CN114106163A

CN114106163A - SARS-CoV-2 virus neutralizing antibody and its use

Info

Publication number: CN114106163A
Application number: CN202010889425.8A
Authority: CN
Inventors: 李强; 孙见宇; 武翠; 张晓峰; 刁家升; 周利; 马心鲁
Original assignee: Anyuan Pharmaceutical Technology Shanghai Co ltd
Current assignee: Anyuan Pharmaceutical Technology Shanghai Co ltd
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2022-03-01

Abstract

The invention relates to the field of therapeutic antibodies and molecular immunology, and particularly provides an antibody for SARS-CoV-2 coronavirus S protein and application thereof in preparing a medicament for treating novel coronavirus pneumonia COVID-19. The antibody is capable of specifically recognizing and binding with high affinity to SARS-CoV-2 coronavirus S protein, which ensures that the antibody is capable of blocking infection of a human cell by SARS-CoV-2.

Description

SARS-CoV-2 virus neutralizing antibody and its use

Technical Field

The present invention relates to the field of therapeutic antibody and molecular immunology, in the concrete, it relates to a recombinant monoclonal antibody of SARS-CoV-2 coronavirus S protein and application of said antibody, in particular, it relates to the application in the treatment, prevention and diagnosis of COVID-19 disease caused by SARS-CoV-2.

Background

The novel coronavirus SARS-CoV-2, a newly emerging human pathogen, causes severe respiratory disease and COVID-19 pneumonia with fever, asthenia, and dry cough as the main manifestations. According to the data of world health organization 2020, 8, 19 days, 21,756,357 cases have been diagnosed globally, resulting in 771,635 deaths. Novel pathogens have been shown to be a novel member of the genus beta coronavirus, based on genomic nucleic acid sequence. The genome sequence similarity of SARS-CoV-2 and bat coronavirus RaTG13 reaches 96.2% (Zhou P et al,2020, Nature,579: 270-. Compared with SARS-CoV, SARS-CoV-2 coronavirus is easier to spread from person to person, WHO has announced COVID-19 disease as a global pandemic disease, and new coronavirus has spread to all over the world now.

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, an RNA dependent RNA polymerase RDRP, and ten more non-structural proteins. Wherein S, M, N and E proteins are used for packaging virus structures, RDRP and more than ten non-structural proteins are used for virus genome RNA replication and synthesis of each protein mRNA. SARS-CoV-2 is similar to SARS-CoV virus, its amino acid sequence homology is high, its S, M, N, E and RDRP protein amino acid number and SARS-CoV homology are 1273 (76%), 222 (91%), 419 (91%), 75 (95%) and 932 (96%), respectively. Similar to SARS-CoV virus, SARS-CoV-2 virus is spherical, enveloped and arranged with coronary spikes. The spike S protein of SARS-CoV-2 forms a trimer (Wrapp D et al,2020, Science,6483:1260-1263) in the form of a mushroom which is embedded in the outer surface membrane of the virus. The S protein is the major antigenic component of the virus and is responsible for binding of the virus to the invaded host cell receptor ACE2 and fusion of the virus to cells. Similar to the SARS-CoV virus S protein (Yuan Y 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-. In the S protein trimer of types such as mushrooms, the three S1 domains form a "mushroom cap" and the three S2 domains form a "mushroom stem". Among them, the RBD domain (amino acid 331-527) in S1 is responsible for binding to the invaded host cell Receptor ACE2, and S2 is responsible for fusion with the host cell. The S2 domain is normally present in a folded or coiled compressed conformation in the overall S protein, and when the virus fuses with host cells following S1 shedding, S2 displays an extended conformation for insertion into the host cell membrane (Walls AC et al,2017, Proc Natl Acad Sci USA,114: 11157-. The binding affinity of the S protein of SARS-CoV-2 virus to human Cell receptors is reported to be much higher than that of the S protein of SARS-CoV virus (Wrapp D et al,2020, Science,6483: 1260-. Another difference from SARS-CoV virus S protein is that SARS-CoV-2S protein has a Furin cleavage site RRAR (amino acid 682-685), which divides the S protein into two parts, S1 and S2, and the S1 and S2 after cleavage are linked together in a non-covalent bond. Due to the Furin enzyme cutting site existing between S1/S2 and the Furin enzyme is widely expressed in eukaryotic tissues and cells; meanwhile, the Furin site containing the multi-basic amino acids can also be degraded by other lysine or arginine targeting enzymes, such as Cell surface enzyme TMPRSS2, endosomal cathepsin L enzyme or possibly Trypsin (Trypsin), etc. (Hoffmann M et al,2020, Cell,181(2):271-280.e 8; Shang J et al,2020, Proc Natl Acad Sci USA,117: 11727-. Therefore, the S1/S2 of SARS-CoV-2 is more easily cleaved, resulting in the S1 domain being more easily shed when the virus is fused with human host cells, thereby increasing the fusion ability of S2 and the infectivity of the virus. Whereas the SARS-CoV virus S1/S2 is linked by only one basic amino acid arginine, where the S protein is cleaved by the cell surface enzyme TMPRSS2 and cathepsin L in endosomes to infect host cells (Beluzard S et al,2012, Viruses,4: 1011-. Therefore, the two differences, i.e., the presence of Furin cleavage site and high affinity to human receptor ACE2, may be responsible for the high infectivity of SARS-CoV-2 coronavirus. Since the S protein is responsible for binding to human host cell receptors and fusion with host cells, the S protein is the primary target for therapeutic neutralizing antibodies against SARS-CoV and SARS-CoV-2 coronavirus.

There is currently no specific drug for the treatment of SARS-CoV-2 coronavirus, and vaccines and neutralizing antibodies appear to be the most promising drugs currently available. The neutralizing antibody prevents the virus from spreading by blocking the virus from invading host cells, thus achieving the purpose of treating diseases. Recently, many reports have been made in the literature on neutralizing antibodies against SARS-CoV-2. For example, the regenerative pharmaceutical company developed a series of SARS-CoV-2 neutralizing antibodies against the RBD domain via transgenic mice and a single B-cell sequencing platform (Hansen J et al,2020, Science 369: 1010-. Another group of researchers also developed a series of neutralizing antibodies against the RBD domain of the S protein of SARS-CoV-2 using a single B Cell sequencing technology platform (Wu Y et al,2020, Science,368, 1274-.

