A sars-cov-2 spike protein antibody and use thereof

Abstract

The present application relates to the field of therapeutic antibodies and molecular immunology, and specifically provides an antibody against SARS-CoV-2 coronavirus S protein and the use thereof in the preparation of a medicament for treating COVID-19. The antibody can specifically recognize and bind to SARS-CoV-2 coronavirus S protein with high affinity, which ensures that the antibody can block the infection of SARS-CoV-2 to human cells.

Description

Technical Field

[0001] This invention relates to the field of therapeutic antibodies and molecular immunology, and more specifically, to a recombinant monoclonal antibody against the SARS-CoV-2 coronavirus S protein, and the use of such an antibody, particularly in the treatment, prevention and diagnosis of COVID-19 disease caused by SARS-CoV-2. Background Technology

[0002] The novel coronavirus SARS-CoV-2, a newly emerging human pathogen, can cause severe respiratory illness and COVID-19 pneumonia, primarily characterized by fever, fatigue, and dry cough. According to data from the World Health Organization on August 19, 2020, there were 21,756,357 confirmed cases globally, resulting in 771,635 deaths. Based on its genomic nucleic acid sequence, the new pathogen has been identified as a novel member of the β-coronavirus genus. SARS-CoV-2 shares 96.2% genomic sequence similarity with bat coronavirus RaTG13 (Zhou P et al, 2020, Nature, 579:270-273), and is closely related to two bat SARS-like coronaviruses, bat-SL-CoVZC45 (88% homology) and bat-SL-CoVZXC21 (87% homology), but is relatively distantly related to SARS-CoV (79% homology) and MERS-CoV (50% homology) (Lu R et al, 2020, Lancet, 395:565-574). Compared to SARS-CoV, SARS-CoV-2 coronavirus is more easily transmitted between humans. The WHO has declared COVID-19 a global pandemic, and the novel coronavirus has now spread worldwide.

[0003] Like other coronaviruses, the novel coronavirus SARS-CoV-2 is a positive-sense RNA virus that encodes several major proteins (S, M, N, and E), the RNA-dependent RNA polymerase RDRP, and more than a dozen non-structural proteins. The S, M, N, and E proteins are used to package the viral structure, while RDRP and the dozen or so non-structural proteins are used for viral genomic RNA replication and the synthesis of various protein mRNAs. SARS-CoV-2 is very similar to SARS-CoV, with high amino acid sequence homology. The amino acid number and homology of its S, M, N, E, and RDRP proteins with SARS-CoV are 1273 (76%), 222 (91%), 419 (91%), 75 (95%), and 932 (96%), respectively. Similar to SARS-CoV, the SARS-CoV-2 virus is spherical, enveloped, and has corona-like spikes arranged on its periphery. The spike protein of SARS-CoV-2 forms a trimer (Wrapp D et al., 2020, Science, 6483:1260-1263), which is mushroom-shaped and embedded in the outer membrane of the virus. The S protein is the main antigenic component of this virus, responsible for binding to the ACE2 receptor on the invaded host cell and for viral fusion with the cell. Similar to the SARS-CoV virus S protein (Yuan Yet et al., 2017, Nat Commun., 8:15092), the SARS-CoV-2 coronavirus S protein is mainly divided into two domains, S1 (1-685) and S2 (686-1122), as well as a short transmembrane region and a cytoplasmic tail. In the mushroom-shaped S protein trimer, the three S1 domains form the "mushroom cap," and the three S2 domains form the "mushroom stem." Specifically, the RBD domain (receptor-binding domain, amino acids 331-527) in S1 is responsible for binding to the ACE2 receptor on the invaded host cell, while S2 is responsible for fusion with the host cell. The S2 domain typically exists in a folded or coiled-up conformation within the overall S protein. However, when the virus fuses with the host cell after S1 detachment, S2 exhibits an extended conformation to insert into the host cell membrane (Walls AC et al., 2017, Proc Natl Acad Sci USA, 114:11157-11162). It has been reported that the binding affinity of the SARS-CoV-2 virus's S protein to human cell receptors is significantly higher than that of the SARS-CoV virus's S protein (Wrapp D et al., 2020, Science, 6483:1260-1263; Walls AC et al., 2020, Cell, 181:281-292).Another difference between the SARS-CoV virus S protein and the SARS-CoV-2 S protein is that the SARS-CoV-2 S protein has a Furin restriction site RRAR (amino acids 682-685). This restriction site divides the S protein into two parts, S1 and S2, which are linked together by non-covalent bonds after restriction. Because of the presence of a Furin cleavage site between S1 and S2, and the widespread expression of Furin enzymes in eukaryotic tissues and cells; and because the Furin site, which contains multiple basic amino acids, can also be degraded by other enzymes targeting lysine or arginine, such as the cell surface enzyme TMPRSS2, endosome cathepsin L enzyme, or possibly trypsin (Hoffmann M et al, 2020, Cell, 181(2): 271-280.e8; Shang J et al, 2020, Proc Natl Acad Sci USA, 117: 11727-11734; Belozard S et al, 2012, Viruses, 4: 1011-1033). Therefore, the S1 / S2 domain of SARS-CoV-2 is more easily cleaved, leading to the easier shedding of the S1 domain during viral fusion with human host cells, thereby increasing the fusion capacity of S2 and viral infectivity. The SARS-CoV virus S1 / S2 is linked by only one basic amino acid, arginine. The S protein is cleaved at this site by the cell surface enzyme TMPRSS2 and the endosome cathepisin L, thus infecting host cells (Belouzard S et al., 2012, Viruses, 4:1011-1033; Belozard S et al., 2009, Proc Natl Acad Sci USA, 106:5871-5876). Therefore, these two differences—the presence of the Furin cleavage site and the high affinity for the human receptor ACE2—may be the reasons for the high infectivity of SARS-CoV-2 coronavirus. Since the S protein is responsible for binding to human host cell receptors and fusing with host cells, it is a major target for therapeutic neutralizing antibodies against SARS-CoV and SARS-CoV-2 coronaviruses.

[0004] Currently, there are no specific drugs for treating SARS-CoV-2 coronavirus. Vaccines and neutralizing antibodies appear to be the most promising treatments. Neutralizing antibodies prevent viral transmission by blocking the virus from invading host cells, thus achieving the goal of treating the disease. Numerous studies have recently reported on neutralizing antibodies against SARS-CoV-2. For example, Regeneron Pharmaceuticals developed a series of SARS-CoV-2 neutralizing antibodies targeting the RBD domain using transgenic mice and a single B-cell sequencing platform (HansenJ et al, 2020, Science, 369:1010-1014). Other researchers have also developed a series of neutralizing antibodies against the RBD domain of the SARS-CoV-2 S protein using a single B-cell sequencing technology platform (Wu Y et al, 2020, Science, 368, 1274-1278; Cao Y et al, 2020, Cell, 182:73-84; Pinto D et al, 2020, Nature, 583:290-295; Ju B et al, 2020, Nature, 584:115-119).

[0005] Currently, three neutralizing antibodies against the SARS-CoV-2 coronavirus S protein have entered clinical trials. On June 1, 2020, Eli Lilly and AbCellera's neutralizing antibody LY-CoV555 completed its first patient dosing and entered Phase I clinical trials. LY-CoV555 is an effective neutralizing antibody against the SARS-CoV-2 spike protein S of the IgG1 subtype. On June 11, Regeneron Pharmaceuticals' dual-antibody cocktail REGN-COV2 entered its first clinical trial phase, and based on favorable safety data from Phase I clinical trials, this study has now directly entered Phase III clinical trials. In June of this year, Junshi Biosciences' recombinant fully human anti-SARS-CoV-2 monoclonal antibody injection (JS016), jointly developed with the Institute of Microbiology, Chinese Academy of Sciences, was approved to enter Phase I clinical trials. JS016 is the earliest COVID-19 neutralizing antibody to enter clinical trials in China. Given the current severe global COVID-19 pandemic, the rapid development of neutralizing antibodies against the SARS-CoV-2 coronavirus S protein, which have higher specificity, better clinical efficacy, and lower treatment costs, will provide more treatment options for patients infected with SARS-CoV-2. Summary of the Invention

[0006] This invention provides an antibody capable of specifically recognizing and binding with high affinity to the SARS-CoV-2 coronavirus S protein. The antibody of this invention can block SARS-CoV-2 infection of host cells. The antibody against the SARS-CoV-2 virus S protein disclosed in this invention can be used (alone or in combination with other agents or treatments) for the treatment, prevention, and / or diagnosis of diseases caused by SARS-CoV-2, such as COVID-19.

[0007] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to the SARS-CoV-2 coronavirus S protein, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) containing at least one, two, or three complementarity-determining regions (CDRs) selected from the group consisting of:

[0008] (i)HCDR1, having a sequence as shown in SEQ ID NO: 1 or 2, or having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 substitutions, deletions or additions) compared to any of the sequences described above;

[0009] (ii) HCDR2 having a sequence as shown in SEQ ID NO: 3 or 4, or having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 substitutions, deletions, or additions) compared to any of the sequences described above; and

[0010] (iii) HCDR3 having a sequence as shown in SEQ ID NO: 5 or 6, or having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 substitutions, deletions or additions) compared to any of the sequences described above;

[0011] And / or, the light chain variable region (VL) it contains includes at least one, two, or three complementary determinant regions (CDRs) selected from the following group:

[0012] (iv) LCDR1, having a sequence as shown in SEQ ID NO: 7 or 8, or having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 substitutions, deletions or additions) compared to any of the sequences described above;

[0013] (v) LCDR2, having a sequence as shown in SEQ ID NO: 9 or 10, or having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 substitutions, deletions, or additions) compared to any of the sequences described above; and

[0014] (vi)LCDR3, having a sequence as shown in SEQ ID NO: 11 or 12, or having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 substitutions, deletions or additions) compared to any of the sequences described above.

[0015] In some preferred embodiments, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0016] In some preferred embodiments, the HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region, and / or the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region, are defined by the Kabat or IMGT numbering system. Table 2 in Example 5 exemplarily shows the CDR amino acid sequences of the murine antibody as defined by the Kabat or IMGT numbering system.

[0017] In some preferred embodiments, the antibody or its antigen-binding fragment comprises three VH variable region CDRs and three VL variable region CDRs, selected from the group consisting of:

[0018] (i) Their HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 each have a sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or have one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or additions) compared to any of the above sequences;

[0019] (ii) Their HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 each have the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12, or have one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or additions) compared to any of the above sequences.

[0020] In (i), the three VH variable area CDRs and three VL variable area CDRs are defined by the Kabat numbering system; in (ii), the three VH variable area CDRs and three VL variable area CDRs are defined by the IMGT numbering system.

[0021] In some embodiments, the antibody or its antigen-binding fragment is murine or chimeric, with its heavy chain variable region comprising the heavy chain FR region of murine IgG1, IgG2, IgG3 or variants thereof; and its light chain variable region comprising the light chain FR region of murine κ, λ chains or variants thereof. Table 3 in Example 5 provides the amino acid sequence numbers of the variable regions of preferred murine antibodies.

[0022] In some preferred embodiments, the VH domain of the murine antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 13, or a sequence substantially identical to the above sequence (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g., conserved substitutions)); and its VL domain comprises an amino acid sequence as shown in SEQ ID NO: 14, or a sequence substantially identical to the above sequence (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g., conserved substitutions)).

[0023] In some embodiments, the antibody or its antigen-binding fragment is humanized. Table 3 in Example 5 provides the variable region amino acid sequence numbers of some preferred humanized antibodies.

[0024] In some preferred embodiments, the VH domain of the humanized antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 15, or a sequence substantially identical to the above sequence (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g., conserved substitutions)); and its VL domain comprises an amino acid sequence as shown in SEQ ID NO: 16, or a sequence substantially identical to the above sequence (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity or having one or more amino acid substitutions (e.g., conserved substitutions)).

[0025] In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region derived from human immunoglobulins.

[0026] More preferably, the antibody comprises the amino acid sequence of the constant region of the human κappa chain (amino acid sequence as shown in SEQ ID NO: 17).

[0027] More preferably, the antibody comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; more preferably, it comprises a heavy chain constant region selected from human IgG1, IgG2, and IgG4; and the heavy chain constant region has a natural sequence or a sequence with one or more amino acid substitutions, deletions, or additions compared to its derived natural sequence. For example, in one embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG1 (amino acid sequence as shown in SEQ ID NO: 18). In another embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG2 (amino acid sequence as shown in SEQ ID NO: 19). In one embodiment, the humanized antibody molecule comprises human IgG2 modified according to EU numbers (e.g., ERKCC deletion, amino acid sequence as shown in SEQ ID NO: 20), see Chinese Patent No. CN104177496B. In another embodiment, the humanized antibody molecule includes human IgG4 (amino acid sequence as shown in SEQ ID NO: 21) with a mutation at position 228 according to EU number (e.g., S to P).

