Sabei coronavirus broad-spectrum neutralizing antibodies and their applications

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to broad-spectrum neutralizing antibodies for Sabei coronavirus and their applications. The antibody is a combination of any one or more of KXD350, KXD352, KXD355, and KXD358, and the KXD350, KXD352, KXD355, and KXD358 all contain a heavy chain variable region and a light chain variable region, and the heavy chain variable region has three heavy chain CDRs, and the light chain variable region has three light chain CDRs. The four broad-spectrum neutralizing antibodies provided by the present invention and the disclosed heavy chain and light chain variable region sequences provide a basis for constructing high-affinity, high-quality, and highly specific Sabei coronavirus therapeutic neutralizing antibodies, and have a high application value for the prevention, control, and treatment of coronaviruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to Sabei coronavirus broad-spectrum neutralizing antibodies and applications thereof. Technical Background

[0002] Coronaviruses can be divided into four genera: α, β, γ, and δ. The coronaviruses of the α and β genera mainly infect mammals, and all coronaviruses that can infect humans are also in these two genera. The β-genus coronavirus is divided into four independent subgroups, A, B, C, and D lineages, of which the B lineage is also called Sarbecovirus. There is only one virus species, SARS-related coronavirus, that is, SARS-related coronavirus, including SARS-CoV-1, SARS-CoV-2 and its variants, and some animal-derived SARS-related coronaviruses (Lan, Q., et al., Pan-coronavirus fusion inhibitors to combat COVID-19 and other emerging coronavirus infectious diseases. J Med Virol, 2023. 95 (1): p. e28143.). SARS-CoV-1 is a representative member of the Sarbeco coronavirus, and the SARS-CoV-2 coronavirus that has broken out in recent years is a newly identified Sarbeco coronavirus.

[0003] The coronavirus genome mainly encodes four structural proteins, spike (S) protein, nucleocapsid (N) protein, membrane (M) protein and envelope (E) protein, as well as non-structural proteins, which play a role in maintaining the structural integrity of enveloped virus particles. The coronavirus S protein forms a trimer on the surface of the virus, which can bind to the ACE2 surface receptor on the host cell and mediate the entry of the coronavirus into the host cell. Therefore, it is the main target of therapeutic neutralizing antibodies and the focus of drug development and vaccine design.

[0004] At present, a total of 7 coronaviruses that can infect humans have been found (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV-1, SARS-CoV-2, MERS-CoV). Since the beginning of the 21st century, coronaviruses SARS-CoV-1, MERS-CoV and SARS-CoV-2 have successively broken out and have spread many times around the world, causing acute respiratory infectious diseases, posing a great threat to public health, and also indicating the possibility of another outbreak of highly pathogenic coronaviruses in the future.

[0005] Therefore, it is necessary to develop broad-spectrum neutralizing antibodies against Sabei coronavirus as a technical reserve to cope with the potential outbreak of coronavirus in the future. Summary of the invention

[0006] In order to solve the problems in the prior art, one of the purposes of the present invention is to provide broad-spectrum neutralizing antibodies against Sabei coronavirus.

[0007] The present invention adopts the following technical solutions:

[0008] Sabei coronavirus broad-spectrum neutralizing antibody, the neutralizing antibody is any one or more combinations of KXD350, KXD352, KXD355 and KXD358, the KXD350, KXD352, KXD355 and KXD358 all comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having three heavy chain CDRs, the light chain variable region having three light chain CDRs, wherein:

[0009] The amino acid sequences of the three CDRs of the KXD350 heavy chain variable region are amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the three CDRs of the KXD350 light chain variable region are amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;

[0010] The amino acid sequences of the three CDRs of the KXD352 heavy chain variable region are, in turn, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; the amino acid sequences of the three CDRs of the KXD352 light chain variable region are, in turn, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12;

[0011] The amino acid sequences of the three CDRs of the KXD355 heavy chain variable region are, in order, amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; the amino acid sequences of the three CDRs of the KXD355 light chain variable region are, in order, amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18;

[0012] The three CDR amino acid sequences of the KXD358 heavy chain variable region are, in turn, amino acid sequences having an identity of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; the three CDR amino acid sequences of the KXD358 light chain variable region are, in turn, amino acid sequences having an identity of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

[0013] The homology sequence is preferably an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. The homology sequence also includes an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in the sequence listing.