Three neutralizing antibodies against SARS-CoV-2 coronavirus S protein are currently in clinical study. On day 1/6 of 2020, the li-shi company developed a neutralizing antibody LY-CoV555 in cooperation with AbCellera completed the first patient dose and entered the phase I clinical study. LY-CoV555 is a potent neutralizing antibody against SARS-CoV-2 spike protein S of the IgG1 subtype. On day 11 of 6 months, the double antibody cocktail REGN-COV2 from the regenerative drug company first entered the clinical study phase and, based on clinically good safety data from phase I, the study is now directly entered phase III. In this 6 th month, the recombinant full-human anti-SARS-CoV-2 monoclonal antibody injection (JS016) developed by Junshi organism and China academy of sciences microorganism is approved to enter phase I clinical test. JS016 is the new coronavirus neutralizing antibody which enters the clinic at the earliest in China. Under the situation of severe new crown epidemic situation, the SARS-CoV-2 coronavirus S protein neutralizing antibody is developed as soon as possible, has higher specificity, better clinical efficacy and lower treatment cost, and provides more medication options for SARS-CoV-2 infected patients.

Disclosure of Invention

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

In a first aspect of the invention, there is provided an antibody or antigen-binding fragment thereof capable of specifically binding to the S protein of SARS-CoV-2 coronavirus, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising at least one, two or three Complementarity Determining Regions (CDRs) selected from the group consisting of:

(i) HCDR1 having the amino acid sequence as set forth in SEQ ID NO: 1 or 2, or a sequence 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 above sequences;

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

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

and/or, it comprises a light chain variable region (VL) comprising at least one, two or three Complementarity Determining Regions (CDRs) selected from the group consisting of:

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

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

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

In certain preferred embodiments, the substitution recited in any one of (i) - (vi) is a conservative substitution.

In certain 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.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises 3 VH variable region CDRs and 3 VL variable region CDRs selected from the group consisting of:

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

(ii) the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 2.4, 6, 8, 10 or 12, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 substitutions, deletions or additions) compared to any of the above sequences.

Wherein the 3 VH variable region CDRs and the 3 VL variable region CDRs in (i) are defined by the Kabat numbering system; (ii) the 3 VH variable region CDRs and 3 VL variable region CDRs in (a) are defined by the IMGT numbering system.

In certain embodiments, the antibody or antigen-binding fragment thereof is murine or chimeric, and the heavy chain variable region thereof comprises the heavy chain FR region of murine IgG1, IgG2, IgG3, or a variant thereof; and a light chain variable region thereof comprising the light chain FR region of a murine kappa, lambda chain or variant thereof. The amino acid sequence numbering of the variable regions of preferred murine antibodies is given in table 3 in example 5.

In certain preferred embodiments, the VH domain of the murine antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 13, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO: 14, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.

In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. The variable region amino acid sequence numbering for some preferred humanized antibodies is given in table 3 in example 5.

In certain preferred embodiments, the VH domain of the humanized antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 15, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO: 16, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.

In certain embodiments, the antibody comprises a heavy chain constant region and a light chain constant region derived from a human immunoglobulin.

More preferably, the antibody comprises a human kappa appa chain constant region amino acid sequence (amino acid sequence shown in SEQ ID NO: 17).

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, a heavy chain constant region selected from the group consisting of human IgG1, IgG2, and IgG 4; and, the heavy chain constant region has a native sequence or a sequence having substitution, deletion or addition of one or more amino acids compared to the native sequence from which it is derived. For example, in one embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG1 (amino acid sequence 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 shown in SEQ ID NO: 19). In one embodiment, the humanized antibody molecule comprises human IgG2 modified at the hinge region according to EU numbering (e.g., deletion of ERKCC, amino acid sequence shown in SEQ ID NO: 20), see Chinese patent No. CN 104177496B. In another embodiment, the humanized antibody molecule comprises human IgG4 (amino acid sequence shown in SEQ ID NO: 21) mutated at position 228 (e.g., S to P) according to EU numbering.

In certain preferred embodiments, the heavy chain of the antibody has the amino acid sequence as set forth in SEQ ID NO: 22; or a sequence having one or several 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 more identity compared to any of the above sequences; and/or the light chain of the antibody has the amino acid sequence shown as SEQ ID NO: 23; or a sequence having one or several 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 more identity compared to any of the above sequences.

In certain preferred embodiments, the substitutions described above are conservative substitutions.

In any of the above embodiments, the antibody or antigen binding fragment thereof of the invention can have a K of 10nM or less_DBinds to SARS-CoV-2 coronavirus S protein, preferably with a K of 1nM or less_D(ii) a binding S protein; more preferably, at a K of 100pM or less_D(ii) a binding S protein; more preferably, at a K of 10pM or less_D(ii) a binding S protein; most preferably, at a K of 1pM or less_DBinds to the S protein.

In a second aspect of the invention, there is provided a DNA molecule encoding the above antibody or antigen binding fragment thereof.

In a preferred embodiment of the invention, the DNA molecule encoding the heavy chain of the above antibody has the amino acid sequence shown in SEQ ID NO: 24; and/or, the DNA molecule encoding the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 25.

In a third aspect of the invention, there is provided a vector comprising the DNA molecule described above.

In a fourth aspect of the present invention, there is provided a host cell comprising the above-described vector; the host cell comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, NS0 cell or other mammalian cell, preferably a CHO cell.

In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, carrier or diluent.

In a sixth aspect of the invention, there is also provided a method of making an antibody or antigen-binding fragment thereof of the invention, comprising: (a) obtaining the gene of the antibody or the antigen binding fragment thereof, and constructing an expression vector of the antibody or the antigen binding fragment thereof; (b) transfecting the expression vector into a host cell by a genetic engineering method; (c) culturing the above host cell under conditions that allow production of the antibody or antigen-binding fragment thereof; (d) isolating and purifying the antibody or antigen-binding fragment thereof produced.

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

wherein, the constructed vector is transfected into a host cell by a genetic engineering method in the step (b), and the host cell comprises prokaryotic cells, yeast or mammalian cells, such as CHO cells, NS0 cells or other mammalian cells, preferably CHO cells.

