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are shown in SEQ ID No. 9 from the 5' end from the 79th to the 105th, the 148th to the 177th, the 292nd to the 324th in turn.

9. The nucleic acid molecule of claim 7, wherein: in the nucleic acid molecule, the nucleotide sequences encoding L-CDR1, L-CDR2 and L-HCDR3 in the light chain variable region are shown in SEQ ID No. 10 from the 5' end from the 67th to the 99th, the 157th to the 186th, the 271st to the 300th.

10. The nucleic acid molecule of claim 7, wherein: in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region is SEQ ID No. 9 from the 5' end from the 1st to the 360th or has at least 90% identity with SEQ ID No. 9 from the 5' end from the 1st to the 360th; the nucleotide sequence encoding the light chain variable region is SEQ ID No. 10 from the 5' end from the 1st to the 330th or has at least 90% identity with SEQ ID No. 10 from the 5' end from the 1st to the 330th.

11. The nucleic acid molecule of claim 7, wherein: In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain is SEQ ID No. 9 or has at least 90% identity with SEQ ID No. 9; the nucleotide sequence encoding the light chain is SEQ ID No. 10 or has at least 90% identity with SEQ ID No.

10.

12. An expression cassette, a recombinant vector, a recombinant cell or a recombinant bacteria comprising the nucleic acid molecule of any one of claims 7-11.

13. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises: (A1) the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6; and (A2) a pharmaceutically acceptable excipient, diluent or carrier.

14. Use, for any one of: (B1) the nucleic acid molecule of any one of claims 7-11 or the expression cassette or the recombinant vector or the recombinant cell or the recombinant bacteria of claim 12 in the manufacture of the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 or the pharmaceutical composition of claim 13; (B2) the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 in the manufacture of the pharmaceutical composition of claim 13; (B3) the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 or the nucleic acid molecule of any one of claims 7-11 or the expression cassette or the recombinant vector or the recombinant cell or the recombinant bacteria of claim 12 or the pharmaceutical composition of claim 13 in the manufacture of a product for preventing and / or treating a disease caused by SARS-CoV-2 infection; (B4) the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 or the nucleic acid molecule of any one of claims 7-11 or the expression cassette or the recombinant vector or the recombinant cell or the recombinant bacteria of claim 12 or the pharmaceutical composition of claim 13 in the manufacture of a product for inhibiting SARS-CoV-2 infection; (B5) the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 or the nucleic acid molecule of any one of claims 7-11 or the expression cassette or the recombinant vector or the recombinant cell or the recombinant bacteria of claim 12 or the pharmaceutical composition of claim 13 in the manufacture of a product for detecting SARS-CoV-2; (B6) the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 or the nucleic acid molecule of any one of claims 7-11 or the expression cassette or the recombinant vector or the recombinant cell or the recombinant bacteria of claim 12 or the pharmaceutical composition of claim 13 in the manufacture of a product for neutralizing SARS-CoV-2; (B7) Use of the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 or the nucleic acid molecule of any one of claims 7-11 or the expression cassette or the recombinant vector or the recombinant cell or the recombinant bacteria or the pharmaceutical composition of claim 13 in the manufacture of a product for detecting the S protein or the RBD of the S protein of SARS-CoV-2; (B8) Use of the antibody of any one of claims 1-5 or the antigen-binding fragment of claim 6 or the nucleic acid molecule of any one of claims 7-11 or the expression cassette or the recombinant vector or the recombinant cell or the recombinant bacteria or the pharmaceutical composition of claim 13 in the manufacture of a product for binding to the S protein or the RBD of the S protein of SARS-CoV-2.

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