[0014] Preferably, the amino acid sequences of the six CDRs of the heavy chain variable region and the light chain variable region of KXD350 are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;

[0015] The amino acid sequences of the six CDRs of the heavy chain variable region and light chain variable region of KXD352 are shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively;

[0016] The amino acid sequences of the six CDRs of the heavy chain variable region and light chain variable region of KXD355 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively;

[0017] The amino acid sequences of the six CDRs of the heavy chain variable region and light chain variable region of KXD358 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

[0018] Specifically, the CDR amino acid sequences of the heavy chain variable region and light chain variable region of KXD350, KXD352, KXD355, and KXD358 are shown in the following table:

[0019]

[0020] In the table, H represents heavy chain, and K represents light chain.

[0021] Preferably, the amino acid sequence of the heavy chain variable region of KXD350 is shown as SEQ ID NO:25, and the amino acid sequence of the light chain variable region of KXD350 is shown as SEQ ID NO:26; the amino acid sequence of the heavy chain variable region of KXD352 is shown as SEQ ID NO:27, and the amino acid sequence of the light chain variable region of KXD352 is shown as SEQ ID NO:28; the amino acid sequence of the heavy chain variable region of KXD355 is shown as SEQ ID NO:29, and the amino acid sequence of the light chain variable region of KXD355 is shown as SEQ ID NO:30; the amino acid sequence of the heavy chain variable region of KXD358 is shown as SEQ ID NO:31, and the amino acid sequence of the light chain variable region of KXD358 is shown as SEQ ID NO:32.

[0022] Preferably, the neutralizing antibody further comprises an Fc domain.

[0023] Preferably, the Fc domain is a human IgG Fc domain, including a human IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain or an IgG4 Fc domain.

[0024] Preferably, the neutralizing antibody further comprises an immune marker coupled thereto, and the immune marker is any one of an enzyme label, a fluorescent label, an isotope label, and a biotin label.

[0025] The present invention also provides a polynucleotide encoding the KXD350, KXD352, KXD355 or KXD358 neutralizing antibody as described above, or encoding the neutralizing antibody comprising an Fc domain as described above;

[0026] Among them, the nucleotide sequence encoding the heavy chain variable region of KXD350 is shown in SEQ ID NO:33, and the nucleotide sequence encoding the light chain variable region of KXD350 is shown in SEQ ID NO:34; the nucleotide sequence encoding the heavy chain variable region of KXD352 is shown in SEQ ID NO:35, and the nucleotide sequence encoding the light chain variable region of KXD352 is shown in SEQ ID NO:36; the nucleotide sequence encoding the heavy chain variable region of KXD355 is shown in SEQ ID NO:37, and the nucleotide sequence encoding the light chain variable region of KXD355 is shown in SEQ ID NO:38; the nucleotide sequence encoding the heavy chain variable region of KXD358 is shown in SEQ ID NO:39, and the nucleotide sequence encoding the light chain variable region of KXD358 is shown in SEQ ID NO:40.

[0027] The present invention also provides an expression vector comprising the polynucleotide as described above, and a host cell comprising the expression vector as described above. Preferably, the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, mammalian cells.

[0028] The present invention provides the use of the neutralizing antibody as described above in the preparation of a medicament for treating and / or preventing Sabei coronavirus infection.

[0029] Preferably, the Sabei coronavirus includes SARS-CoV-1 and SARS-CoV-2 and their variants, and the variants include Delta, BA.1, BA.4 / 5, BA.2.75, BQ.1, XBB.1.5, EG.5.1, HK.3, BA.2.86, JN.1, KP.2, and KP.3.

[0030] The present invention provides a pharmaceutical composition, which comprises any one neutralizing antibody or a combination of at least two neutralizing antibodies as described above, and further comprises a pharmaceutically acceptable carrier.

[0031] The present invention provides the use of the neutralizing antibody as described above in preparing a kit for detecting Sabei coronavirus.