[0028] In some preferred embodiments, the heavy chain of the antibody has an amino acid sequence as shown in SEQ ID NO: 22; or has one or more substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 substitutions, deletions, or additions) compared to any of the above sequences; or has a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher identity compared to any of the above sequences; and / or, the light chain of the antibody has an amino acid sequence as shown in SEQ ID NO: 22. The amino acid sequence shown in NO:23; or a sequence having one or more substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the above sequences; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher identity compared to any of the above sequences.

[0029] In some preferred embodiments, the above-described substitution is a conservative substitution.

[0030] In any of the above embodiments, the antibody or its antigen-binding fragment of the present invention can be delivered at 10 nM or lower K. D Preferred to bind to the SARS-CoV-2 coronavirus S protein, at 1 nM or lower KD Binds to the S protein; preferably, with 100 pM or lower K D Binds to the S protein; more preferably, with 10 pM or less K D Binds to the S protein; most preferably, with 1 pM or less K D Binds to the S protein.

[0031] In a second aspect, the present invention provides a DNA molecule encoding the aforementioned antibody or an antigen-binding fragment thereof.

[0032] In a preferred embodiment of the present invention, the DNA molecule encoding the antibody heavy chain has a nucleotide sequence as shown in SEQ ID NO: 24; and / or, the DNA molecule encoding the antibody light chain has a nucleotide sequence as shown in SEQ ID NO: 25.

[0033] A third aspect of the present invention provides a vector comprising the aforementioned DNA molecule.

[0034] In a fourth aspect, the present invention provides a host cell comprising the above-described carrier; said host cell comprising prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells.

[0035] A fifth aspect of the present invention provides a pharmaceutical composition comprising the antibody described above or an antigen-binding fragment thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0036] A sixth aspect of the present invention also provides a method for preparing the antibody or antigen-binding fragment thereof, comprising: (a) obtaining a gene for the antibody or antigen-binding fragment thereof, and constructing an expression vector for the antibody or antigen-binding fragment thereof; (b) transfecting the expression vector into a host cell by a genetic engineering method; (c) culturing the host cell under conditions that allow the production of the antibody or antigen-binding fragment thereof; and (d) isolating and purifying the produced antibody or antigen-binding fragment thereof.

[0037] In step (a), the expression vector is selected from one or more of plasmids, bacteria and viruses, and preferably, the expression vector is pcDNA3.1;

[0038] In step (b), the constructed vector is transfected into a host cell using genetic engineering methods. The host cell includes prokaryotic cells, yeast cells, or mammalian cells, such as CHO cells, NSO cells, or other mammalian cells, preferably CHO cells.

[0039] Step (d) involves using conventional immunoglobulin purification methods, including protein A affinity chromatography and ion exchange, hydrophobic chromatography, or molecular sieving to separate and purify the antibody or its antigen-binding fragment.

[0040] A seventh aspect of the invention provides the use of the antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating and preventing a disease caused by the SARS-CoV-2 coronavirus; preferably, the disease is COVID-19.

[0041] An eighth aspect of the present invention provides an immunoassay method for detecting or determining the presence or quantification of SARS-CoV-2 virus or its antigen in a biological sample using the aforementioned antibody; the method includes incubating the biological sample to be tested with the anti-SARS-CoV-2 virus S protein monoclonal antibody or its antigen-binding fragment of the present invention to form an antigen-antibody complex, and qualitatively and quantitatively detecting and determining the formed binding complex, wherein the presence or quantity of the complex indicates the presence or content of SARS-CoV-2 virus; specifically, the method includes the following steps:

[0042] (1) Incubate the biological sample to be tested with at least one monoclonal antibody or its antigen-binding fragment of the present invention under suitable conditions;

[0043] (2) Detect the presence of the binding complex in the above steps.

[0044] The monoclonal antibody or its antigen-binding fragment according to the present invention can be used in the above-mentioned immunoassay methods without depending on the label used (e.g., enzyme, fluorescence, etc.) and without depending on the detection mode (e.g., fluorescence immunoassay, enzyme-linked immunosorbent assay, or chemiluminescence assay, etc.) or the principle of the assay (e.g., sandwich assay, competitive assay, etc.); wherein, examples of the antigen-binding fragment include, but are not limited to, F(ab')2, Fab', Fab and Fv.

[0045] The aforementioned immunoassay methods include enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, Western blotting, immunochromatography, latex agglutination assay, etc.; and all of the above immunoassay methods can be used to determine target antigens in biological samples by using labeled antigens or antibodies in a competitive or sandwich manner.

[0046] The above-described competitive method is based on a quantitative competitive binding reaction between the SARS-CoV-2 virus in the sample and a known amount of labeled SARS-CoV-2 virus S protein and the monoclonal antibody or its antigen-binding fragment of the present invention. Specifically, the competitive method includes: embedding a predetermined amount of the monoclonal antibody or its antigen-binding fragment against the SARS-CoV-2 virus S protein of the present invention onto a solid-phase support; then adding the biological sample containing SARS-CoV-2 virus to be detected and a predetermined amount of labeled SARS-CoV-2 virus S protein, and incubating under appropriate conditions for a sufficient time; after the reaction, thoroughly washing the solid phase and detecting the signal value of the label retained on the support or not retained on the support; then comparing the measured signal value with the signal value of a predetermined amount of control sample measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample; preferably, the labeled antigen and the biological sample to be detected are added almost simultaneously.

[0047] The sandwich method described above is based on the fact that the monoclonal antibody or its antigen-binding fragment of the present invention, as a capture antibody (or solid-phase antibody), and the labeled antibody that can be used in conjunction can specifically bind to the SARS-CoV-2 virus in biological samples. The content of SARS-CoV-2 virus in the sample is determined by quantifying the labeled antibody. Specifically, the sandwich method includes: binding the specific monoclonal antibody against the SARS-CoV-2 virus S protein of the present invention or its antigen-binding fragment to a solid-phase carrier to form a solid-phase antibody (also called a capture antibody or a first antibody); then adding the biological sample to be tested and the control sample to the coated solid-phase carrier respectively and incubating under appropriate conditions for a sufficient time; after the reaction, thoroughly washing the solid phase and adding an appropriate amount of labeled second antibody that can bind to the SARS-CoV-2 virus S protein and incubating again; after the reaction, thoroughly washing the solid phase and detecting the signal value of the label bound to the second antibody using a suitable method; comparing the measured signal value with the signal value of a predetermined amount of control sample measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample.

[0048] The second antibody can also be other polyclonal antibodies; preferably, the second antibody is a monoclonal antibody.

[0049] More preferably, the second antibody is selected from any monoclonal antibody or antigen-binding fragment thereof that can be used in conjunction with the first antibody according to the present invention.

[0050] The marker can be a radioactive isotope (e.g.) 125 I) Enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin, and coloring substances.

[0051] Preferably, the markers used in this invention include alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, and glucose oxidase; the markers can also be fluorescent substances, such as fluorescein derivatives and rhodamine derivatives; additionally, the markers can be rare earth elements or rare earth element complexes that allow time-resolved fluorescence measurement, such as europium or europium complexes; furthermore, the markers can be phosphorescent substances, such as acridine esters and isoluminol; or radioactive isotopes such as... 125 I, 3 H, 14 C and 32 P; Additionally, the marker can be a coloring substance, such as latex particles and colloidal gold. That is, this invention includes the qualitative or quantitative determination of the presence or content of SARS-CoV-2 virus in biological components by measuring color, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemical fluorescence, or radioactivity.

[0052] When performing SARS-CoV-2 immunoassays using the competitive and sandwich methods described above, the solid phase must be thoroughly washed, and the activity bound to the label must be measured. When the label is a radioactive isotope, a well counter or liquid chromatography scintillation counter is used for measurement. When the label is an enzyme, a substrate is added, and enzyme activity is measured colorimetrically or fluorescently after color development. When the label is a fluorescent, phosphorescent, or coloring substance, it can be measured using methods known in the art.

[0053] The biological samples mentioned above were selected from plasma, whole blood, mouthwash, throat swabs, urine, feces, and bronchial irrigation fluid.

[0054] The solid-phase carriers mentioned above include, but are not limited to, nitrocellulose membranes, latex particles, magnetic particles, colloidal gold, beads, or materials such as glass, fiberglass, polymers (e.g., polystyrene or polyvinyl chloride), or fiber optical sensors.

[0055] A ninth aspect of the present invention provides the use of the above-described antibody or its antigen-binding fragment in the preparation of a SARS-CoV-2 virus detection kit.

[0056] In a tenth aspect, the present invention provides a SARS-CoV-2 virus detection kit comprising at least one monoclonal antibody or antigen-binding fragment thereof of the present invention; the monoclonal antibody used to prepare the detection kit is not particularly limited, and may be any of the monoclonal antibodies or antigen-binding fragments thereof of the present invention (such as F(ab')2, Fab', Fab and scFv) used alone as a solid-phase antibody or a labeled antibody; or two monoclonal antibodies or antigen-binding fragments thereof targeting different antigenic epitopes of the present invention may be used in combination as solid-phase antibodies or labeled antibodies respectively.

[0057] In a preferred embodiment of the present invention, the detection kit comprises:

[0058] (1) Selected from any of the following:

[0059] a. Solid-phase support and primary antibody;

[0060] b. A solid-phase carrier coated with a primary antibody;

[0061] The first antibody is selected from any monoclonal antibody or its antigen-binding fragment of the present invention;

[0062] (2) Second antibody;

[0063] The second antibody is optionally appropriately labeled, and the second antibody is selected from monoclonal antibodies or antigen-binding fragments thereof that are capable of cooperating with the first antibody in (1) according to the present invention.

[0064] The monoclonal antibody or its antigen-binding fragment contained in the above-mentioned detection reagent can be pre-immobilized on a solid-phase support to form a solid-phase antibody. The solid-phase support includes, but is not limited to, nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, beads, or such as glass, fiberglass, or polymers (such as polystyrene or polyvinyl chloride) or fiber optical sensors. In a preferred embodiment of the present invention, the solid-phase support is a microtiter plate.

[0065] The monoclonal antibody or its antigen-binding fragment contained in the above-described immunoassay reagents can be pre-labeled with a marker to form labeled antibodies. The markers include, but are not limited to, radioactive isotopes (e.g., radioactive isotopes). 125 I) Enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin, and coloring substances; preferably, the enzymes include alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, and glucose oxidase; the fluorescent substances include fluorescein derivatives and rhodamine derivatives, as well as rare earth elements or rare earth element complexes, such as europium or europium complexes; the phosphorescent substances include acridine esters and isoluminol; the radioactive isotopes include... 125 I, 3 H, 14 C and 32 P; the coloring substance includes, for example, latex particles and colloidal gold; in a preferred embodiment of the invention, the marker is biotin.

[0066] In an eleventh aspect, the present invention provides the use of the above-described immunoassay reagent in diagnosing diseases caused by SARS-CoV-2 virus infection; preferably, the disease is novel coronavirus pneumonia.

[0067] The technical solution disclosed in this invention has achieved beneficial technical effects, which are summarized as follows:

[0068] 1. Using a mouse hybridoma platform, mice were immunized with the S protein (amino acids 326-685) as an immunogen to obtain mouse antibodies against the SARS-CoV-2 coronavirus S protein. These antibodies can specifically recognize and bind to the S protein with high affinity, and the KD value reaches the pM level.

[0069] 2. In vitro ACE2 competition assay results showed that these murine antibodies competed with the human ACE2 receptor for the binding site of the S protein, with an IC50 concentration of 100%. 50 Values ​​as low as nM.

[0070] 3. We humanized the murine antibody, reducing its immunogenicity. The humanized antibody retained the affinity and pseudovirus inhibitory activity of the murine antibody, and its binding affinity K... D The values ​​reach the pM level, while the pseudovirus inhibitory activity is equivalent to the nM level. These characteristics lay the foundation for the clinical application of the antibody.

[0071] 4. The antibody provided by the present invention can also be used to detect the presence of SARS-CoV-2 virus or its corresponding antigen in a sample, wherein the detection sensitivity of the antibody is less than 100 pg / ml; more preferably, less than 10 pg / ml. Invention Details

[0073] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, approaches, and reagents described herein. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0074] Abbreviations and Definitions

[0075] CDR (Complementarity-Determining Region) is the complementarity-determining region within the immunoglobulin variable region, defined using the Kabat, IMGT, Chothia, or AbM numbering system (see terms “hypervariate region”, “CDR region”, or “complementarity-determining region”).

[0076] EC 50 To produce 50% efficacy or a combined concentration

[0077] ELISA (Enzyme-Linked Immunosorbent Assay)

[0078] FR (Framework Region) is the immunoglobulin variable region that excludes the CDR (Cellular Defibrillator) region.

[0079] HRP (Hordeum peroxidase)

[0080] IC 50 A concentration that produces 50% inhibition.

[0081] IgG Immunoglobulin G

[0082] Kabat is an immunoglobulin amino acid sequence alignment and numbering system pioneered by Elvin A. Kabat.