Wherein step (d) separates, purifies the antibody or antigen-binding fragment thereof by conventional immunoglobulin purification methods, including protein a affinity chromatography and ion exchange, hydrophobic chromatography, or molecular sieve methods.

In a seventh aspect of the invention, there is provided the use of the antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment and prevention of a disease caused by SARS-CoV-2 coronavirus; preferably, the disease is COVID-19.

The eighth aspect of the present invention provides an immunoassay method for detecting or measuring the presence of SARS-CoV-2 virus or an antigen thereof in a biological sample or quantifying the amount thereof using the above-mentioned antibody; the method comprises incubating a biological sample to be tested with the anti-SARS-CoV-2 virus S protein monoclonal antibody or antigen binding fragment thereof of the invention to form an antigen-antibody complex, and performing qualitative detection and quantitative determination on the formed binding complex, wherein the existence or amount of the complex indicates the existence or content of SARS-CoV-2 virus; specifically, the method comprises the following steps:

(1) incubating a biological sample to be tested with at least one monoclonal antibody or antigen-binding fragment thereof of the invention under suitable conditions;

(2) detecting the presence of the bound complex in the above step.

The monoclonal antibody or antigen-binding fragment thereof according to the present invention can be used in the above immunoassay method independently of the label used (e.g., enzyme, fluorescence, etc.) and independently of the detection mode (e.g., fluorescence immunoassay, enzyme-linked immunosorbent assay, chemiluminescence assay, etc.) or assay principle (e.g., sandwich method, competition method, etc.); examples of such antigen-binding fragments include, but are not limited to, F (ab')₂Fab', Fab and Fv.

The above immunoassay methods, including enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, western blot, immunochromatography, latex agglutination assay, etc.; furthermore, the above-mentioned immunoassay methods can be used for measuring a target antigen in a biological sample by a competitive method or a sandwich method using an antigen or an antibody labeled with a labeling substance.

The competitive method is based on the quantitative competitive binding reaction of SARS-CoV-2 virus in the detected specimen and labeled SARS-CoV-2 virus S protein and the monoclonal antibody or antigen binding fragment thereof of the invention; specifically, the competition method includes: embedding a predetermined amount of the monoclonal antibody of the present invention against SARS-CoV-2 virus S protein or an antigen-binding fragment thereof on a solid phase carrier, then adding a biological sample containing SARS-CoV-2 virus to be detected and a predetermined amount of SARS-CoV-2 virus S protein labeled with a labeling substance, and incubating for a sufficient period of time under appropriate conditions; washing said solid phase extensively after the reaction and detecting the signal value of the label retained on the support or not retained on the support; the measured signal value is then compared to a predetermined amount of a 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 at approximately the same time.

The sandwich method is based on the fact that the monoclonal antibody or antigen-binding fragment thereof of the present invention, which is a capture antibody (or a solid phase antibody), and a labeled antibody that can be used in combination both specifically bind to SARS-CoV-2 virus in a biological sample, and the amount of SARS-CoV-2 virus in the sample is measured by quantifying the labeled antibody; specifically, the above sandwich method comprises: binding the specific monoclonal antibody or antigen binding fragment thereof aiming at SARS-CoV-2 virus S protein to a solid phase carrier to form a solid phase antibody (also called capture antibody or first antibody), then respectively adding a biological sample to be detected and a control sample to the coated solid phase carrier and incubating for a sufficient time under proper conditions; after the reaction, fully washing the solid phase, adding a second antibody which is marked by a proper amount of a marker and can be combined with S protein of SARS-CoV-2 virus, and incubating again; after the reaction, washing the solid phase sufficiently and detecting a signal value of the label bound to the second antibody by an appropriate method; the measured signal value is compared to a signal value of a control sample of a predetermined amount measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample.

The second antibody may also be other polyclonal antibodies; preferably, the second antibody is a monoclonal antibody.

More preferably, the second antibody is selected from any of the monoclonal antibodies or antigen-binding fragments thereof of the present invention that can be used in conjunction with the first antibody.

Wherein the label may be a radioisotope (e.g., a radioisotope)¹²⁵I) Enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin and coloring substances.

Preferably, the markers used in the present invention include, for example, alkaline phosphatase, horseradish peroxidase, β -galactosidase, urease and glucose oxidase; the label may also be a fluorescent substance, such as fluorescein derivatives and rhodamine derivatives; alternatively, the label may be a rare earth element or rare earth element complex, such as europium or europium complexes, which allow time-resolved fluorescence measurements; in addition, the label may be a phosphorescent substance, such as acridinium ester and isoluminol; or a radioactive isotope such as¹²⁵I、³H、¹⁴C and³²p; in addition, the labeling substance may be a coloring substance such as latex particles and colloidal gold. That is, the present invention includes qualitative or quantitative determination of the presence or amount of SARS-CoV-2 virus in a biological component by measuring color, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemical fluorescence, or radioactivity.

When SARS-CoV-2 immunoassay is carried out by the above competition method and sandwich method, the solid phase needs to be sufficiently washed to measure the activity of binding to the label. When the label is a radioisotope, the measurement is performed using a well counter or a liquid scintillation counter. When the label is an enzyme, a substrate is added and the enzyme activity is measured colorimetrically or by fluorescence after color development. When the labeling substance is a fluorescent substance, a phosphorescent substance, or a coloring substance, the measurement can be performed by a method known in the art, respectively.

The above-mentioned biological sample is selected from the group consisting of plasma, whole blood, mouthwash, throat swab, urine, stool, and bronchial perfusate.

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

The ninth aspect of the invention provides the use of the above antibody or antigen binding fragment thereof in the preparation of a SARS-CoV-2 virus detection kit.

In a tenth aspect of the present invention, there is provided a SARS-CoV-2 virus detection kit comprising at least one monoclonal antibody or an antigen-binding fragment thereof of the present invention; the monoclonal antibody used for preparing the detection reagent is not particularly limited, and may be any of the monoclonal antibodies of the present invention described above or an antigen-binding fragment thereof (e.g., F (ab')₂Fab', and scFv) are used alone as one of a solid phase antibody or a labeled antibody; two monoclonal antibodies or antigen-binding fragments thereof against different epitopes of the present invention as described above may be used in combination as a solid phase antibody or a labeled antibody, respectively.