[0032] The present invention provides a kit for detecting Sabae coronavirus, which comprises the neutralizing antibody as described above.

[0033] The present invention provides the use of the neutralizing antibody as described above in preparing a vaccine for preventing Sabei coronavirus infection.

[0034] The present invention also provides a vaccine comprising the neutralizing antibody described above.

[0035] The beneficial effects of the present invention are:

[0036] The present invention screened out 4 groups of antibodies KXD350, KXD352, KXD355, and KXD358 with potent and broad-spectrum neutralizing activity against Sabei coronavirus (including SARS-CoV-1 and SARS-CoV-2 and their variants, including the latest SARS-CoV-2 epidemic variants EG.5.1, HK.3, JN.1, KP.2, and KP.3), cloned their light chain and heavy chain variable regions, and obtained their nucleotide sequences after sequencing, respectively, providing a basis for constructing high-affinity, high-quality, and highly specific Sabei coronavirus therapeutic neutralizing antibodies, and having high application value for the prevention, control, and treatment of Sabei coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Antigen-specific flow sorting of single B cells;

[0038] Figure 2 It is the ELISA reading of the supernatant of SARS-CoV-2WT Spike and SARS-CoV-1Spike double-positive antibodies;

[0039] Figure 3 Neutralization IC values ​​of 4 antibodies against various pseudoviruses 50 value. DETAILED DESCRIPTION

[0040] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments:

[0041] Example 1

[0042] Antibody discovery and production

[0043] 1.1 Acquisition of antigens

[0044] The SARS-CoV-2WT Spike extracellular region (1-1208) and the SARS-CoV-1Spike extracellular region (1-1195) were respectively constructed with the trimer tag of bacteriophage T4 fibritin and the 8×His, Twin-StrepⅡtag, and Flagtag at the C-terminus into the vector pCAGGS to form an expression plasmid. The plasmid was gently mixed with the transfection reagent PEI, incubated at room temperature for 20 minutes, and transfected into 293F suspension cells to obtain recombinant cells for protein expression. The recombinant cells were cultured in SMM 293-TII complete medium for 96 hours, then centrifuged at 4°C and 3000rpm for 30 minutes, and the supernatant was collected. The target protein WT Spike was purified by Strep-Tactin magnetic bead affinity chromatography. Specific purification process: 100 ml of supernatant was mixed with Strep-Tactin magnetic beads, incubated overnight at 4°C, and the solution and magnetic beads were separated by a magnetic stand. Add 30ml PBS solution to the magnetic beads, rotate to mix, wash the magnetic beads for 5min, collect the magnetic beads using a magnetic stand and discard the supernatant; repeat the washing step three times. Add 10ml elution buffer (5mM D-Desthiobiotin in PBS) to the magnetic beads, flip and mix at room temperature, and incubate for 15min. Collect the magnetic beads using a magnetic separation stand and transfer the eluate containing the target protein to a clean centrifuge tube. Use a 100KDa cutoff ultrafiltration tube for concentration and replace the solution with PBS buffer.

[0045] 1.2 Antigen-specific flow cytometry sorting of single B cells

[0046] One person with SARS-CoV-2 Omicron breakthrough infection was recruited as a volunteer. Before infection, the volunteer received two doses of mRNA vaccine in 2021, and was infected with the Omicron variant for the first time in December 2022. The dominant strains at this time were BA.5 and BF.7. Nine months after recovery from Omicron breakthrough infection, 5 ml of peripheral blood was drawn and centrifuged to obtain plasma and blood cells. Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation using lymphocyte separation fluid (Ficoll-Paque PLUS density gradient centrifugation medium, Si Tuofan, catalog number: 17144002). PBMCs were subjected to cell flow cytometry staining and antigen-specific sorting was performed using a flow cytometer. Flow sorting and gating strategies are as follows: Figure 1 As shown, DAPI - CD3 - CD19 + CD27 + IgD - Memory B cells and plasmablasts that were double-positive for His and Flag of the antigen SARS-CoV-2WT Spike were sorted, and single B cells were sorted into a 96-well plate containing cell lysate.