[0083] mAb monoclonal antibody

[0084] PCR Polymerase Chain Reaction

[0085] Region V is a segment of the IgG chain whose sequence varies between different antibodies. It extends to the Kabat residue at position 109 of the light chain and the residue at position 113 of the heavy chain.

[0086] VH Immunoglobulin Heavy Chain Variable Region

[0087] VL Immunoglobulin Light Chain Variable Region

[0088] K D Equilibrium dissociation constant

[0089] k a Binding rate constant

[0090] k d Dissociation rate constant

[0091] The term "EC" 50 "" refers to the concentration of the antibody or its antigen-binding fragment that induces a 50% response in an in vitro or in vivo assay using the antibody or its antigen-binding fragment, i.e., the concentration at which the maximum response is half of the baseline.

[0092] The term "EU Numbering System" (or Scheme) refers to Eu, the first human IgG1 immunoglobulin isolated and purified by Gerald M. Edelman et al. in the late 1960s (1968-1969). They named it Eu, determined its amino acid sequence, and assigned it a number (Edelman GM et al, 1969, Proc Natl Acad USA, 63:78-85). The amino acid sequences of the heavy chain constant regions of other immunoglobulins are compared with Eu, and the corresponding amino acid position is the Eu number. The Eu numbering system primarily targets the heavy chain constant regions of immunoglobulins, including CH1, CH2, CH3, and the hinge region.

[0093] The term "Kabat Numbering System" (or Scheme) refers to a standardized numbering scheme for variable regions of human immunoglobulins first proposed by Kabat et al. in 1979 (Kabat EA, Wu TT, Bilofsky H, Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain and β2-Microglobulins. 1979. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health). In the book "Sequences of Immunologically Related Proteins" (Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C. 1991. Sequences of Proteins of Immunological Interest, 5th edition. Bethesda, MD: US Department of Health and Human Services, National Institutes for Health), Kabat et al. compared and numbered the amino acid sequences of antibody light and heavy chains. They found that the analyzed sequences exhibited variable lengths, with default and inserted amino acids or amino acid fragments appearing only at specific positions. Interestingly, insertion sites were mostly located inside the CDR, but could also appear at certain positions within the frame region. In the Kabat numbering scheme, the variable region of the light chain is numbered up to position 109, and the variable region of the heavy chain is numbered up to position 113. Inserted amino acids in both the light and heavy chains are identified and annotated with letters (e.g., 27a, 27b...). All Lambda light chains do not contain residue 10, and the Lambda and Kappa light chains are encoded by two different genes located on different chromosomes. The Lambda and Kappa light chains can be distinguished by the differences in their constant region amino acid sequences. Unlike the EU numbering system, which only targets the heavy chain constant region, the Kabat numbering system covers the full-length immunoglobulin sequence, including both the variable and constant regions of the immunoglobulin light and heavy chains.

[0094] The term "binding" defines the affinity interaction between a specific epitope on an antigen and its corresponding antibody, and is generally understood as "specific recognition." "Specific recognition" means that the antibody of this invention does not cross-react with, or substantially does not cross-react with, any polypeptide other than the target antigen. The degree of specificity can be determined using immunological techniques, including but not limited to Western blotting, immunoaffinity chromatography, and flow cytometry. In this invention, specific recognition is preferably determined using flow cytometry, while the criteria for specific recognition in specific cases can be determined by those skilled in the art based on their general knowledge in the field.

[0095] The term "antigen" refers to a foreign substance that can induce the production of antibodies in an organism or a human. It can be any substance that can trigger an immune response, such as bacteria or viruses. Foreign antigen molecules are recognized and processed by B cells or antigen-presenting cells (such as macrophages, dendritic cells, endothelial cells, and B cells), and bind to the major histocompatibility complex (such as MHC II molecules) to form a complex that reactivates T cells, triggering a continuous immune response.

[0096] The term "antigenic epitope" or "antigenic determinant" refers to a specific chemical group or peptide sequence on a molecule that possesses antigenicity (i.e., the ability to induce a specific immune response). It is a site on an antigen (e.g., the S protein of SARS-CoV-2) that is specifically bound to immunoglobulins or antibodies. Epitope-determining regions typically consist of chemically active surface groups (such as amino acids or glycosidic side chains) of the molecule and usually have specific three-dimensional structural properties and specific charge properties. There are two types of epitopes or antigenic determinants: B-cell epitopes and T-cell epitopes, recognized by B cells and T cells, respectively. When we usually refer to antigenic epitopes, we generally mean B-cell epitopes. B-cell epitopes are located on the surface of antigen molecules and are antigenic sites that bind to B-cell receptors (BCRs, antibodies located on the B-cell membrane). B-cell epitopes can be directly recognized by B cells without processing. Then, B cells engulf the antigen molecule, process it into small peptides (about 15 amino acids in size, T-cell epitopes), and present them to Th cells (helper T cells). Simultaneously, antigen molecules can also be processed into small peptides and presented to Th cells via another pathway, such as phagocytosis by macrophages. Th cells are co-stimulated by B cells and macrophages, and the three cell types interact. Th cells send feedback signals to B cells, instructing them to proliferate and differentiate into plasma cells and memory cells. Plasma cells have the function of secreting antibodies, mediating humoral adaptive immunity. Antibodies bind to antigen molecules through their variable region Fv and to receptors FcR on various immune cells through their constant region Fc, thereby guiding various immune cells to kill antigen molecules and performing ADCC (via NK cells), CDC (via complement), and ADCP (via macrophages). Each type of B cell is specific and can only secrete one type of antibody. B cell epitopes can be divided into continuous epitopes and conformational epitopes (or discontinuous epitopes) based on their continuity in the protein amino acid sequence. B cell antigenic epitopes vary in size, ranging from 5 to 20 amino acids. T-cell antigenic epitopes are recognized by T cells. Unlike B-cell epitopes, T-cell epitopes can be located anywhere on an antigen molecule (such as a viral protein), thus spanning the entire protein sequence. T-cell epitopes are continuous determinants, typically 10-20 amino acids in size. They bind to either class I (MHC I) or class II (MHC II) MHC molecules and are presented on the cell surface, where they are detected by two distinct subsets of T cells, CD8+. + T cells (cytotoxic T cells) and CD4 + T cell (helper Th cell) recognition. Therefore, T cell epitopes include CD8. + and CD4 +There are two types of T cell epitopes. MHC I molecules are expressed by almost all cells and can provide information about intracellular conditions. For example, if a cell is infected with a virus, small peptide molecules of viral fragments will be expressed on the cell surface via MHC I, which can be used to target the killer CD8 inhibitor. + T cells and other cells recognize and kill MHC II molecules. Most MHC II molecules are located on antigen-presenting cells, such as macrophages. These MHC II molecules provide information about the extracellular environment (e.g., body fluids). For example, if bacteria invade tissues, macrophages engulf the bacteria and then use MHC II to signal helper Th cells, initiating an immune response. B cells and T cells can only recognize and bind to epitopes of foreign antigen molecules. They do not bind to antigen fragments originating from the organism itself, such as protein molecules and their fragments. This is because during the differentiation, development, and maturation of B cells and T cells, B cells and T cells with high affinity for their own protein molecules or fragments are inhibited from maturing or undergo apoptosis.

[0097] The term "antibody" generally refers to a protein-binding molecule that functions as a class of immunoglobulins. Typical examples of antibodies are immunoglobulins, as well as their derivatives or functional fragments, provided they exhibit the desired binding specificity. Techniques for preparing antibodies are well known in the art. "Antibodies" include different classes of natural immunoglobulins (e.g., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, IgA1, IgA2, etc.). "Antibodies" also include non-natural immunoglobulins, including, for example, single-chain antibodies, chimeric antibodies (e.g., humanized mouse antibodies), and heteroconjugated antibodies (e.g., bispecific antibodies), as well as their antigen-binding fragments (e.g., Fab', F(ab')2, Fab, Fv, and rIgG). See also, for example, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, 11); Kuby J, Immunology, 3rd Ed., WH Freeman & Co., New York, 1997. Antibodies can bind to one antigen, called "monospecific"; to two different antigens, called "bispecific"; or to more than one different antigen, called "multispecific". Antibodies can be monovalent, divalent, or polyvalent, meaning they can bind to one, two, or more antigen molecules at a time. An antibody binds "monovalently" to a specific protein, meaning one molecule of antibody binds to only one protein molecule, but it can also bind to different proteins. When an antibody binds to each molecule of two different proteins, it is "monovalently" bound to each protein, and it is "bispecific" and "monovalently" bound to each of the two different proteins. Antibodies can be "monomers," meaning they contain a single polypeptide chain. Antibodies can contain multiple polypeptide chains ("multimers") or can contain two ("dimers"), three ("trimers"), or four ("tetramers") polypeptide chains. If the antibody is a polymer, it can be a homomultimer, meaning the antibody contains more than one molecule of a single polypeptide chain, including homodimers, homotrimers, or homotetramers. Optionally, a multimeric antibody can be a heteromultimer, meaning the antibody contains more than one different polypeptide chain, including heterodimers, heterotrimers, or heterotetramers.

[0098] The term "monoclonal antibody (mAb)" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that, apart from possibly minor mutations such as naturally occurring mutations, the individual antibodies in the population are identical. Therefore, the qualifier "monoclonal" indicates that the antibody is characterized as not being a mixture of discrete antibodies. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by fusing myeloma cells and immune spleen cells to prepare hybrid antibody-producing cells. Synthesis is achieved through hybridoma culture, preventing contamination by other immunoglobulins. Monoclonal antibodies can also be obtained using techniques such as recombinant technology, phage display technology, synthetic technology, or other existing technologies.

[0099] The term "intact antibody" refers to an antibody composed of two antibody heavy chains and two antibody light chains. An "intact antibody heavy chain" consists of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3) in the N-to-C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3; and in the case of IgE subclass antibodies, optionally also including an antibody heavy chain constant domain 4 (CH4). Preferably, an "intact antibody heavy chain" is a polypeptide composed of VH, CH1, HR, CH2, and CH3 in the N-to-C-terminal direction. An "intact antibody light chain" is a polypeptide composed of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the N-to-C-terminal direction, abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). Intact antibody chains are linked together by interpeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the intact antibody heavy chain. Typical examples of intact antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE.

[0100] The term "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogue that retains the ability to specifically bind to an antigen (e.g., the S protein of the SARS-CoV-2 coronavirus). It typically includes at least a portion of the antigen-binding region or variable region of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when expressed in molar units (K... DWhen the activity is expressed as ), the antibody fragment retains at least 10% of the parent antibody binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% of the parent antibody's binding affinity to the target. Antibody fragments include, but are not limited to: Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, complementarity-determining region (CDR) fragments, disulfide bond-stabilized proteins (dsFv), etc.; linear antibodies, single-chain antibodies (e.g., scFv monoclonal antibodies), monoclonal antibodies (Unibody, technology from Genmab), bivalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (e.g., VH domain antibodies), domain antibodies (Domantis, technology from Domantis), nanobodies (Ablynx); multispecific antibodies formed from antibody fragments (e.g., triple-chain antibodies, quadruple-chain antibodies, etc.); and engineered antibodies such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies, etc. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and their applicability is screened using the same methods as for intact antibodies.

[0101] The term "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment containing the VH and VL domains of the antibody. It is a recombinant protein in which the heavy chain variable region (VH) and light chain variable region (VL) are linked by a linker. The linker allows these two domains to cross-link to form an antigen-binding site. The linker sequence is generally composed of a flexible peptide, such as, but not limited to, G2(GGGGS)3. The size of an scFv is generally 1 / 6 that of a complete antibody. A single-chain antibody is preferably a single amino acid sequence encoded by a single nucleotide chain. For a review of scFv, see Pluckthun A, 1994. Antibodies from Escherichia coli, in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenberg M and Moore GP (EDs.), Springer-Verlag, New York, pp 269-315. See also International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0102] The term "VL domain" refers to the amino-terminal variable region domain of the immunoglobulin light chain.

[0103] The term "VH domain" refers to the amino-terminal variable region domain of the immunoglobulin heavy chain.

[0104] The term "hinge region" refers to the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding domains to move independently. The hinge region can be divided into three distinct domains: the upper, middle, and lower hinge domains (Roux KH et al., 1998, J Immunol, 161:4083-4090).

[0105] The term "functional domain" refers to a three-dimensional structure capable of specifically recognizing and / or binding to an epitope, such as an antibody or antibody fragment, including native intact antibodies, single-chain antibodies (scFv), Fd fragments, Fab fragments, F(ab')2 fragments, single-domain antibody fragments, isolated CDR fragments, and their derivatives. Here, "single-chain" means that the first and second functional domains are covalently linked and can be represented by a collinear amino acid sequence encoded by a single nucleic acid molecule.

[0106] The term "Fab fragment" consists of a variable region and a CH1 region of a heavy chain and a light chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. A "Fab antibody" is one-third the size of a complete antibody and contains only one antigen-binding site.

[0107] The term "Fab fragment" contains a light chain, a heavy chain, a VH domain and a CH1 domain, and a constant region between the CH1 and CH2 domains.