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

(1) selected from any one of:

a. a solid support and a first antibody;

b. a solid support coated with a first antibody;

the first antibody is selected from any one of the monoclonal antibodies or antigen binding fragments thereof;

(2) a second antibody;

the second antibody is optionally labeled appropriately and is selected from the group consisting of a monoclonal antibody or an antigen-binding fragment thereof of the present invention that can be used in combination with the first antibody of (1).

The monoclonal antibody or antigen-binding fragment thereof of the present invention contained in the above detection reagent may be immobilized on a solid phase carrier in advance to form a solid phase antibody, wherein the solid phase carrier includes, but is not limited to, nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, beads, or optical fiber sensors such as glass, fiberglass, or polymers (e.g., polystyrene or polyvinyl chloride); in a preferred embodiment of the present invention, the solid support is a microtiter plate.

The monoclonal antibody or antigen-binding fragment thereof of the present invention contained in the above-mentioned immunoassay reagent may be labeled with a label in advance to form a labeled antibody, the label including, but not limited to, a radioisotope (e.g., radioisotope)¹²⁵I) Enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin and coloring substances; preferably, the enzymes include, for example, alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease, and glucose oxidase; the fluorescent substance includes, for example, fluorescein derivatives and rhodamine derivatives, and rare earth elements or rare earth element complexes, such as europium or europium complexes; the phosphorescent substances include such as acridine ester and isoluminol; the radioactive isotopes include¹²⁵I、³H、¹⁴C and³²p; the coloring matter includes, for example, latex particles and colloidal gold; in a preferred embodiment of the present invention, the marker is biotin.

The eleventh aspect of the present invention provides the use of the above immunoassay reagent for the diagnosis of a disease caused by SARS-CoV-2 virus infection; preferably, the disease is a novel coronavirus pneumonia.

The technical scheme disclosed by the invention achieves beneficial technical effects, and is summarized as follows:

1. by using a mouse hybridoma platform, the mouse is immunized by taking S protein (amino acid 326-685) as an immunizing antigen, and mouse antibodies aiming at the S protein of SARS-CoV-2 coronavirus are obtained, the antibodies can specifically recognize and bind the S protein with high affinity, and the KD value reaches pM level.

2. Results of in vitro competitive experiments with ACE2 show that these murine antibodies compete with human ACE2 receptor for binding site, IC, of S protein₅₀Values down to nM range.

3. We have performed humanization of murine antibodies,reducing its immunogenicity. The humanized antibody retains the affinity of murine antibody and pseudovirus inhibitory activity, and binds to affinity K_DThe value reaches pM level, and the pseudovirus inhibition activity is equivalent to nM level. The characteristics lay a foundation for the clinical application of the antibody.

4. The antibody provided by the invention can also be used for detecting the existence of SARS-CoV-2 virus or corresponding antigen in a sample, and the detection sensitivity of the antibody is lower than 100 pg/ml; more preferably, less than 10 pg/ml.

Detailed Description

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, 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 belongs.

Abbreviations and Definitions

CDR complementary-Determining Region, the Complementarity Determining Region in an immunoglobulin variable Region, is defined using the Kabat, IMGT, Chothia or AbM numbering system (see the terms "hypervariable Region" or "CDR Region" or "Complementarity Determining Region").

EC₅₀Concentrations that give 50% efficacy or binding

ELISA enzyme-linked immunosorbent assay

FR antibody Framework region (Framework), immunoglobulin variable region excluding CDR region

HRP horse radish peroxidase

IC₅₀Concentration giving 50% inhibition

IgG immunoglobulin G

The alignment of immunoglobulin amino acid sequences and numbering system advocated by Elvin A Kabat by Kabat.

mAb monoclonal antibodies

PCR polymerase chain reaction

V regions are IgG chain segments with variable sequences between different antibodies. It extends to Kabat residue 109 of the light chain and residue 113 of the heavy chain.

VH immunoglobulin heavy chain variable region

VL immunoglobulin light chain variable region

K_DEquilibrium dissociation constant

k_aConstant of binding rate

k_dOff rate constant

The term "EC₅₀"refers to the concentration of an antibody or antigen-binding fragment thereof that induces a 50% response in an in vitro or in vivo assay using the antibody or antigen-binding fragment thereof, i.e., the concentration half-way between the maximal response and baseline.

The term "EU Numbering System" (EU Numbering System or Scheme): eu refers to the first human IgG1 immunoglobulin isolated and purified by Gerald M Edelman et al, named Eu, at the end of the last 60 th century (1968-1969), and its amino acid sequence was determined and numbered (Edelman GM et al,1969, Proc Natl Acad USA,63: 78-85). The heavy chain constant regions of other immunoglobulins are aligned with the amino acid sequence of Eu, and the corresponding amino acid position is the Eu number. The Eu numbering system is primarily directed to immunoglobulin heavy chain constant regions, including CH1, CH2, CH3, and the hinge region.

The term "Kabat Numbering System" (Kabat Numbering System or Scheme): in 1979, Kabat et al first proposed a standardized numbering scheme for human Immunoglobulin variable regions (Kabat EA, Wu TT, Bilofsky H, Sequences of Immunoglobulin Chains: tasks and Analysis of Amino Acid Sequences of syndromes, V-regions, C-regions, J-Chains and β₂Microglobulins.1979.department of Health, Edutation, and Welfare, Public Health Service, National Institutes of Health). In the "immunologically relevant protein sequences" (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 for the amino acids of antibody light and heavy chainsThe amino acid sequences are aligned and numbered. They found that these analyzed sequences exhibited variable lengths, and that the default and inserted amino acids or amino acid fragments were only present at specific positions. Interestingly, the insertion point is mostly located within the CDRs, but may also occur at certain positions in the framework regions. In the Kabat numbering scheme, the light chain variable region is numbered to position 109, the heavy chain variable region is numbered to position 113, and the inserted amino acids of the light and heavy chains are identified and annotated by letters (e.g., 27a, 27 b.). All Lambda light chains do not contain the residue at position 10, whereas Lambda and Kappa light chains are encoded by two different genes, located on different chromosomes. Lambda and Kappa light chains can be distinguished by differences in their constant region amino acid sequences. Unlike the EU numbering system for the heavy chain constant region only, the numbering range of the Kabat numbering system covers the full-length immunoglobulin sequences, including the variable and constant regions of the immunoglobulin light and heavy chains.