[0047] 1.3 Antibody variable region gene amplification

[0048] Using single B cell gene amplification technology, the cleaved mRNA was reverse transcribed to obtain cDNA, and single B cell antibody gene amplification primers were used to obtain the gene products of the heavy chain and light chain variable regions by PCR and sequenced. The sequences were analyzed using the IgBlast tool.

[0049] 1.4 Construction of recombinant plasmid

[0050] The heavy chain variable region DNA molecule was seamlessly cloned into the expression vector hIgG to obtain the heavy chain expression plasmid.

[0051] The light chain variable region DNA molecule is seamlessly cloned into the expression vector hIgκ or hIgλ to obtain the light chain expression plasmid.

[0052] The coding regions of the heavy chain and light chain each consist of three parts: a signal peptide, a variable region, and a constant region.

[0053] 1.5 Antibody Screening

[0054] Finally, multiple antibodies were cloned successfully and paired. The paired heavy chain expression plasmid and light chain expression plasmid were co-transfected into HEK-293T cells and cultured for 48 hours to express antibodies and obtain the cultured antibody supernatant. The antibody supernatant was used to perform preliminary screening of antibody affinity and neutralizing activity.

[0055] 1.5.1 ELISA binding assay to screen positive clones

[0056] 1) Antigen coating: Dilute SARS-CoV-2 WT Spike antigen and SARS-CoV-1 Spike antigen in PBS to a final concentration of 1 ng / μl. Coat each well of the ELISA plate with 100 μl at 4°C overnight.

[0057] 2) Blocking: Discard the solution, wash each well with 200 μl PBST for 3 times, add 200 μl blocking solution to each well, and block at 37°C for 2 h.

[0058] 3) Washing: discard the supernatant, add 200 μl PBST to each well, wash three times, and pat dry.

[0059] 4) Add cell culture antibody supernatant: dilute the antibody supernatant 3 times in a gradient manner, add 100 μl of antibody solution to each well, the first well is the undiluted stock solution, and the negative control is PBS. Incubate at 37°C for 1 hour.

[0060] 5) Add secondary antibody: discard the supernatant, add 200 μl PBST to each well, wash 3 times, pat dry. Add 100 μl HRP-labeled goat anti-human IgG (H+L) secondary antibody to each well. Incubate at 37°C for 1 hour.

[0061] 6) Color development: discard the supernatant, add 200 μl PBST to each well, wash 4 times, pat dry, add 100 μl TMB color development solution, and after the color changes, add 50 μl 10% sulfuric acid stop solution to each well to terminate the reaction.

[0062] 7) Reading: Read the absorbance at 450nm using an ELISA reader. Set up one set of duplicate wells for each cell culture antibody supernatant, and repeat each experiment once independently. The average absorbance value higher than 2.0 is selected as a positive antibody.

[0063] The experimental results are shown in Table 1. It can be seen that after the initial screening of the cell culture antibody supernatant by ELISA binding experiment, 28 strains of double-positive antibodies against SARS-CoV-2WT Spike and SARS-CoV-1Spike were obtained. Figure 2 ELISA readings of antibody supernatant binding to antigens SARS-CoV-2 WT Spike and SARS-CoV-1 Spike.

[0064] 1.5.2 Preliminary screening of antibody neutralization activity

[0065] The antibody supernatants from the above 28 ELISA double-positive cell cultures were further screened for neutralizing activity against SARS-CoV-2WT and SARS-CoV-1 pseudoviruses.

[0066] 1) Prepare fake viruses

[0067] SARS-CoV-2WT and SARS-CoV-1 pseudoviruses were taken out from -80°C and thawed at room temperature, and the relative titer of the pseudoviruses was adjusted to about 100,000 (the reading value of the luciferase reporter gene).

[0068] 2) Take a 96-well cell culture plate, add 200μl PBS to the wells around the 96-well plate, add 100μl culture medium to each of the remaining wells, add 50μl of antibody supernatant of different cell cultures to the first and fifth columns respectively, mix well, take 50μl to the next column of wells and mix well, perform 3-fold gradient dilution, a total of 4 gradients, and the dilution of the first well is 4.5 times; finally, add 50μl of SARS-CoV-2 pseudovirus solution to each well and incubate in a 37°C cell culture incubator for 1 hour. The last two columns are cell control and virus control, respectively. Replace the antibody supernatant with an equal volume of PBS buffer as a virus control. Replace the pseudovirus solution with an equal volume of DMEM culture medium containing 10% fetal bovine serum as a cell control.