[0108] The term "F(ab')2 segment" contains the VH and CH1 domains of two light chains and two heavy chains, as well as a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0109] The term "Fd fragment" consists of a variable region of a heavy chain and CH1, and is the heavy chain portion remaining after the light chain of the Fab fragment is removed.

[0110] The term "Fv region" contains variable regions from both the heavy and light chains, but lacks constant regions; it is the smallest fragment containing complete antigen recognition and binding sites.

[0111] The term "disulfide bond stabilizing protein (dsFv)" introduces a cysteine ​​mutation at each of the VH and VL regions, thereby forming a disulfide bond between VH and VL to achieve structural stability. The term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds, at positions 239 and 242 (positions 226 or 229 in the EU numbering system) corresponding to positions 239 and 242 in the Kabat numbering system.

[0112] The term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide used in this application may comprise a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this application comprises a polypeptide chain having a CH3 domain. Additionally, the antibody used in this application may lack at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). As described above, but those skilled in the art should understand that the heavy chain constant regions may be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0113] The term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain and a constant lambda domain.

[0114] The term "Fc region" or "Fc fragment" refers to the C-terminal region of the immunoglobulin heavy chain, containing at least a portion of the hinge region, a CH2 domain, and a CH3 domain, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. The Fc region includes native sequence Fc regions and variant Fc regions.

[0115] Typically, the Fc region of the human IgG heavy chain is the segment from the amino acid residue at its Cys 226 or Pro 230 position to the carboxyl terminus, but its boundary may vary. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may or may not be present. Fc can also refer to this region as it exists independently, or in the case of a protein polypeptide containing Fc, such as "a binding protein containing the Fc region," and is also called "Fc fusion protein" (e.g., an antibody or an immunoadhesive). The native sequence Fc region in the antibody of the present invention is derived from mammalian (e.g., human) IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the amino acid sequence of the two Fc polypeptide chains comprises substitutions, insertions, and / or deletions of approximately 10 amino acids per 100 amino acids relative to the mammalian Fc polypeptide amino acid sequence. In some implementations, the aforementioned amino acid differences in the Fc region may be Fc alterations that prolong half-life, alterations that increase FcRn binding, alterations that enhance Fcγ receptor (FcγR) binding, and / or alterations that enhance ADCC, ADCP, and / or CDC.

[0116] In IgG, IgA, and IgD antibody isotypes, the Fc region contains the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the Fc region of IgM and IgE contains the three heavy chain constant domains (CH2-4 domains) of each polypeptide chain.

[0117] The term "chimeric antibody" refers to a fragment of an antibody whose heavy and / or light chain portion is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, provided that they exhibit the desired biological activity (US Patent US4816567; Morrison SL et al, 1984, Proc Natl AcadSci USA, 81:6851-6855). For example, the term "chimeric antibody" can include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0118] The term "human" antibody refers to an antibody having a variable region, wherein both the frame region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of this invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence.

[0119] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with that of a human antibody. Most or all amino acids outside the CDR domain of the non-human antibody, such as mouse antibodies, are replaced with corresponding amino acids from human immunoglobulins, while most or all amino acids within one or more CDR regions remain unchanged. Addition, deletion, insertion, substitution, or modification of amino acids is permitted as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies retain antigen specificity similar to the original antibody. There are no particular restrictions on the source of the CDR; it can be derived from any animal. For example, CDR regions derived from mouse antibodies, rat antibodies, rabbit antibodies, or non-human primate antibodies (e.g., cynomolgus monkeys) can be used. The frame region can be searched using the IMGT antibody germline database (…). http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi To obtain the human antibody germline sequence, the human germline antibody sequence with high homology to the non-human antibody being modified is generally selected as the framework region of the humanized antibody.

[0120] The terms "hypervariant region," "CDR region," or "complementarity-determining region" refer to the amino acid residues of the antibody responsible for antigen binding, which are non-continuous amino acid sequences. The CDR region sequence can be defined by the methods of Kabat, Chothia, IMGT (Lefranc et al., 2003, DevComparat Immunol, 27:55-77) and AbM (Martin ACR et al., 1989, Proc Natl Acad Sci USA, 86:9268–9272) or identified by any CDR region sequence determination method well known in the art. For example, the hypervariable region contains the following amino acid residues: amino acid residues from the “complementarity-determining region” or “CDR” defined by sequence alignment (Kabat numbering system), such as residues at positions 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of the light chain variable domain and residues at positions 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of the heavy chain variable domain; see Kabat et al, 1991, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health. Health, Bethesda, Md.; and / or residues from the structurally defined “hypervariant loop” (HVL) (Chothia numbering system), such as residues at positions 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) of the light chain variable domain and residues at positions 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) of the heavy chain variable domain, see Chothia C and LeskAM, 1987, J Mol Biol, 196:901-917; Chothia C et al, 1989, Nature, 342:878-883. “Frame” residues or “FR” residues are variable domain residues other than those defined herein as hypervariant region residues. In some embodiments, the CDRs contained in the antibody or antigen-binding fragment of the present invention are preferably determined by the Kabat, IMGT, or Chothia numbering system. Those skilled in the art can explicitly assign each numbering system to any variable domain sequence without relying on any experimental data beyond the sequence itself. For example, the numbering of Kabat residues for a given antibody can be determined by comparing the antibody sequence with homologous regions of each “standard” numbering sequence.Based on the sequence numbering scheme provided in this paper, determining the numbering of any variable region sequence in the sequence list is entirely within the scope of conventional techniques for those skilled in the art.

[0121] The term “recombination”, when referring to polypeptides or polynucleotides, refers to a form of polypeptide or polynucleotide that does not exist in its natural state. One non-limiting example is that it can be achieved by combining polynucleotides or polypeptides that do not normally appear together.

[0122] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are effectively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), also serve equivalent functions.

[0123] The term "isolated antibody molecule" refers to an antibody molecule that has been identified, isolated, and / or recovered from components of its natural environment. These natural environmental contaminants are substances that can interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein- or non-protein solutes.

[0124] As used in this article, the term "isolated" for nucleic acids (such as DNA or RNA) refers to molecules isolated from other naturally occurring macromolecules of DNA or RNA. The term "isolated" also refers to nucleic acids or peptides produced using recombinant DNA technology that are substantially free of cellular material, viral material, or culture medium, or prepared chemically that are substantially free of chemical precursors or other chemicals. Furthermore, "isolated nucleic acids" refers to nucleic acid fragments that are not naturally occurring and cannot be found in their natural state. The term "isolated" also refers to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides include both purified and recombinant peptides.

[0125] The term "cross-reactivity" refers to the ability of the antibody described herein to bind antigens from different species. For example, the antibody described herein that binds to the SARS-CoV-2 coronavirus S protein may also bind to S proteins from other species (e.g., the SARS-CoV S protein). Cross-reactivity can be measured by detecting a specific reactivity with a purified antigen in a binding assay (e.g., SPR, ELISA), or by binding to cells physiologically expressing the antigen, or by other functional interactions with cells physiologically expressing the antigen. Examples of analyses known in the art for measuring binding affinity include surface plasmon resonance (e.g., Biacore) or similar techniques (e.g., Kinexa or Octet).

[0126] The terms "immunobinding" and "immunobinding property" refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen (specific to that antigen). The strength or affinity of the immunobinding interaction can be expressed by the equilibrium dissociation constant (K0) of the interaction. D ) indicates that K D A smaller value indicates higher affinity. The immunobinding properties of the selected peptide can be determined using methods known in the art. One assay involves measuring the rate of antigen / antibody complex formation and dissociation. The "binding rate constant" (Kbinding rate constant) is used. a or K on ) and "dissociation rate constant" (K d or K off Both can be calculated from concentration and the actual rates of association and dissociation (see Malmqvist M, 1993, Nature, 361: 186-187). d / k a The ratio is equal to the equilibrium dissociation constant K. D (See Davies DR et al, 1990, Annual RevBiochem, 59:439-473). K can be measured by any effective method. D k a and k d value.

[0127] The term "immunogenicity" refers to the ability of a particular substance to elicit an immune response.

[0128] The term "host cell" refers to a cell in which the vector can proliferate and whose DNA can be expressed; said cell can be a prokaryotic or eukaryotic cell. The term also includes any progeny of the tested host cell. It should be understood that not all progeny are identical to the parent cell, as mutations can occur during replication; such progeny are included. Host cells include prokaryotic cells, yeast cells, or mammalian cells such as CHO cells, NSO cells, or other mammalian cells.

[0129] The term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules occupied by adenine, or a position in each of two polypeptides occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For instance, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum possible identity. Such comparisons can be conveniently performed using computer programs such as the Align program (DNAstar, Inc.), or by using the method of Needleman and Wunsch (Needleman SB and Wunsch CD, 1970, J Mol Biol, 48:443-453).

[0130] The terms “mutated,” “mutant,” and “mutation” refer to the substitution, deletion, or insertion of one or more nucleotides or amino acids compared to a natural nucleic acid or polypeptide (i.e., a reference sequence that can be used to define the wild type).

[0131] Antibodies with conserved modifications

[0132] The term "conservative modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of antibodies containing that amino acid sequence. Such conserved modifications include the substitution, addition, and deletion of amino acids. Modifications can be introduced into the antibodies of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are described in detail in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced with other amino acid residues from the same side chain family.

[0133] Therapeutic applications of antibodies against the SARS-CoV-2 coronavirus S protein

[0134] The term "prevention" refers to methods implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a tumor or infection) in a subject or to minimize its effect if it occurs.

[0135] The term "treatment" refers to a method implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reducing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis, whether detectable or undetectable. The amount of a therapeutic agent that effectively relieves symptoms of any particular disease can vary depending on factors such as the patient's disease state, age, weight, and the drug's ability to elicit the desired response in the subject. Whether disease symptoms are relieved can be assessed by any clinical measurement typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptom.

[0136] The antibodies of the present invention (including bispecific, polyclonal, monoclonal, and humanized antibodies) can be used as therapeutic agents. These agents are commonly used to treat or prevent COVID-19 in subjects, enhance vaccine efficacy, or improve innate immune responses. The antibody formulation (preferably an antibody formulation with high specificity and high affinity for its target antigen S protein) is administered to the subject and is generally effective due to its binding to the target. Administration of the antibody can eliminate, inhibit, or interfere with the activity of the SARS-CoV-2 coronavirus S protein. When using antibody fragments, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequence of the antibody, which retains the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or prepared by recombinant DNA technology (see, for example, Marasco WA et al, 1993, Proc Natl Acad Sci USA, 90:7889-7893).

[0137] The antibody or fragment thereof that specifically binds to the SARS-CoV-2 coronavirus S protein of the present invention can be administered in the form of a pharmaceutical composition. The formulation may contain more than one active compound, preferably those with complementary activities that do not adversely affect each other, depending on the specific indication for treatment. Alternatively or additionally, the composition may contain an agent that enhances its function.

[0138] Pharmaceutical Composition

[0139] The antibodies of this invention, or nucleic acids or polynucleotides encoding the antibodies of this application, can be used to prepare pharmaceutical compositions or sterile compositions, for example, by mixing the antibody with a pharmaceutically acceptable carrier, excipient, or stabilizer. Pharmaceutical compositions may include one or a combination (e.g., two or more different) of the antibodies of this invention. For example, a pharmaceutical composition of this invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on a target antigen. Formulations of therapeutic and diagnostic agents may be prepared by mixing with a pharmaceutically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized powder, slurry, aqueous solution, or suspension. The term "pharmaceutically acceptable" means that when the molecular bulk, molecular fragment, or composition is properly administered to an animal or human, it will not produce adverse, allergic, or other adverse reactions. Specific examples of substances that may serve as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginate, etc. The antibodies of the present invention, or the nucleic acids or polynucleotides encoding the antibodies of this application, may be used alone or in combination with one or more other therapeutic agents, such as vaccines.

[0140] The term "pharmaceutically acceptable carriers and / or excipients and / or stabilizers" refers to carriers and / or excipients and / or stabilizers that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, and that are non-toxic to the cells or mammals exposed to them at the doses and concentrations used. This includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, delayed absorption agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Delayed absorption agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0141] Diagnostic uses of antibodies against the SARS-CoV-2 coronavirus S protein

[0142] The monoclonal antibody or its antigen-binding fragment of the present invention can be used for the detection or quantification of SARS-CoV-2 virus using immunoassay. Immunoassay methods are well-known, and any known immunoassay method can be used. That is, if classified by the form of assay, there are sandwich assays, competitive assays, agglutination assays, Western blotting, etc.; if classified by the label used, there are fluorescence assays, enzymatic assays, radiometric assays, biotinylate assays, etc., all of which can be used. Diagnosis can also be performed by immunohistochemical staining. When labeled antibodies are used in the immunoassay method, the antibody labeling method itself is well-known, and any known method can be used.