The term "binding" defines the affinity interaction between a particular epitope on an antigen and its corresponding antibody, also commonly understood as "specific recognition". By "specifically recognizes" is meant that the antibody of the invention does not cross-react or does not substantially cross-react with any polypeptide other than the antigen of interest. The degree of specificity can be determined by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like. In the present invention, the specific recognition is preferably determined by flow cytometry, and the standard of specific recognition in a specific case can be judged by a person of ordinary skill in the art based on his or her knowledge in the art.

The term "antigen" is a foreign substance capable of eliciting antibodies from an organism itself or a human, and is any substance capable of inducing an immune response, such as bacteria, viruses, etc. The foreign antigen molecules are identified and processed by B cells or antigen presenting cells (such as macrophages, dendritic cells, endothelial cells, B cells, and the like), and combined with major histocompatibility complex (such as MHC II molecules) to form complexes to reactivate T cells, thereby triggering continuous immune response.

The term "antigenic epitope" or "antigenic determinant" refers to a molecule that is antigenic (i.e., capable of eliciting a specific immunity)Immune response) is a site on an antigen (e.g., the S protein of SARS-CoV-2) to which an immunoglobulin or antibody specifically binds. Epitopic determinants are typically composed of chemically active surface groups of the molecule (e.g., amino acids or glycosyl side chains) and typically have specific three-dimensional structural properties as well as specific charge properties. There are two epitopes or antigenic determinants (epitopes) of an antigen, a B cell epitope and a T cell epitope, recognized by B cells and T cells, respectively. By epitope we generally refer to B cell epitopes. B cell epitopes are located on the surface of antigenic molecules, and are antigenic sites that bind to B Cell Receptors (BCRs), an antibody located on the B cell membrane, and B cell epitopes are recognized directly by B cells without the need for processing. The B cells then phagocytose antigenic molecules, process them into small peptides (about 15 amino acids in size, antigenic T cell epitopes) and present them on Th cells (helper T cells). Meanwhile, the antigenic molecules can also be processed into small peptides through another route, such as phagocytosis by macrophages, and presented to Th cells. Th is co-stimulated by B cells and macrophages, the three cells interact together, and the Th cells send feedback signals to the B cells to indicate the B cells to proliferate, differentiate into plasma cells and memory cells. The plasma cells have the function of secreting antibodies and mediate humoral adaptive immunity. Antibodies bind to antigen molecules through their variable region Fv portion and bind to receptor fcrs on various immune cells through their constant region Fc portion, thereby directing the various immune cells to kill the antigen molecules and perform ADCC (by NK cells), CDC (by complement) and ADCP (by macrophages) functions. Each B cell is specific and secretes only one antibody. B cell epitopes can be classified into continuous epitopes and conformational epitopes (or discontinuous epitopes) according to their continuity in the amino acid sequence of the protein. The size of the B cell epitope is variable and has 5-20 amino acids. T cell epitopes are recognized by T cells and, unlike B cell epitopes, T cell epitopes can be located anywhere in an antigenic molecule (e.g., a viral protein) and thus are within the sequence of the entire protein. T cell epitopes are continuous determinants, typically 10-20 amino acids in size. T cell epitopes and class I (MHC I) or class II (MHC II) MHC moleculesBound and presented on the cell surface by two different subsets of T cells, CD8⁺T cells (killer T cells) and CD4⁺T cell (helper Th cell) recognition. Thus, the T cell epitope is CD8⁺And CD4⁺T cell epitopes are two. MHC I molecules are expressed by almost all cells and can provide some conditions in the cells, for example, when the cells are infected by virus, small peptide molecules of virus fragments are prompted on the cell surface through MHC I and can be used for killing CD8⁺T cells and the like for killing. MHC II molecules are mostly located on antigen presenting cells, such as macrophages and the like. Such MHC II molecules provide extra-cellular (e.g. humoral) conditions, such as bacterial invasion of tissues, and subsequent phagocytosis by macrophages, bacterial debris is presented to helper Th cells using MHC II, initiating an immune response. B cells and T cells recognize and bind only the epitope of a foreign antigen molecule, and do not have binding ability to antigen fragments derived from the organism itself, such as protein molecules and fragments thereof, because B cells and T cells having high affinity for self protein molecules or fragments are inhibited from developmental maturation or from apoptosis during the differentiation, development and maturation of B cells and T cells.

The term "antibody" generally refers to a protein-binding molecule having a function such as an immunoglobulin. Typical examples of antibodies are immunoglobulins, as well as derivatives or functional fragments thereof, as long as they exhibit the desired binding specificity. Techniques for making antibodies are well known in the art. "antibodies" include natural immunoglobulins of different classes (e.g., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, lgG2, IgA1, IgA2, etc.). "antibody" also includes non-natural immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies), as well as antigen-binding fragments thereof (e.g., Fab ', F (ab')₂Fab, Fv and rIgG). See also, e.g., Pierce Catalog and Handbook, 1994-; kuby J, Immunology,3rd Ed, WH Freeman&Co, New York, 1997). Antibodies can bind to an antigen, termed "monospecific"; or combined into twoThe same antigen, termed "bispecific"; or bind to more than one different antigen, referred to as "multispecific". Antibodies can be monovalent, bivalent, or multivalent, i.e., an antibody can bind to one, two, or more antigen molecules at a time. An antibody binds "monovalent" to a particular protein, i.e., a molecule of antibody binds only to one molecule of protein, but the antibody may also bind to a different protein. When an antibody binds to only each molecule of two different proteins, the antibody is "monovalent" binding to each protein, and the antibody is "bispecific" and "monovalent" binding to each of the two different proteins. An antibody may be "monomeric," i.e., it comprises a single polypeptide chain. An antibody can comprise multiple polypeptide chains ("multimeric") or can comprise two ("dimeric"), three ("trimeric") or four ("tetrameric") polypeptide chains. If the antibody is multimeric, the antibody may be homomultimeric (homomulitmer), i.e. the antibody comprises more than one molecule of only one polypeptide chain, including homodimers, homotrimers or homotetramers. Alternatively, the multimeric antibody may be a heteromultimer, i.e., the antibody comprises more than one different polypeptide chain, including a heterodimer, a heterotrimer, or a heterotetramer.