[0069] 3) Take the above cell culture plate and add 50 μl of ACE2-HEK293T cell suspension to each of the 60 wells in the middle, with 2×10 4 ACE2-HEK293T cells were cultured in a 37°C cell culture incubator for 48 hours.

[0070] 4) Take the above cell culture plate, discard the supernatant, add 100 μl PBS and 20 μl luciferase reporter gene detection reagent to each well, and incubate at room temperature in the dark for 2 minutes.

[0071] 5) Take the above cell culture plate and detect the luciferase activity.

[0072] One set of duplicate wells was set for each antibody supernatant. The neutralization experiment was repeated twice independently.

[0073] Neutralization activity (%) = [1-(fluorescence intensity of the test group-fluorescence intensity of the cell control) / (fluorescence intensity of the virus control-fluorescence intensity of the cell control)] x 100%.

[0074] The experimental results are shown in Table 1.

[0075] Broad-spectrum neutralizing activity was defined as a neutralization activity >80% against SARS-CoV-2 WT pseudovirus and >60% against SARS-CoV-1 pseudovirus when the cell culture antibody supernatant was diluted 4.5 times. Four strains achieved broad-spectrum neutralizing activity, named KXD350, KXD352, KXD355, and KXD358.

[0076] Table 1 ELISA binding and pseudovirus neutralization activity antibody screening

[0077]

[0078]

[0079] 1.6 Antibody Preparation

[0080] 1.6.1 The heavy chain expression plasmids and light chain expression plasmids of the four antibodies screened above were co-transfected into 293F cells to obtain recombinant cells. The cells were cultured in SMM 293-TII complete medium for 96 h, then centrifuged at 4°C and 3000 rpm for 30 min, and the supernatant was collected.

[0081] 1.6.2 Protein A magnetic bead affinity chromatography to purify antibodies

[0082] Filler for affinity chromatography: Protein A magnetic beads; elution buffer: 0.1 M glycine, pH 3.0; neutralization buffer: 1 M Tris, pH 8.5.

[0083] Mix 50 ml of supernatant with Protein A magnetic beads and incubate at 4 °C for 16 h; separate the solution and beads using a magnetic stand. Add 20 ml of PBS solution to the magnetic beads, rotate to mix, wash the beads for 5 min, collect the beads using a magnetic stand and discard the supernatant; repeat the washing step three more times. Add 9 ml of elution buffer to the magnetic beads, quickly resuspend, mix evenly, flip and mix at room temperature, and incubate for 5 min. Collect the magnetic beads using a magnetic separation stand and transfer the supernatant containing the eluted antibody to a clean centrifuge tube. Immediately add 1 ml of neutralization buffer to the 9 ml of elution solution to neutralize the pH to maintain the biological activity of the antibody and avoid antibody inactivation.

[0084] 1.6.3 Solution replacement

[0085] The antibody solution obtained in step 2 was concentrated using an ultrafiltration tube, and the solution was replaced with PBS buffer to obtain an antibody solution with a protein concentration of 1 mg / ml.

[0086] Example 2

[0087] Broad spectrum neutralization test

[0088] 1 Preparation of pseudoviruses of SARS-CoV-1, SARS-CoV-2 and their variants

[0089] Here are the steps:

[0090] S1. The genes of artificially synthesized SARS-CoV-1 Spike, SARS-CoV-2 WT Spike and its variants, including Delta, BA.1, BA.4 / 5, BA.2.75, BQ.1, XBB.1.5, EG.5.1, HK.3, BA.2.86, JN.1, KP.2 and KP.3, were cloned into the pcDNA3.1 expression vector.

[0091] S2. Co-transfect HEK-293T cells with the recombinant plasmid pcDNA3.1 / Spike, plasmid pLenti-CMV-puro-Luciferase and plasmid psPAX2 to obtain recombinant cells.