[0143] These immunoassay methods are well-known and need not be elaborated upon in this specification. In simple terms, for example, the sandwich method involves immobilizing the antibody or antigen-binding fragment of this invention as a first antibody onto a solid phase, reacting it with the biological sample to be tested, rinsing it, reacting it with a second antibody, rinsing it again, and then measuring the second antibody bound to the solid phase. The second antibody can be labeled with enzymes, fluorescent substances, radioactive substances, biotin, etc., to measure the second antibody bound to the solid phase. By measuring multiple standards of known concentrations using the above methods, a standard curve can be constructed based on the relationship between the measured labeled amount and the standard content. By comparing the measurement results of test samples of unknown concentrations with this standard curve, the SARS-CoV-2 viral antigen in the test sample can be quantified. The first and second antibodies can also be substituted as described above. In the agglutination method, the antibody or its antigen-binding fragment of this invention is immobilized on particles such as latex, reacts with the sample, and the absorbance is measured. By measuring multiple standards of known concentrations using the above method, and constructing a standard curve based on the relationship between the measured labeling amount and the standard content, the measurement results of the test sample with unknown concentration can be compared with the standard curve to quantify the SARS-CoV-2 virus antigen in the test sample.

[0144] The biological samples supplied for the above-mentioned immunoassay method are any samples containing the S protein of the SARS-CoV-2 virus, without any particular limitation. For example, they can be serum, plasma, whole blood from humans and animals. In addition, there are body fluid extracts such as nasal swabs, nasal aspiration fluid, and pharyngeal swabs, as well as saliva, respiratory secretions, urine, feces, cell or tissue homogenates, etc.

[0145] By using the monoclonal antibody of the present invention described above, the antibody can be used as at least one of a solid-phase antibody and a labeled antibody to prepare an immunoassay reagent for SARS-CoV-2 virus. The solid phase bound to the monoclonal antibody can be any solid phase used in conventional immunoassays, such as ELISA plates, latex, gelatin particles, magnetic particles, polystyrene, glass, beads, insoluble carriers such as liquid-transferable matrices, etc. Alternatively, labeled antibodies can be prepared by labeling them with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances, etc. Combining these solid-phase antibodies and / or labeled antibodies can prepare reagents used in enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence immunoassays, etc. These assay reagents are used to detect the target antigen in a sample using a sandwich method or a competitive binding assay.

[0146] The reagents used for the sandwich immunoassay described above can be as follows: for example, two monoclonal antibodies of the present invention are prepared, one of which is the labeled antibody described above, and the other is a solid-phase antibody bound to the solid phase described above. First, a sample containing the antigen to be measured is reacted with the solid-phase antibody, and then the labeled antibody (second antibody) is reacted with the antigen captured on the solid-phase antibody. Immunoassay can be performed by detecting the presence or activity of the label bound to the insoluble carrier. Similarly, a sample containing the antigen to be measured is reacted with the solid-phase antibody, and then the labeled antibody (second antibody) is reacted with the antigen captured on the solid-phase antibody. Immunoassay can be performed by measuring the presence or activity of the label bound to the insoluble carrier, that is, by quantifying the amount of the antigen to be measured by measuring the amount of the labeled antibody. In the sandwich immunoassay reagent, one monoclonal antibody can be used as both the solid-phase antibody and the labeled antibody (e.g., when the antigen is a polymer), but it is generally preferred to use two or more antibodies that can respectively recognize two different epitopes of the antigen to be measured. Furthermore, for any solid-phase antibody and labeled antibody, a combination of two or more monoclonal antibodies can be selected and used.

[0147] As an immunoassay reagent employing a competitive binding assay, it can be prepared, for example, by labeling a certain amount of viral antigen with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances, etc. Using this reagent, a competitive reaction can be performed with, for example, a sample containing a certain amount of the monoclonal antibody of this invention, the aforementioned labeled viral antigen, and the antigen to be measured. The amount of labeled viral antigen bound or unbound by the antibody is used to quantify the amount of antigen in the sample to be measured, thereby performing an immunoassay.

[0148] In this invention, when the above-mentioned antibody or antigen is bound to the solid phase or label, physical adsorption method, chemical binding method or other methods can be used (see "Protein Nucleic Acid Enzymes", Supplement No. 31, 37-45 (1987)).

[0149] The aforementioned labeled anti-SARS-CoV-2 virus monoclonal antibodies can be prepared by binding the anti-SARS-CoV-2 virus monoclonal antibody to a label. The label can be an enzyme, colloidal metal particles, stained latex particles, fluorescent latex particles, luminescent substances, fluorescent substances, etc. The enzyme can be any enzyme used in enzyme-linked immunosorbent assays (EIA), such as alkaline phosphatase, peroxidase, β-D-galactosidase, etc.; colloidal metal particles can be, for example, colloidal gold particles, colloidal selenium particles, etc.

[0150] The binding of the marker to the anti-SARS-CoV-2 monoclonal antibody can be achieved using known methods for generating covalent or non-covalent bonds. Examples of binding methods include: glutaraldehyde method, periodic acid method, maleimide method, dithiodipyridine method, and methods using various cross-linking agents (e.g., "Protein Nucleases," Supplementary No. 31, 37-45 (1985)). In binding methods using cross-linking agents, examples of cross-linking agents include N-succinimino-4-maleiminobutyric acid (GMBS), N-succinimino-6-maleiminohexanoic acid, and N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid. In covalent bonding methods, functional groups present in the antibody can be utilized. Alternatively, conventional methods can be used to introduce functional groups such as thiohydroxy, amino, carboxyl, and hydroxyl groups into the antibody, followed by the aforementioned bonding methods to bind these functional groups to the label, thereby preparing labeled monoclonal antibodies against SARS-CoV-2. Non-covalent bonding methods include physical adsorption.

[0151] The substrates can be various chromogenic substrates, fluorescent substrates, luminescent substrates, etc., corresponding to the labeled enzymes and shown below.

[0152] (a) Chromogenic substrates: 2,2'-azono-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), and diaminobenzidine (DAB) in combination with hydrogen peroxide were used for peroxidase; 5-bromo-4-chloro-3-indole phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), and sodium 5-bromo-4-chloro-3-indole phosphate (BCIP·Na) were used for alkaline phosphatase.

[0153] (b) Fluorescent substrates: 4-methylumbelliferyl phenyl phosphate (4-MUP) for alkaline phosphatase; 4-methylumbelliferyl phenyl-β-D-galactoside (4MUG) for β-D-galactosidase.

[0154] (c) Luminescent substrates: 3-(2'-spiroadamantane)-4-methoxy-4-(3”-phosphoryloxy)phenyl-1,2-dioxane·2 sodium salt (AMPPD) for alkaline phosphatase; 3-(2'-spiroadamantane)-4-methoxy-4-(3”-β-D-galactopyranosyl)phenyl-1,2-dioxane-1 (AMGPD) for β-D-galactosidase; and luminol and isoluminol obtained by combining with hydrogen peroxide for peroxidase.

[0155] Diagnosis of SARS-CoV-2 virus infection can be performed by using the monoclonal antibody against the S protein of the SARS-CoV-2 virus of this invention to test various biological samples from humans or animals. Attached Figure Description

[0156] Figure 1 Serum titers of mice immunized with SARS-CoV-2S1 antigen.

[0157] Figure 2 Neutralizing antibody titers in the serum of mice immunized with SARS-CoV-2S1 antigen.

[0158] Figure 3 Determination of the binding ability of purified murine antibody S1B-91-3 to SARS-CoV-2S1.

[0159] Figure 4 Cross-reactivity assay of purified murine antibody S1B-91-3 with SARS-CoV S.

[0160] Figure 5 The murine monoclonal antibody S1B-91-3 competes with ACE2-HRP for the ability to bind to SARS-CoV-2S1.

[0161] Figure 6 The murine antibody S1B-91-3 blocks the binding of the spike S protein to 293T-ACE2 cells.

[0162] Figure 7 A superimposed diagram of three structures: the molecular docking model of the mouse antibody S1B-91-3 / RBD, the ACE2 / RBD structure (PDB 6M0J), and the antibody CR3022 / RBD structure (PDB 6W41).

[0163] Figure 8 The results of the amino acid sequence alignment of the heavy chain variable region of the humanized antibody hS1B-91-3 with its parent mouse antibody.

[0164] Figure 9 The results of amino acid sequence alignment of the light chain variable region of humanized antibody hS1B-91-3 and its parental mouse antibody.

[0165] Figure 10 The binding ability of humanized antibody hS1B-91-3 to SARS-CoV-2S trimer antigen was determined by indirect ELISA.

[0166] Figure 11 The ability of humanized antibody hS1B-91-3 to block the binding of SARS-CoV-2S trimer to human ACE2 was determined by competitive ELISA.

[0167] Figure 12 The humanized antibody hS1B-91-3 blocks the binding of the spike S protein to 293T-ACE2 cells.

[0168] Figure 13 , In vitro assay of the activity of humanized antibody hS1B-91-3 in inhibiting pseudoviruses. Detailed Implementation

[0169] Example 1: Preparation of mouse monoclonal antibody against SARS-CoV-2S1 protein

[0170] Antigen Preparation: SARS-CoV-2 S1 Antigen Preparation Process: Based on the full-length amino acid sequence of the novel coronavirus S protein published in Uniprot (Uniprot Entry P0DTC2), the 326-685 aa region (labeled S1B) was selected as the antigen for antibody screening in this embodiment. To obtain highly efficient expression of the target protein, the coding gene of S1B was artificially modified and optimized, and a eukaryotic expression vector pcDNA3.1-S1B was constructed according to conventional molecular biology methods. The correctly sequenced recombinant expression plasmid was transfected into CHO cells, and expression and purification were performed according to conventional methods to obtain the purified antigen for immunization.

[0171] Animal Immunization: The SARS-CoV-2S1 protein antigen described above was fully emulsified with complete Freund's adjuvant and then used to immunize male Balb / C mice (Shanghai Silex Laboratory Animal Co., Ltd.) at multiple sites, 50 μg / mouse, with an immunization cycle of once every three weeks. On day 10 after the third immunization, blood was collected from the orbital sinus, and the serum anti-SARS-CoV-2S1 antibody titer was tested using the indirect ELISA method described in Example 2.1 to monitor the degree of immune response in mice. The results showed... Figure 1 The results of competitive ELISA testing of serum SARS-CoV-2S1 neutralizing antibody levels showed that... Figure 2 Three days before fusion, mice that produced the highest titers of anti-SARS-CoV-2S1 antibodies and the highest levels of neutralizing antibodies were given a booster immunization. Three days later, the mice were sacrificed and their spleens were fused with mouse myeloma Sp2 / 0 cell lines.

[0172] Cell fusion and antibody screening: Mixed 2×10 8 Sp2 / 0 cells and 2×10 8 Splenic cells were fused in a solution of 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO). The spleen cell number was adjusted to 5 × 10⁶ cells / mL using Iscove medium (containing 10% fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine). 5 / mL, 0.3mL was added to the wells of a 96-well culture plate and incubated at 37°C in a 5% CO2 incubator. After 10 days of culture, the ability of the antibody in the supernatant to competitively bind SARS-CoV-2S1 with HRP-labeled hACE2-Fc (ACE2(18-740)-Fc) (ACRO Biosystems, hereinafter the same source) was detected by high-throughput ELISA to screen out positive wells that compete with hACE2-Fc (see Example 2.3 for the method). The fusion cells in the wells containing the monoclonal antibody that can inhibit the binding of hACE2-Fc to SARS-CoV-2S1 were then subcloned, and the hybridoma cell line #S1B-91-3 expressing a high-affinity mouse monoclonal antibody was obtained by competitive ELISA screening. The clones that produced specific antibodies were cultured in RPMI 1640 medium supplemented with 10% FCS. When the cell density reached approximately 5×10 5 When the concentration of cells / mL is reached, replace the medium with serum-free medium. After 2 to 4 days, centrifuge the culture medium and collect the culture supernatant. Purify the antibody using a protein A column, and then dialyze the monoclonal antibody elution buffer with 150 mM NaCl. Filter the dialyzed solution through a 0.2 μm filter to obtain the purified mouse monoclonal antibody S1B-91-3 for testing.

[0173] Example 2: Functional identification of murine monoclonal antibodies against SARS-CoV-2S1

[0174] 2.1 Determination of the binding ability of murine antibodies to SARS-CoV-2S1 antigen by indirect ELISA method

[0175] The ELISA plate was coated with SARS-CoV-2S1 (ACRO Biosystems) and incubated overnight at room temperature. The coating solution was discarded, and the plate was blocked with skim milk powder dissolved in PBS buffer for 1 hour. The plate was then washed 3-4 times with PBST (PBS containing 0.05% Tween-20, pH 7.4). Then, 100 μL of purified anti-SARS-CoV-2S1 mouse antibody S1B-91-3 and human antibody CR3022 against SARS-CoV and SARS-CoV-2S1 (whose heavy chain variable region and light chain variable region are published in GenBank (Accession numbers: DQ168569 and DQ168570) were added to each well as a positive control. The plates were incubated at room temperature for 1 hour, washed with PBS containing 0.05% Tween 20, and then 100 μL of HRP-labeled goat anti-mouse IgG polyclonal antibody (Jackson Laboratory) was added to each well as the detection antibody. The plates were then washed 3–4 times with PBST, incubated with substrate TMB for 10 minutes, and the reaction was terminated with 0.2 M H2SO4. The absorbance values ​​(OD values) were then read. The results are shown in [Figure / Table / Insert Table ...Insert Table / Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Figure 3 .