The term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprised by the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the phrase "monoclonal" means 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 produce hybrid antibody producing cells. Synthesized by hybridoma culture, and is not contaminated by other immunoglobulins. Monoclonal antibodies can also be obtained using, for example, recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art.

The term "whole antibody" refers to an antibody consisting of two antibody heavy chains and two antibody light chains. An "intact antibody heavy chain" is composed of an antibody heavy chain variable domain (VH), an antibody constant heavy chain 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-terminal to C-terminal direction, abbreviated VH-CH1-HR-CH2-CH 3; and, in the case of antibodies of the IgE subclass, optionally also antibody heavy chain constant domain 4(CH 4). Preferably a "whole antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. An "intact antibody light chain" is a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated VL-CL, in the N-terminal to C-terminal direction. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). Intact antibody chains are linked together by interpeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light and heavy chains) and between the hinge region of the intact antibody heavy chain. Examples of typical whole antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE.

The term "Antibody fragment" or "antigen-binding fragment" refers to antigen-binding fragments and Antibody analogs of antibodies that retain the ability to specifically bind to an antigen (e.g., the S protein of SARS-CoV-2 coronavirus), which typically include at least a portion of the antigen-binding or variable region of a parent Antibody (partial Antibody). Antibody fragments retain at least some of the binding specificity of the parent antibody. Usually, when molar units (K) are used_D) When active, the antibody fragment retains at least 10% of the parent binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% of the binding affinity of the parent antibody to the target. Antibody fragments include, but are not limited to: fab fragment, Fab 'fragment, F (ab')₂Fragments, Fv fragments, Fd fragments, Complementarity Determining Region (CDR) fragments, disulfide bond stability proteins (dsFv), and the like; linear antibodies (Linear antibodies), Single chain antibodies (e.g., scFv Single 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 (domanis, technology from domanis), nanobodies (technology from Ablynx); multispecific antibodies formed from antibody fragments (examples)Such as three-chain antibodies, four-chain antibodies, etc.); and engineered antibodies such as Chimeric antibodies (e.g., humanized murine antibodies), Heteroconjugate antibodies (Heteroconjugate antibodies), and the like. These antibody fragments are obtained by conventional techniques known to those skilled in the art and are screened for utility in the same manner as are intact antibodies.

The term "single chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, a recombinant protein having a heavy chain variable region (VH) and a light chain variable region (VL) joined by a linker (linker) that crosslinks the two domains to form an antigen binding site, the linker sequence typically consisting of a flexible peptide, such as, but not limited to, G2(GGGGS)₃. The size of the scFv is typically 1/6 for a whole antibody. Single chain antibodies are preferably a sequence of amino acids 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. 4946778 and 5260203.

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

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

The term "hinge region" includes that portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises about 25 residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be divided into three distinct domains: upper, middle, and lower hinge domains (Roux KH et al,1998, J Immunol,161: 4083-.

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 a natural intact antibody, a single chain antibody (scFv), an Fd fragment, an Fab fragment, an F (ab')₂Fragments, single domain antibody fragments, isolated CDR fragments and methods of making the sameAnd (3) derivatives. By "single-stranded" is meant herein that the first and second domains are covalently linked and may be represented by a co-linear amino acid sequence encoded by a single nucleic acid molecule.

The term "Fab fragment" consists of the variable region of one heavy chain together with the CH1 region and one light chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fab antibody" is 1/3 for the size of a whole antibody, which contains only one antigen binding site.

The term "Fab' fragment" contains one light chain, the VH domain and the CH1 domain of one heavy chain, and the constant region portion between the CH1 and CH2 domains.

The term "F (ab')₂A fragment "contains the VH and CH1 domains of two light and two heavy chains and the constant region portion between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F (ab')₂The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The term "Fd fragment" consists of the variable region of one heavy chain and CH1, and is the heavy chain portion remaining after the Fab fragment has removed the light chain.

The term "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant region, and is the smallest fragment that comprises the entire antigen recognition and binding site.

The term "disulfide-bond stability protein (dsFv)" introduces a cysteine mutation point in the VH and VL regions, respectively, to form a disulfide bond between VH and VL for structural stability. The term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a sulfhydryl group which may form a disulfide bond or bridge with a second sulfhydryl 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, EU numbering system) corresponding to the use of the Kabat numbering system.

The term "heavy chain constant region" includes amino acid sequences from immunoglobulin heavy chains. A polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge (e.g., upper hinge region, middle hinge region, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide as used herein can 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 structure, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present application comprises a polypeptide chain having a CH3 domain. In addition, an antibody used in the present 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 as will be appreciated by those of ordinary skill in the art, the heavy chain constant regions may be modified such that they differ in amino acid sequence from the naturally occurring immunoglobulin molecule.

The term "light chain constant region" includes amino acid sequences from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain and a constant lambda domain.

The term "Fc region" or "Fc fragment" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region, the CH2 domain, and the CH3 domain, which mediates binding of the immunoglobulin 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 of the classical complement system (C1 q). The Fc region includes a native sequence Fc region and a variant Fc region.

Typically, the human IgG heavy chain Fc region is the carboxy-terminal stretch from the amino acid residue at position Cys 226 or Pro 230, but the boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc may also refer to this region, either independently, or in the case of a protein polypeptide comprising Fc, such as "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). The native sequence Fc region in the antibodies of the invention is derived from IgG1, IgG2(IgG2A, IgG2B), IgG3 and IgG4, including mammalian (e.g., human). In certain embodiments, the amino acid sequences of the two Fc polypeptide chains have single amino acid substitutions, insertions, and/or deletions of around 10 amino acids per 100 amino acids relative to the amino acid sequence of a mammalian Fc polypeptide. In some embodiments, the above-described Fc region amino acid differences may be Fc alterations that extend half-life, alterations that increase FcRn binding, alterations that enhance fcgamma receptor (fcyr) binding, and/or alterations that enhance ADCC, ADCP and/or CDC.

In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the IgM and IgE Fc regions comprise three heavy chain constant domains (CH2-4 domains) in each polypeptide chain.

The term "chimeric antibody" refers to a portion of the heavy and/or light chain that is identical or homologous to corresponding sequences in antibodies 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 corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,4816567; Morrison SL et al,1984, Proc Natl Acad Sci USA,81: 6851-. For example, the term "chimeric antibody" can include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).