[0092] The packaging plasmid psPAX2 is a plasmid that can express the lentiviral capsid, and its expression product can more easily pass through the cell membrane through the adhesion mechanism. The plasmid pLenti-CMV-puro-Luciferase is a lentiviral vector plasmid that expresses the reporter luciferase gene in the host cell. Replacing the envelope glycoprotein in the lentiviral vector with the new coronavirus S protein can form a pseudovirus that simulates the new coronavirus infection. The pseudovirus infects the target cell through the surface S protein and expresses the reporter luciferase gene.

[0093] S3. The recombinant cells obtained in step S2 were cultured in DMEM medium containing 10% fetal bovine serum for 60 hours.

[0094] S4. Collect the culture supernatant and centrifuge to obtain the supernatant, which is the virus liquid of the pseudovirus of the original strain of SARS-CoV-1, SARS-CoV-2 and its variants.

[0095] S5. Detect the virus titer in each pseudovirus solution using a luciferase reporter gene detection reagent.

[0096] 2 Antibody neutralization activity detection

[0097] Neutralizing antibodies can block the binding of pseudovirus Spike protein and receptor ACE2, thereby preventing pseudovirus from infecting host cell ACE2-HEK293T. By detecting the expression of reporter gene luciferase, the degree of virus blocking can be inferred, thereby screening or verifying neutralizing antibodies.

[0098] The test antibody solutions were the antibody solutions KXD350, KXD352, KXD355, and KXD358 prepared in Example 1, respectively.

[0099] Here are the steps:

[0100] S1. Prepare antibodies and pseudoviruses

[0101] The purified monoclonal antibodies and pseudovirus solutions were taken out from -80°C and thawed at room temperature. The relative titer of the pseudovirus was adjusted to about 100,000 (the reading value of the luciferase reporter gene). The antibodies were sterilized by filtration and the concentration was adjusted to 0.5 mg / ml.

[0102] S2. Take a 96-well cell culture plate, add 200μl PBS to the wells around the 96-well plate, add 100μl culture medium to each of the remaining wells, add 45.6μl culture medium to each well in the first column, add 4.4μl antibody to each well, mix well, take 50μl to the next column of wells and mix well, perform 3-fold dilution, a total of 8 dilutions; finally, add 50μl pseudovirus solution to each well. The starting concentration of the antibody is 10ng / μl, and it is incubated in a 37°C cell culture incubator for 1 hour. The last two columns are cell control and virus control, respectively. Replace the antibody diluent with an equal volume of PBS buffer (pH 7.4, 10mM) as a virus control. Replace the SARS-CoV-2 virus solution with an equal volume of DMEM medium containing 10% fetal bovine serum as a cell control.

[0103] S3. Take out the cell culture plate described in step S2, add 50 μl of ACE2-HEK293T cell suspension to all the middle 60 wells, and add 2×10 4 ACE2-HEK293T cells were cultured in a 37°C cell culture incubator for 48 hours.

[0104] S4. Take out the cell culture plate after completing step 3, discard the supernatant, add 100 μl PBS and 20 μl luciferase reporter gene detection reagent to each well, and incubate at room temperature in the dark for 2 minutes.

[0105] S5. Take out the cell culture plate after completing step 4 and detect the luciferase activity.

[0106] One set of duplicate wells was set for each antibody, and the neutralization experiment was repeated once independently.

[0107] Neutralization activity (%) = [1-(fluorescence intensity of the test group-fluorescence intensity of the cell control) / (fluorescence intensity of the virus control-fluorescence intensity of the cell control)] x 100%.

[0108] The antibody concentration at which the neutralization activity is 50% is calculated using Prism 8 software, i.e., the IC of the antibody. 50 value.

[0109] The results of neutralization IC50 values ​​are shown in Table 2 and Figure 3 .

[0110] Table 2 Neutralization IC of antibodies against pseudoviruses 50 Value (ng / μl)

[0111]

[0112]

[0113] The results of the neutralization test showed that the IC values ​​of the four antibodies KXD350, KXD352, KXD355, and KXD358 were higher for SARS-CoV-2 WT, Delta, BA.1, BA.4 / 5, BA.2.75, BQ.1, XBB.1.5, EG.5.1, HK.3, BA.2.86, JN.1, KP.2, and KP.3 pseudoviruses as well as SARS-CoV-1 pseudoviruses. 50 The values ​​are all small, and they have strong and broad-spectrum neutralizing activity.