[0176] 2.2 Cross-reactivity assay of anti-SARS-CoV-2S1 murine antibodies

[0177] The binding ability of the obtained murine monoclonal antibody S1B-91-3 to SARS-CoV S was determined.

[0178] SARS-CoV S1 protein (ACRO Biosystems) was diluted to 0.1 μg / mL with PBS buffer and added to 96-well plates at a volume of 100 μL / well. The plates were incubated at 4°C for 16–20 h. The supernatant was removed, and the plates were washed once with PBST buffer. Then, 200 μL of PBST containing 1% skim milk powder (PBST / 1% skim milk powder) was added to each well for blocking at room temperature for 1 h. The blocking buffer was removed, and the plates were washed three times with PBST buffer. Then, 100 μL of the aforementioned mouse antibody S1B-91-3 was added to each well, and the plates were incubated at room temperature for 1.5 h. The reaction mixture was removed, and the plates were washed three times with PBST. Then, 50 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:4000 (The Jackson Laboratory) was added to each well, and the plates were incubated at room temperature for 1 h. Finally, the plates were washed three times with PBST, and 100 μL of TMB was added to each well, and the plates were incubated at room temperature for 5–10 min. Finally, 50 μL of 0.2 M H2SO4 was added to each well to stop the reaction, and the OD value was read at dual wavelengths of 450 / 620 nm using a microplate reader.

[0179] like Figure 4 As shown, the murine antibody S1B-91-3 does not cross-react with SARS-CoV.

[0180] 2.3. Determination of the ability of murine antibodies to block the binding of SARS-CoV-2S1 to ACE2 using competitive ELISA.

[0181] Dilute SARS-CoV-2S1 protein (ACRO Biosystems) to 0.1 μg / mL with PBS buffer and add 100 μL / well to a 96-well plate overnight at room temperature. Discard the coating solution and add 200 μL of PBST / 1% skim milk powder to each well for blocking at room temperature for 1 h. Remove the blocking solution, wash the plate three times with PBST buffer, and then add 100 μL of a mixture of horseradish peroxidase (HRP)-labeled hACE2-Fc and antibody S1B-91-3 to each well. Use PBST as a blank control. After sufficient incubation, wash away unbound HRP-labeled hACE2-Fc with PBS and incubate at room temperature for 1 h. Wash the plate three times with PBST again, and then add 100 μL of TMB to each well and incubate at room temperature for 5-10 min. Finally, 50 μL of 0.2 M H2SO4 was added to each well to stop the reaction, and the OD value was read at dual wavelengths of 450 / 620 nm using a microplate reader.

[0182] Figure 5 The study showed that the murine monoclonal antibody S1B-91-3 can competitively bind to SARS-CoV-2S1 with ACE2, thus exerting its function by blocking the binding of SARS-CoV-2S1 and ACE2.

[0183] 2.4 Determination of kinetic constants and affinity of anti-SARS-CoV-2S1 murine antibodies using biofilm interferometry

[0184] Biofilm interferometry (BLI) was used to determine the binding affinity constants of purified murine antibody S1B-91-3, control antibody CR3022, and hACE2-Fc with SARS-CoV-2S1 (ACRO Biosystems). During the assay, biotinylated SARS-CoV-2S1 was immobilized on the surface of a Streptavidin (SA) sensor, and the murine antibody against SARS-CoV-2S1 was used as the analyte. Data were processed and fitted using a 1:1 binding model in analytical software. The fitted data largely overlapped with the experimental data, yielding the binding and dissociation rate constants k. a and k d , using k d Divide by k a The equilibrium dissociation constant K is obtained. D (See Table 1). The results showed that the murine antibody S1B-91-3 binds to SARS-CoV-2S1 at K... D The value reaches the pM level, which is three orders of magnitude lower than human ACE2.

[0185] Table 1. Kinetic constants and affinity determination results of murine antibodies

[0186]

[0187] Example 3: Anti-SARS-CoV-2S1 murine antibody blocks the binding of spike S protein to 293T-ACE2 cells.

[0188] At the cellular level, the binding of the test antibody to the spike S protein-mouse Fc fusion protein (S-mFc) with 293T-ACE2 cells was determined by FACS. The average fluorescence intensity of the bound S-mFc on the cells was detected by fluorescently labeled goat anti-mouse secondary antibody, and the IC50 of the test antibody in blocking the binding of S protein to ACE2 on the cell surface was calculated. 50 To evaluate the blocking effect of the antibody to be tested.

[0189] S-mFc antigen preparation process: Based on the full-length amino acid sequence of the novel coronavirus S protein published in Unirot (Uniprot Entry P0DTC2), a full-length segment of the S protein was selected, and a mouse IgG2a Fc fragment (Uniprot Entry P01863(107-330aa)) was ligated to the C-terminus of the S protein to construct the S-mFc fusion protein used to evaluate the neutralizing antibody in this embodiment. To obtain the target protein with high efficiency expression, the encoding gene of S-mFc was artificially modified and optimized, and the eukaryotic expression vector pcDNA3.1-S-mFc of the target gene was constructed according to conventional molecular biology methods. The recombinant expression plasmid with correct sequencing was transfected into CHO cells, and expression and purification were performed according to conventional methods.

[0190] The specific steps for the in vitro blocking experiment with mouse antibodies are as follows. 293T-ACE2 cells were trypsinized and added to 96-well U-plates at a cell density of 1×10⁶ cells / mL, 100 μL / well, and incubated at 4°C for 30 min. S-mFc was diluted to a certain concentration with 1% PBSB. The mouse antibody sample S1B-91-3 was diluted to 8 μg / mL, serially diluted 4-fold (9 gradients), and then the diluted S-mFc and antibodies of different concentration gradients were mixed 1:1, pre-incubated at room temperature for 30 min, and added to the above 96-well U-plates, and incubated at 4°C for 1 h. The supernatant was removed by centrifugation, and the cells were washed 3 times with 1% PBSB. AF647-goat anti-mouse IgG Fc (Jackson Immuno) was diluted 1:400 with 1% PBSB, 100 μL / well, and incubated at 4°C for 1 h. The supernatant was removed by centrifugation, and the cells were washed 3 times with 1% PBSB. The cells were resuspended in 1% PBSB, 150 μL / well, and the signal intensity was detected by flow cytometry. Then, using average fluorescence intensity as the Y-axis and antibody concentration as the X-axis, the analysis was performed using GraphPad Prism 6 software to calculate the IC50 of the anti-mouse antibody S1B-91-3 blocking the binding of S-mFc protein to 293T-ACE2 cells. 50 value.

[0191] like Figure 6 As shown, at the cellular level, the murine antibody S1B-91-3 against SARS-CoV-2S1 can effectively competitively block the binding of the S protein to ACE2, with an IC50 concentration of 100%. 50 The concentration was 13.70 ng / mL.

[0192] Example 4: Evaluation of the effect of mouse antibody against S1 protein on the binding affinity between RBD / ACE2 using computer molecular docking technology.

[0193] The structure of the murine antibody S1B-91-3 was modeled using the computer software Discovery Studio. The molecular docking spatial conformation of these murine antibodies with their antigen RBD domain was simulated to predict the binding site of the murine S protein antibody on the RBD domain and to evaluate the effect of the antibody on the binding ability between RBD and ACE2.

[0194] A three-dimensional structural model of the murine antibody S1B-91-3 was constructed using Discovery Studio software. The modeling process involved three steps: 1. Searching for three-dimensional structural templates with high amino acid sequence similarity to the variable regions of the light and heavy chains of the murine antibody. Searching for three-dimensional structural templates with high amino acid sequence similarity to the overall variable region of the murine antibody (light and heavy chains combined) was also necessary to determine the relative orientation of the light and heavy chains within the variable region; 2. Using the three structural model templates obtained in step 1 and the amino acid sequences of the light and heavy chains in the variable region of the murine antibody, a structural model of the murine antibody backbone region was constructed; 3. Based on step 2, structural models of the six CDR loop regions were constructed. The RBD structural model used in the molecular docking simulation calculations was derived from a protein database. High-resolution RBD structure (PDB ID 6M0J). By comparing the two RBD structures (PDB ID 6M0J and 6W41), the side chain conformation of the F486 residue in 6M0J was adjusted to be consistent with that in 6W41, i.e., the rotamer1 conformation. This conformation has the highest occupancy, and in 6M0J, the F486 side chain does not exist in this conformation due to ACE2 binding. The molecular docking software used was ZDOCK software in the Discovery Studio package. The parameters used in the molecular docking simulation experiment were all set to default values. The mouse antibody was used as the molecular docking receptor, and the RBD was used as the molecular docking ligand. Receptor blocked residues were selected from variable region amino acids that are far from the CDR region and located in the opposite direction of the CDR region. Receptor binding site residues were selected from the top amino acid of the HCDR3 loop exposed on the protein surface. The molecular docking results of the mouse antibody S1B-91-3 with the RBD are shown in [reference needed]. Figure 7 The structures of ACE2 (PDB 6M0J, Lan J et al, 2020, Nature, 581:215-220) and antibody CR3022 (PDB 6W41, Yuan M et al, 2020, Science, 368:630-633) were introduced through RBD structural superposition. As shown in the figure, there is a small overlap between the binding sites of mouse anti-S1B-91-3 and ACE2 in the RBD domain, indicating that S1B-91-3 directly competes with ACE2 for RBD binding.

[0195] Molecular docking results showed that the murine antibody S1B-91-3 competitively binds to RBD with ACE2, thereby blocking the binding between RBD and ACE2. This provides a reasonable molecular explanation for the ability of these antibodies to inhibit SARS-CoV-2 virus infection of host cells.

[0196] Example 5: Humanization of mouse antibodies against SARS-CoV-2 coronavirus S1 protein

[0197] We used CDR grafting to humanize murine antibodies. The basic principle of CDR grafting is to transplant the CDR region of the murine antibody onto the human antibody template, while simultaneously introducing several or more key FR region residues of the murine antibody—important for stabilizing the CDR conformation and antigen-antibody binding—into the human antibody template (backmutations). This achieves the goal of reducing the immunogenicity of the murine antibody while maintaining its affinity. In addition to the CDR grafting operation, we further calculated the isoelectric point (PI), hydrophobic aggregation, post-translational modifications (PTMs, such as glycosylation, breakage, and isomerization sites), and immunogenicity of the humanized antibody after CDR grafting. We mutated amino acids that caused these issues to ensure the humanized antibody fully exerts its therapeutic effect in clinical use.

[0198] The specific process for antibody humanization is as follows. Search the IMGT human antibody germline database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) to obtain a human antibody template with high similarity to the mouse antibody (VH is IGHV1-69*02, VL is IGKV2-28*01). Annotate the CDR regions of the mouse antibody and human antibody templates using Discovery Studio, defining the CDR regions according to the Kabat or IMGT scheme (Table 2). Replace the six CDR regions of the human antibody template with the six CDR regions of the mouse antibody. Each individual CDR region among the six transplanted CDR regions can be an amino acid region defined according to Kabat or IMGT. After CDR transplantation, perform reversion mutations from the mouse antibody to the FR region of the humanized template. The amino acids in the key mouse antibody FR region, which are crucial for stabilizing the antibody CDR region conformation and for antigen-antibody binding, include four classes of amino acid residues: 1) CDR region... 1) Amino acids embedded under the surface of the antibody; 2) CDR region The four key mouse antibody FR region residues were identified by establishing a three-dimensional structural model of the mouse antibody. These residues consisted of: 1) amino acids exposed on the antibody surface; 2) interfacial amino acids between the antibody light and heavy chain domains; and 3) vernier zone residues that stabilize the antibody CDR region conformation (Foote Jand Winter G, 1992, J Mol Biol, 224:487-499). For these four types of amino acids in the human template that were inconsistent with the mouse antibody sequence, three-dimensional structural analysis was used to select amino acids important for maintaining the CDR conformation and antigen-antibody binding, and amino acid transplantation or replacement was performed from the mouse antibody to the human template. Then, the isoelectric point, hydrophobic aggregation, post-translational modifications, and immunogenicity of the humanized antibody generated after the transplantation of these four types of amino acids were further calculated. The problematic amino acids were mutated to obtain the final humanized antibody sequence (Table 3). Figure 8 The results of the alignment of the heavy chain variable region amino acid sequence of the humanized antibody hS1B-91-3 with its parental mouse antibody are shown. Figure 9 The results show the alignment of the amino acid sequence of the light chain variable region of the humanized antibody hS1B-91-3 with its parental mouse antibody.