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

The term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Most or all of the amino acids outside the CDR domain of a non-human antibody, e.g., a mouse antibody, are replaced with corresponding amino acids from a human immunoglobulin, while most or all of the amino acids within one or more CDR regions are unchanged. Amino acid additions, deletions, insertions, substitutions or modifications are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. "humanized" antibodies retain antigen specificity similar to the original antibody. The source of the CDR is not particularly limited and may be derived from any animal. For example, CDR regions derived from a mouse antibody, a rat antibody, a rabbit antibody, or a non-human primate (e.g., cynomolgus monkey) antibody can be utilized. Framework regions human antibody germline sequences can be obtained by searching for IMGT antibody germ line database (http:// www.imgt.org/3D structure-DB/cgi/DomainGapAlign. cgi), and generally human germline antibody sequences with high homology to the engineered non-human antibody are selected as framework regions of the humanized antibody.

The term "hypervariable region" or "CDR region" or "complementarity determining region" refers to the amino acid residues of an antibody which are responsible for antigen binding and are non-contiguous amino acid sequences. CDR region sequences can be defined by the methods of Kabat, Chothia, IMGT (Lefranc et al,2003, Dev company at Immunol,27:55-77) and AbM (Martin ACR et al,1989, Proc Natl Acad Sci USA, 86: 9268-. For example, the hypervariable region comprises the following amino acid residues: amino acid residues (Kabat numbering system) from "complementarity determining regions" or "CDRs" defined by sequence alignment, e.g., residues 24-34(LCDR1), 50-56(LCDR2) and 89-97(LCDR3) of the light chain variable domain and residues 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, Bethesda, Md.; and/or residues from the "hypervariable loops" (HVLs) defined by structure (Chothia numbering system), e.g., residues 26-32(LCDR1), 50-52(LCDR2) and 91-96(LCDR3) of the light chain variable domain and residues 26-32(HCDR1), 53-55(HCDR2) and 96-101(HCDR3) of the heavy chain variable domain, see Chothia C and Lesk AM,1987, J Mol Biol,196:901 917; chothia C et al,1989, Nature,342: 878-883. "framework" residues or "FR" residues are variable domain residues other than the hypervariable region residues defined herein. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, IMGT, or Chothia numbering system. The skilled person can explicitly assign each numbering system to any variable domain sequence without relying on any experimental data beyond the sequence itself. For example, the Kabat residue numbering for a given antibody can be determined by aligning the antibody sequences to the regions of homology for each "standard" numbered sequence. The determination of the numbering of any variable region sequence in a sequence listing is well within the routine skill of those in the art based on the sequence numbering scheme provided herein.

The term "recombinant," when referring to a polypeptide or polynucleotide, refers to a form of the polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which may be achieved by combining polynucleotides or polypeptides that do not normally occur together.

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 a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "isolated antibody molecule" refers to an antibody molecule that has been recognized and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

The term "isolated" as used herein with respect to a nucleic acid (e.g., DNA or RNA) refers to a molecule that is isolated from other DNA or RNA, respectively, that is present as a macromolecule from natural sources. The term "isolated" as used herein also refers to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when produced by chemical synthesis. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring fragments and are not found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are isolated from other cellular proteins or tissues. Isolated polypeptides are meant to include both purified and recombinant polypeptides.

The term "cross-reactive" refers to the ability of an antibody described herein to bind to an antigen from a different species. For example, an antibody described herein that binds to the S protein of SARS-CoV-2 coronavirus can also bind to an S protein from other species (e.g., the S protein of SARS-CoV). Cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or binding to or otherwise interacting with the function of a cell that physiologically expresses the antigen. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (e.g., Biacore) or similar techniques (e.g., Kinexa or Octet).

The terms "immunological binding" and "immunological binding properties" refer to a non-covalent interaction that occurs betweenBetween an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction may be determined by the equilibrium dissociation constant (K) of the interaction_D) Is represented by the formula, wherein K_DSmaller values indicate higher affinity. The immunological binding properties of the selected polypeptide may be determined using methods well known in the art. One assay involves measuring the rate of antigen/antibody complex formation and dissociation. "binding Rate constant" (K)_aOr K_on) And "dissociation rate constant" (K)_dOr K_off) Both can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M,1993, Nature,361: 186-187). k is a radical of_d/k_aIs equal to the equilibrium dissociation constant K_D(see Davies DR et al,1990, Annual Rev Biochem,59: 439-. K can be measured by any effective method_D、k_aAnd k_dThe value is obtained.

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

The term "host cell" refers to a cell, which may be prokaryotic or eukaryotic, in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that not all progeny may be identical to the parent cell, since mutations may occur during replication and such progeny are included. The host cell comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, NS0 cell or other mammalian cell.

The term "identity" is used to refer to the match in sequence between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent 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 x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such an alignment can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.), by using the method of Needleman and Wunsch (Needleman SB and Wunsch CD,1970, J Mol Biol,48: 443-.

The terms "mutated", "mutant" and "mutation" refer to the substitution, deletion or insertion of one or more nucleotides or amino acids, respectively, as compared to the native nucleic acid or polypeptide (i.e., a reference sequence that may be used to define the wild type).

Antibodies with conservative modifications

The term "conservative modification" is intended to mean that the amino acid modification does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions refer to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been described in detail in the art. These families include amino acids with 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the invention can be replaced with other amino acid residues from the same side chain family.

Therapeutic uses of antibodies directed against SARS-CoV-2 coronavirus S protein

The term "prevention" refers to a method performed in order to prevent or delay the onset of a disease or disorder or condition (e.g., tumor or infection) in a subject or if its effects are minimized.

The term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, decreasing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis, whether detectable or undetectable. The amount of therapeutic agent effective to alleviate any particular disease symptom may vary depending on factors such as the disease state, age and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a symptom of a disease is alleviated can be assessed by any clinical measure that is typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptom.

Antibodies of the invention (which include bispecific, polyclonal, monoclonal, humanized antibodies) may be used as therapeutic agents. These agents may be used generally to treat or prevent novel coronary pneumonia COVID-19, increase vaccine efficacy or enhance innate immune response in a subject. Antibody preparations, preferably with high specificity and high affinity for their target antigen S protein, are administered to a subject and generally have an effect on their binding to the target. Administration of the antibody can eliminate or inhibit or interfere with the activity of the SARS-CoV-2 coronavirus S protein. In the case of 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 sequences of the antibody, which retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and/or prepared by recombinant DNA techniques (see, e.g., Marasco WA et al,1993, Proc Natl Acad Sci USA,90: 7889-.