[0114] In general, the experimental results show that the four antibodies KXD350, KXD352, KXD355, and KXD358 have strong neutralizing efficacy against SARS-CoV-1 and SARS-CoV-2 and their different variants, including the latest popular strains EG.5.1, HK.3, JN.1, KP.2, and KP.3, and all have strong broad-spectrum neutralizing activity. The coronaviruses that can infect humans among the Sabei coronavirus are SARS-CoV-1 and SARS-CoV-2 and their variants. It can be seen that these four antibodies are strong broad-spectrum neutralizing antibodies for Sabei coronavirus, which are worth further research and development into therapeutic drugs. See the sequence table for the antibodies and sequences involved in the embodiments.

[0115] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Sabei coronavirus broad-spectrum neutralizing antibodies, characterized in that, The neutralizing antibody is a combination of any one or more of KXD350, KXD352, KXD355 and KXD358, wherein KXD350, KXD352, KXD355 and KXD358 all comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has three heavy chain CDRs, and the light chain variable region has three light chain CDRs, wherein: The amino acid sequences of the three CDRs of the KXD350 heavy chain variable region are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the three CDRs of the KXD350 light chain variable region are the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; The amino acid sequences of the three CDRs in the heavy chain variable region of KXD352 are the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; the amino acid sequences of the three CDRs in the light chain variable region of KXD352 are the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively; The amino acid sequences of the three CDRs in the heavy chain variable region of KXD355 are the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; the amino acid sequences of the three CDRs in the light chain variable region of KXD355 are the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; The amino acid sequences of the three CDRs in the heavy chain variable region of KXD358 are the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; the amino acid sequences of the three CDRs in the light chain variable region of KXD358 are the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

2. The broad-spectrum neutralizing antibody against Sabella coronavirus according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of KXD350 is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region of KXD350 is shown in SEQ ID NO: 26; the amino acid sequence of the heavy chain variable region of KXD352 is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region of KXD352 is shown in SEQ ID NO: 28; the amino acid sequence of the heavy chain variable region of KXD355 is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region of KXD355 is shown in SEQ ID NO: 30; the amino acid sequence of the heavy chain variable region of KXD358 is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of KXD358 is shown in SEQ ID NO:

32.

3. The neutralizing antibody according to any one of claims 1 to 2, characterized in that Also included is the Fc domain.

4. The neutralizing antibody according to claim 3, characterized in that The Fc domain is a human IgG Fc domain.

5. The antibody according to claim 3, characterized in that It also includes an immune marker coupled to the neutralizing antibody, wherein the immune marker is any one of an enzyme marker, a fluorescent marker, an isotope marker, and a biotin marker.

6. A polynucleotide encoding the neutralizing antibody according to any one of claims 1 to 4. An expression vector comprising the polynucleotide according to claim 6 . A host cell comprising the expression vector according to claim 7 .

9. Use of the neutralizing antibody described in any one of claims 1 to 4 in the preparation of a medicament for treating and / or preventing Sabei coronavirus infection.

10. The use according to claim 9, characterized in that: The Sabei coronavirus includes SARS-CoV-1 and SARS-CoV-2 and their variants, and the variants include Delta, BA.1, BA.4 / 5, BA.2.75, BQ.1, XBB.1.5, EG.5.1, HK.3, BA.2.86, JN.1, KP.2, and KP.

3.

11. A pharmaceutical composition comprising the neutralizing antibody according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

12. Use of the neutralizing antibody described in any one of claims 1 to 5 in the preparation of a kit for detecting Sabei coronavirus.

13. A kit for detecting Sabae coronavirus, comprising the neutralizing antibody according to any one of claims 1 to 5.

14. Use of the neutralizing antibody described in any one of claims 1 to 4 in the preparation of a vaccine for Sabei coronavirus infection.

15. A vaccine comprising the antibody according to any one of claims 1 to 4.

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