[0199] Table 2. CDR region in the variable region of murine antibody

[0200]

[0201]

[0202] To obtain a full-length antibody sequence consisting of two heavy chains and two light chains, the VH and VL sequences shown in Table 3 are spliced ​​or assembled with the antibody heavy chain constant region (preferably from human IgG1, IgG2, or IgG4) and light chain constant region (preferably from the human κ light chain, amino acid sequence as shown in SEQ ID NO: 17) sequences using conventional techniques. For example, in one embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG1 (amino acid sequence as shown in SEQ ID NO: 18). In another embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG2 (amino acid sequence as shown in SEQ ID NO: 19). Alternatively, a modified human IgG2 constant region sequence may be used; in one embodiment, the humanized antibody molecule includes human IgG2 modified according to the hinge region of the EU number (e.g., ERKCC deletion, amino acid sequence as shown in SEQ ID NO: 20). In another embodiment, the humanized antibody molecule includes human IgG4 with a mutation at position 228 according to the EU number (e.g., S to P) (amino acid sequence as shown in SEQ ID NO: 21).

[0203] Table 3. Humanized antibodies corresponding to murine antibodies and their variable region amino acid sequences

[0204]

[0205] Example 6: Construction and protein expression of humanized antibody expression vector

[0206] The cDNA encoding the heavy and light chains of the humanized antibody obtained by the above method is inserted into PcDNA3.1 or its derivative plasmid, or other eukaryotic expression vectors, to construct a humanized antibody expression vector. Preferably, the vector plasmid used should contain the cytomegalovirus early gene promoter-enhancer required for high-level expression in mammalian cells. Simultaneously, the vector plasmid contains a selectable marker gene to confer resistance to ampicillin in bacteria and G418 resistance in mammalian cells. Furthermore, the vector plasmid contains the DHFR gene, which, in suitable host cells, can co-amplify the humanized antibody gene and the DHFR gene with methotrexate (MTX, Sigma) (see, for example, patent CN103333917B).

[0207] The recombinant expression vector plasmid constructed above was transfected into a mammalian host cell line to express the humanized antibody. For stable high-level expression, the preferred host cell line is dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells (see, for example, U.S. Patent 4,818,679 to Chasin, L. et al.). Electroporation is the preferred transfection method, but other methods, including calcium phosphate co-precipitation, lipid transfection, and protoplasmic fusion, can also be used. In electroporation, using a Gene Pulser (Bio-Rad Laboratories) set to a 250V electric field and a 960μFd capacitance, 2 × 10⁻⁶ cells were added to the cuvette. 7 Cells were suspended in 0.8 mL of PBS containing 10 μg of expression vector plasmid DNA linearized with PvuI (Takara). Two days after transfection, cells were added containing 0.2 mg / mL G418 and 200 nM methotrexate (MTX). To achieve higher expression levels, the transfected humanized antibody gene was co-amplified using the DHFR gene, which was inhibited by MTX. The secretion rate of each cell line was determined by limiting dilution of subclones and ELISA, and cell lines expressing high levels of humanized antibodies were selected. Conditioned culture media containing humanized antibodies were collected for the determination of their in vitro and in vivo biological activities.

[0208] For example, the nucleotide sequences of the heavy and light chains encoding the humanized antibody hS1B-91-3 shown in Table 4 were inserted into the expression vector constructed above. After pressure screening, subcloning stabilization, and high expression of the target antibody in cell lines, the target antibodies were obtained through culture and purification.

[0209] Table 4. Amino acid sequences and their encoding nucleotide sequences of the heavy and light chains of humanized antibodies.

[0210] Antibody number HC amino acid sequence LC amino acid sequence HC nucleotide sequence LC nucleotide sequence hS1B-91-3 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25

[0211] Example 7: Functional Identification of Humanized Antibodies

[0212] 7.1. Indirect ELISA method for determining the binding ability of humanized antibodies to SARS-CoV-2S trimer antigen.

[0213] The ELISA plate was coated with SARS-CoV-2S trimer (ACRO BioSystem) and incubated overnight at room temperature. The coating solution was discarded, and the plate was blocked with skim milk powder dissolved in PBS buffer for 1 hour. The plate was then washed 3-4 times with PBST (pH 7.4, PBS containing 0.05% Tween-20). Then, 100 μl of purified anti-SARS-CoV-2S1 RBD humanized antibody hS1B-91-3 and receptor hACE2-Fc (ACRO BioSystem) against SARS-CoV-2S1 RBD were added to each well, and the mixture was incubated at room temperature for 1 h. The wells were washed with PBS containing 0.05% Tween 20, and then 100 μl of HRP-labeled goat anti-human IgG polyclonal antibody (Jackson Laboratory) was added to each well as the detection antibody. The plate was washed 3–4 times with PBST, and the substrate TMB was added for color development for 10 minutes. The reaction was then terminated by adding 0.2 M H₂SO₄. The absorbance (OD) value was then read. The results are shown below. Figure 10 .

[0214] 7.2. Determination of the ability of humanized antibodies to block the binding of SARS-CoV-2S trimer to ACE2 by competitive ELISA.

[0215] Dilute SARS-CoV-2S trimer protein (ACRO BioSystem) to 0.1 μg / ml with PBS buffer and add 100 μl / well to each well of a 96-well plate. Incubate overnight at room temperature. Discard the coating solution and add 200 μl of PBST / 1% skim milk powder to each well for blocking. Remove the blocking solution, wash the plate three times with PBST buffer, and then add 100 μl of a mixture of horseradish peroxidase (HRP)-labeled hACE2-Fc, humanized antibody hS1B-91-3, and receptor hACE2-Fc (ACRO BioSystem) against SARS-CoV-2S1 RBD to each well. Use PBST as a blank control. After sufficient incubation, wash away unbound HRP-labeled hACE2-Fc with PBS and incubate at room temperature for 1 hour. Wash the plate three times with PBST, and then add 100 μl of TMB to each well. Incubate at room temperature for 5-10 minutes. Finally, 50 μl of 0.2 M H₂SO₄ was added to each well to stop the reaction, and the OD values ​​were read using a microplate reader at dual wavelengths of 450 / 620 nm. The results are shown below. Figure 11 .

[0216] 7.3. Determination of the ability of humanized antibodies to block the binding of SARS-CoV-2S1 to ACE2 using competitive ELISA.

[0217] The experimental method for determining the ability of humanized antibody hS1B-91-3 to block the binding of SARS-CoV-2S1 to ACE2 using competitive ELISA is described in Example 2.3.

[0218] The results are shown in Table 5. The humanized antibody hS1B-91-3 can competitively bind to SARS-CoV-2S1 with hACE2, that is, it functions by blocking the binding of SARS-CoV-2S1 and hACE2. 50 The value was 0.07377 μg / ml.

[0219] Table 5. Competitive binding ability of humanized antibodies to hACE2

[0220] <![CDATA[EC 50 (μg / ml)]]> hS1B-91-3 0.07377 hACE2-Fc 1.31470

[0221] 7.4 Determination of kinetic constants and affinity of humanized antibodies against SARS-CoV-2S1 using biofilm interferometry

[0222] The experimental methods for determining the kinetic constant and affinity equilibrium dissociation constant of the humanized antibody are described in Example 2.4. The experimental results are shown in Table 6. The affinity of the humanized antibody hS1B-91-3 is similar to that of the parental mouse antibody S1B-91-3, both reaching the pM level.

[0223] Table 6. Kinetic constants and affinity determination results of humanized antibodies

[0224]

[0225] 7.5. Anti-SARS-CoV-2S1 humanized antibody blocks the binding of the spike S protein to 293T-ACE2 cells.

[0226] The experimental method for blocking the binding of the spike S protein to 293T-ACE2 cells is described in Example 3. Experimental results are shown below. Figure 12 At the cellular level, the humanized anti-SARS-CoV-2S1 antibody hS1B-91-3 effectively competitively blocks the binding of the S protein to ACE2, with an IC50 concentration of [missing information]. 50 The concentration was 8.13 ng / mL. Compared with the murine antibody, the blocking effect of the humanized antibody did not change significantly.

[0227] 7.6. Experiment on the in vitro inhibition of pseudoviruses by humanized monoclonal antibodies against SARS-CoV-2S1

[0228] 293T-ACE2 cells were infected with a pseudovirus containing the SARS-CoV-2 S protein after incubation with the test antibody. The Luciferase fluorescence intensity (RLU) was measured using chemiluminescence immunoassay. The pseudovirus inhibition rate of the test antibody was calculated based on the RLU readings to evaluate the neutralizing effect of the antibody. The SARS-CoV-2 S protein pseudovirus genome encodes firefly luciferase. After viral genome integration into cells, the expression and activity of firefly luciferase are directly proportional to the number of transduced cells. Compared to the true virus, the pseudovirus can only infect cells once.

[0229] The specific steps of the inhibition experiment of humanized antibody against pseudovirus are as follows: The antibody sample to be tested was diluted to 20 μg / mL with DMEM, serially diluted 3-fold in 8 steps; the pseudovirus was removed from -80℃, thawed at 4℃, and the thawed pseudovirus (Shanghai Yisheng Biotechnology Co., Ltd.) was diluted 50-fold to prepare the working solution; the diluted antibody and pseudovirus working solutions were added to 25 μL / well of each well in a 96-well plate, mixed, and incubated at room temperature for 1 hour in duplicate; 293T-ACE2 cells (Shanghai Yisheng Biotechnology Co., Ltd.) were cultured in DMEM + 10% FBS + 0.75 μg / mL puromycin to the logarithmic growth phase, digested with trypsin, resuspended in DMEM + 10% FBS, mixed, and counted at a cell density of 2 × 10⁶ cells / well. 5Pseudovirus / mL, 50 μL / well, was seeded into a white plate and incubated at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours, 50 μL of DMEM + 10% FBS medium was added to each well, and the plate was incubated for another 24 hours. The supernatant was carefully aspirated with a pipette, and 50 μL of freshly prepared luciferase chromogenic solution was immediately added. The plate was incubated at room temperature for 5 minutes, and the chemiluminescence signal of each well was read using a microplate reader. Positive control: 25 μL of pseudovirus and DMEM medium were added to each well of the 96-well white plate and mixed well, then incubated at 37°C for 1 hour. Negative control: DMEM medium was added to each well of the 96-well white plate and incubated at 37°C for 1 hour. Inhibition rate (%) = 1 - (sample RLU signal value - negative control RLU signal value) / (positive control RLU signal value - negative control RLU signal value). Then, using the inhibition rate as the Y-axis and the antibody concentration as the X-axis, the analysis was performed using GraphPad Prism 6 software to obtain the dose-response curve of the antibody. Figure 13 Table 7 shows the inhibition rate of the humanized antibody hS1B-91-3 against the SARS-CoV-2 S protein pseudovirus at various concentrations. Figure 13 As shown in Table 7, the humanized monoclonal antibody hS1B-91-3 significantly and in a dose-dependent manner inhibited the infection of SARS-CoV-2 pseudovirus into 293T-ACE2 cells, indicating that hS1B-91-3 can block the early invasion of host cells by SARS-CoV-2 infection and play a protective role.

[0230] Table 7. Results of pseudovirus inhibition by humanized monoclonal antibody against SARS-CoV-2S1

[0231]

[0232] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Huang Jingwen <120> An antibody against the SARS-CoV-2 spike protein and its application <130> S1B-91-3 <160> 25 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> The amino acid sequence of murine antibody S1B-91-3 HCDR1-1 (as defined by Kabat). <400> 1 Asp Tyr Trp Leu His 1 5 <210> 2 <211> 8 <212> PRT <213> Amino acid sequence of murine antibody S1B-91-3 HCDR1-2 (as defined by IMGT) <400> 2 Gly Tyr Ser Phe Thr Asp Tyr Trp 1 5 <210> 3 <211> 17 <212> PRT <213> The amino acid sequence of murine antibody S1B-91-3 HCDR2-1 (according to Kabat definition). <400> 3 Met Leu Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 4 <211> 8 <212> PRT <213> The amino acid sequence of the murine antibody S1B-91-3 HCDR2-2 (as defined by IMGT). <400> 4 Leu Asp Pro Ser Asp Gly Glu Thr 1 5 <210> 5 <211> 9 <212> PRT <213> The amino acid sequence of murine antibody S1B-91-3 HCDR3-1 (according to Kabat definition). <400> 5 Arg His Phe Gly Tyr Asp Phe Asp Tyr 1 5 <210> 6 <211> 11 <212> PRT <213> The amino acid sequence of murine antibody S1B-91-3 HCDR3-2 (as defined by IMGT). <400> 6 Ala Arg Arg His Phe Gly Tyr Asp Phe Asp Tyr 1 5 10 <210> 7 <211> 16 <212> PRT <213> Amino acid sequence of murine antibody S1B-91-3 LCDR1-1 (according to Kabat definition) <400> 7 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15 <210> 8 <211> 11 <212> PRT <213> Amino acid sequence of murine antibody S1B-91-3 LCDR1-2 (as defined by IMGT) <400> 8 Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 9 <211> 7 <212> PRT <213> Amino acid sequence of murine antibody S1B-91-3 LCDR2-1 (as defined by Kabat) <400> 9 Arg Met Ser Asn Leu Ala Ser 1 5 <210> 10 <211> 3 <212> PRT <213> Amino acid sequence of murine antibody S1B-91-3 LCDR2-2 (as defined by IMGT) <400> 10 Arg Met Ser 1 <210> 11 <211> 9 <212> PRT <213> Amino acid sequence of murine antibody S1B-91-3 LCDR3-1 (according to Kabat definition) <400> 11 Met Gln His Leu Glu Tyr Pro Leu Thr 1 5 <210> 12 <211> 9 <212> PRT <213> Amino acid sequence of murine antibody S1B-91-3 LCDR3-2 (as defined by IMGT) <400> 12 Met Gln His Leu Glu Tyr Pro Leu Thr 1 5 <210> 13 <211> 118 <212> PRT <213> The amino acid sequence of the variable region of the heavy chain of the mouse antibody S1B-91-3 () <400> 13 Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Trp Leu His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Leu Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Phe Gly Tyr Asp Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 14 <211> 112 <212> PRT <213> The amino acid sequence of the light chain variable region of the murine antibody S1B-91-3 () <400> 14 Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys 100 105 110 <210> 15 <211> 118 <212> PRT <213> The amino acid sequence of the heavy chain variable region of the humanized antibody hS1B-91-3 () <400> 15 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Leu Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Phe Gly Tyr Asp Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 16 <211> 112 <212> PRT <213> The amino acid sequence of the light chain variable region of the humanized antibody hS1B-91-3 () <400> 16 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Lys Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 17 <211> 107 <212> PRT <213> Human κ constant region amino acid sequence () <400> 17 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 18 <211> 330 <212> PRT <213> Amino acid sequence of human IgG1 wild-type constant region() <400> 18 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 19 <211> 326 <212> PRT <213> Amino acid sequence of human IgG2 wild-type constant region() <400> 19 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 20 <211> 321 <212> PRT <213> IgG2 (ERKCC deletion mutant constant region amino acid sequence) <400> 20 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 100 105 110 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 115 120 125 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 130 135 140 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 145 150 155 160 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 165 170 175 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu 180 185 190 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys 195 200 205 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 210 215 220 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 225 230 235 240 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu 245 250 255 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 260 265 270 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 275 280 285 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 290 295 300 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 305 310 315 320 Lys <210> twenty one <211> 327 <212> PRT <213> Human IgG4 (S228P mutant constant region amino acid sequence) <400> twenty one Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 22 <211> 439 <212> PRT <213> Amino acid sequence of the heavy chain of humanized antibody hS1B-91-3() <400> 22 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Leu Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Phe Gly Tyr Asp Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Thr Val Val Glu Cys Pro Pro Cys Pro Ala 210 215 220 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 225 230 235 240 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 245 250 255 Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 260 265 270 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 275 280 285 Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp 290 295 300 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 305 310 315 320 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg 325 330 335 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 340 345 350 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 355 360 365 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 370 375 380 Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 385 390 395 400 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 405 410 415 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 420 425 430 Leu Ser Leu Ser Pro Gly Lys 435 <210> 23 <211> 219 <212> PRT <213> People <400> 23 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Lys Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 24 <211> 1317 <212> DNA <213> Humanized antibody hS1B-91-3 heavy chain nucleic acid sequence() <400> 24 gaagtgcagc tggtccaatc cggcgctgaa gtcaaaaagc ctggctcctc cgtgaaggtg 60 tcttgcagg cctctggcta ctccttcacc gactactggc tgcactgggt gcggcaggct 120 cctggccagg gcctggaatg gatcggcatg ctggaccct ccgacggcga gatagactg 180 aaccagaagt tcaggacaa ggctaccctg accgtggaca agtcaactac caccgcttac 240 atgcagctgt cctctctgag gtccgaggac accgctgtgt actattgcgc cagaagacac 300 ttcggctacg acttcgatta ctggggccaa ggcaccaccg tgacagtc ctccgcaagc 360 accaagggac cttctgtgtt tcctctggcc ccatgtagtc gctcaacttc cgagagcacc 420 gcagcactgg gatgcctggt gaaggattac ttcccagac ccgtcacagt gtcttggaac 480 agtggggccc tgacaagcgg tgtcagctg tgctggcctc atccgactg 540 tactctctga gctctgtggt cactgtgccc agttcaaatt tcgggaccca gatatact 600 tgtaacgtgg accataagcc ttccaatacc aaggtcgata aacagtggt ggaatgtcca 660 ccttgcccag ctccaccagt cgcaggacct agcgtgttcc tgtttcctcc aaagcccaaa 720 gandacactga tgatctcacg cacacctgag gtcacttgcg tggtcgtgga cgtgtcccac 780 gaggaccctg aagtccagtt taactggtac gtggatggcg tcgaagtgca taatgccaag 840 accaaaccaa gagaggaaca gttcaactcc acatttcgcg tcgtgagcgt gctgactgtc 900 gtgcaccagg actggctgaa cggcaaggag tataagtgta aagtgagcaa taagggcctg 960 cctgctccaa tcgagaaaac catttctaag acaaaaggcc agcccagaga acctcaggtg 1020 tacacactgc ccccttcccg cgaggaaatg actaagaacc aggtcagcct gacctgcctg 1080 gtgaaaggtt tttatcccag tgacatcgcc gtggagtggg aatcaaatgg ccagcctgag 1140 aacaattaca agactacccc acccatgctg gactcagatg gttccttctt tctgtattca 1200 aagctgaccg tggataaatc caggtggcag cagggcaacg tcttctcttg tagtgtgatg 1260 catgaggctc tgcacaatca ttacacacag aagtcactgt ccctgagccc aggcaag 1317 <210> 25 <211> 657 <212> DNA <213> Humanized antibody hS1B-91-3 light chain nucleic acid sequence() <400> 25 gatatcgtga tgacccagtc ccctagcagc ctgcctgtga cccctggaca gcccgcctcc 60 atcagctgca gatcctccaa gtccctgctg cactccaacg gcaacaccta cctgtactgg ttcctgcaga agcccggcaa gagccctcag ctgctgatat accggatgtc caacctggct tctggcgtgc ctgaccggtt ttctggcagc gggtcagga cagattttac actgaagatt tcaagagtgg aagccgagga tgtggggcgtg tactactgca tgcagcacct ggagtacccc 300 ctgaccttcg gcggcggcac caaggtgga atcaagcgta ctgtcgctgc accaagcgtg ttcatttttc ctccatctga cgaacagctg aagtctgga ccgctagtgt cgtgtgcctg 420 ctgaacaatt tttaccccag ggaggcaaag gtccagtgga aagtggataa cgccctgcag agcggcaatt ctcaggagag tgtgaccgaa caggactcaa aggattccac atatagcctg tcatccactc tgaccctgag caaagctgac tacgagaagc acaaagtcta tgcatgcgaa gtgacccatc agggactgag ctctcctgtg acaaagtctt tcaaccgggg ggagtgc 657

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to the SARS-CoV-2 coronavirus S protein, characterized in that, The antibody or its antigen-binding fragment comprises three VH variable region CDRs and three VL variable region CDRs. The amino acid sequences of the CDRs are defined using the Kabat or IMGT method and are selected from the following groups: (i) HCDR1, HCDR2, and HCDR3 are composed of SEQ ID NO: 1, 3, and 5, respectively; and LCDR1, LCDR2, and LCDR3 are composed of SEQ ID NO: 7, 9, and 11, respectively; (ii) HCDR1, HCDR2, and HCDR3 are composed of SEQ ID NO: 2, 4, and 6, respectively; and LCDR1, LCDR2, and LCDR3 are composed of SEQ ID NO: 8, 10, and 12, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody or its antigen-binding fragment is murine or chimeric, and its heavy chain variable region includes a heavy chain FR region of murine IgG1, IgG2, IgG3 or variants thereof; and its light chain variable region includes a light chain FR region of murine κ, λ chains or variants thereof.

3. The antibody or its antigen-binding fragment as described in claim 2, characterized in that, The VH domain of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO: 13, or a sequence having at least 95% identity with SEQ ID NO: 13; and its VL domain contains the amino acid sequence shown in SEQ ID NO: 14, or a sequence having at least 95% identity with SEQ ID NO:

14.

4. The antibody or its antigen-binding fragment as described in claim 3, characterized in that, The antibody or its antigen-binding fragment is humanized.

5. The antibody or its antigen-binding fragment as described in claim 4, characterized in that, The VH domain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 15, or a sequence having at least 95% identity with SEQ ID NO: 15; and its VL domain contains an amino acid sequence as shown in SEQ ID NO: 16, or a sequence having at least 95% identity with SEQ ID NO:

16.

6. The antibody or its antigen-binding fragment as described in claim 5, characterized in that, The antibody comprises a heavy chain constant region and a light chain constant region derived from human immunoglobulins; the heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; and the heavy chain constant region has a natural sequence or a sequence with one or more amino acid substitutions, deletions or additions compared to the natural sequence from which it is derived.

7. The antibody or its antigen-binding fragment as described in claim 6, characterized in that, The antibody contains a heavy chain constant region selected from the following group: (i) The heavy chain constant region of wild-type human IgG1 as shown in SEQ ID NO: 18; (ii) The heavy chain constant region of wild-type human IgG2 as shown in SEQ ID NO: 19; (iii) The hinge region of human IgG2 modified with the constant heavy chain region as shown in SEQ ID NO: 20; (iv) Heavy chain constant region of human IgG4 containing the Ser228Pro mutation, as shown in SEQ ID NO:

21.

8. The antibody or its antigen-binding fragment as described in claim 7, characterized in that, The heavy chain of the antibody has an amino acid sequence as shown in SEQ ID NO: 22; or a sequence having 1-5 conserved amino acid substitutions compared to SEQ ID NO: 22, or a sequence having at least 95% identity with SEQ ID NO: 22; and its light chain has an amino acid sequence as shown in SEQ ID NO: 23; or a sequence having 1-5 conserved amino acid substitutions compared to SEQ ID NO: 23, or a sequence having at least 95% identity with SEQ ID NO:

23.

9. The antibody or antigen-binding fragment thereof as described in any one of claims 1-8, characterized in that, The antibody or its antigen-binding fragment binds to the S protein at a KD of 10 nM or lower.

10. A DNA molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

11. The DNA molecule as claimed in claim 10, characterized in that, The DNA molecule encoding the antibody heavy chain has a nucleotide sequence as shown in SEQ ID NO: 24; and the DNA molecule encoding the antibody light chain has a nucleotide sequence as shown in SEQ ID NO:

25.

12. A vector comprising the DNA molecule as described in claim 10 or 11.

13. A host cell comprising the vector as described in claim 12; said host cell comprising a prokaryotic cell, yeast, or mammalian cell.

14. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9 and a pharmaceutically acceptable carrier.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-9, comprising: (a) Obtain the gene of the antibody or its antigen-binding fragment, and construct an expression vector of the antibody or its antigen-binding fragment; (b) Transfecting the expression vector into host cells using genetic engineering methods; (c) culturing the host cells under conditions that allow the production of the antibody or its antigen-binding fragment; (d) isolating and purifying the produced antibody or its antigen-binding fragment; Wherein, the expression vector mentioned in step (a) is the PXY1A1M vector; In step (b), the constructed vector is transfected into a host cell using genetic engineering methods. The host cell may include prokaryotic cells, yeast cells, or mammalian cells. Step (d) involves separating and purifying the antibody or its antigen-binding fragment using conventional immunoglobulin purification methods, including protein A affinity chromatography, ion exchange chromatography, hydrophobic chromatography, or molecular sieving methods.

16. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-9 in the preparation of a medicament for treating and preventing disease caused by SARS-CoV-2 coronavirus.

17. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-9 in the preparation of a SARS-CoV-2 virus detection kit.

18. A test kit comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

19. The detection kit of claim 18, wherein the kit comprises: (1) Selected from any of the following: a. Solid-phase support and primary antibody; b. A solid-phase carrier coated with a primary antibody; The first antibody is selected from the antibody or antigen-binding fragment thereof according to any one of claims 1-9; (2) Second antibody; The second antibody is optionally appropriately labeled, and the second antibody is selected from the antibody or antigen-binding fragment of any one of claims 1-9, and the second antibody can be used in combination with the first antibody.

20. The detection kit of claim 19, wherein the solid support is selected from nitrocellulose membrane, latex particles, colloidal gold, glass, polystyrene, polyvinyl chloride, or fiber optic sensor.

21. The detection kit of claim 19, wherein the labeling agent is a radioactive isotope, an enzyme, an enzyme substrate, a phosphorescent substance, a fluorescent substance, biotin, and a coloring substance; wherein, The enzymes include alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, and glucose oxidase; the fluorescent substances include fluorescein derivatives and rhodamine derivatives, as well as rare earth elements or rare earth element complexes; the phosphorescent substances include acridine esters and isoluminol; the radioactive isotopes include 125I, 3H, 14C, and 32P; and the coloring substances include latex particles and colloidal gold.

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