The antibody or fragment thereof of the present invention that specifically binds to SARS-CoV-2 coronavirus S protein can be administered in the form of a pharmaceutical composition. The formulations may contain more than one active compound, preferably those with complementary activities that do not adversely affect each other, as required for the particular indication being treated. Alternatively or additionally, the composition may comprise an agent that enhances its function.

Pharmaceutical composition

The antibodies of the invention, or nucleic acids or polynucleotides encoding the antibodies of the application, may be used to prepare pharmaceutical or sterile compositions, for example, by mixing the antibodies with a pharmaceutically acceptable carrier, excipient or stabilizer. The pharmaceutical composition may comprise one or a combination (e.g. two or more different) of the antibodies of the invention. For example, the pharmaceutical compositions of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activity that bind to different epitopes on the target antigen. Formulations of the therapeutic and diagnostic agents may be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions or suspensions. The term "pharmaceutically acceptable" means that the molecular entity, molecular fragment, or composition does not produce an adverse, allergic, or other untoward reaction when properly administered to an animal or human. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof include sugars (e.g., lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (e.g., propylene glycol), alginic acid, and the like. The antibodies of the invention or nucleic acids or polynucleotides encoding the antibodies of the application may be used alone or may be used in conjunction with one or more other therapeutic agents, such as vaccines.

The term "pharmaceutically acceptable carrier and/or excipient and/or stabilizer" refers to a carrier and/or excipient and/or stabilizer that is pharmacologically and/or physiologically compatible with the subject and active ingredient and that is non-toxic to the cells or mammal to which it is exposed at the dosages and concentrations employed. Including but not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents 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, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament, including, but not limited to, sodium 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 degradation products thereof (such as lactalbumin hydrolysate), and the like.

Diagnostic use of antibodies against SARS-CoV-2 coronavirus S protein

The monoclonal antibody or antigen-binding fragment thereof of the present invention can be used for the detection or quantification of SARS-CoV-2 virus by immunoassay. The immunoassay method itself is well known, and any known immunoassay method can be used. That is, there are a sandwich method, a competition method, an aggregation method, a western blot method and the like if classification is performed in a measurement format, and there are a fluorescence method, an enzymatic method, a radiation method, a biotin method and the like if classification is performed with a label used, and these methods can be used. Diagnosis can also be made by immunohistological staining. When a labeled antibody is used in the immunoassay method, the method of labeling the antibody is known per se, and any known method can be used.

These immunoassays are known per se, and needless to say in this specification, for example, the sandwich method is a method in which an antibody or an antigen-binding fragment of the present invention is immobilized as a first antibody on a solid phase, reacted with a biological sample to be tested, rinsed, reacted with a second antibody, and rinsed, followed by measurement of the second antibody bound to the solid phase. The second antibody can be labeled with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like, and the second antibody bound to the solid phase can be measured. By measuring a plurality of standards of known concentrations by the above method, preparing a standard curve based on the relationship between the amount of the measured marker and the content of the standard, and comparing the measurement result of the test sample of unknown concentration with the standard curve, the SARS-CoV-2 virus antigen in the test sample can be quantified. The first antibody and the second antibody may also be substituted in the above description. In the agglutination method, the antibody or antigen-binding fragment thereof of the present invention is immobilized on particles such as latex, and reacted with a sample to measure the absorbance. By measuring a plurality of standards of known concentrations by the above method, preparing a standard curve based on the relationship between the amount of the measured marker and the content of the standard, and comparing the measurement result of the test sample of unknown concentration with the standard curve, the SARS-CoV-2 virus antigen in the test sample can be quantified.

The biological sample to be supplied to the immunoassay method is not particularly limited as long as it contains the S protein of SARS-CoV-2 virus, and examples thereof include human-and animal-derived serum, plasma, and whole blood, as well as body fluid extracts such as nasal swab (nasal swab), nasal aspirate, and pharyngeal swab (pharyngeal swab), saliva, respiratory secretions, urine, feces, cells, and tissue homogenate.

By using the monoclonal antibody of the present invention, a reagent for measuring SARS-CoV-2 virus immunity can be produced by using the antibody as at least one of a solid-phase antibody and a labeled antibody. The solid phase to which the monoclonal antibody is bound can be any of various solid phases used in conventional immunoassays, and examples thereof include: ELISA plate, latex, gelatin particle, magnetic particle, polystyrene, glass and other various solid phase, beads, liquid-transmitting matrix and other insoluble carrier. The labeled antibody can be prepared by labeling an antibody with an enzyme, colloidal metal particles, colored latex particles, a luminescent substance, a fluorescent substance, a radioactive substance, or the like. By combining these reagents such as the solid-phase antibody and/or the labeled antibody, a reagent used in enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, or the like can be prepared. These assay reagents are reagents for assaying a target antigen in a sample by a sandwich method or a competitive binding assay.

The reagent for immunoassay by the sandwich method may be the following reagents: for example, two kinds of the monoclonal antibodies of the present invention are prepared, one of which is the labeled antibody and the other is a solid phase antibody bound to the solid phase. First, a sample containing an antigen to be measured is reacted with the solid-phase antibody, and then a labeled antibody (second antibody) is reacted with the antigen captured by the solid-phase antibody, whereby the presence or activity of the label bound to the insoluble carrier is detected, whereby immunoassay can be performed. Similarly, an immunoassay can be performed by reacting a sample containing an antigen to be measured with a solid-phase antibody, subsequently reacting a labeled antibody (second antibody) with the antigen captured on the solid-phase antibody, and determining the presence or activity of the label bound to the insoluble carrier, that is, quantifying the amount of the antigen to be measured by the amount of the labeled antibody. In the immunoassay reagent of the sandwich method, one monoclonal antibody may be used as the solid phase antibody and the labeled antibody (for example, when the antigen is a polymer), but it is generally preferable to use 2 or more antibodies that can recognize two different epitopes of the antigen to be measured, respectively. Further, any of the solid-phase antibody and the labeled antibody may be used in combination selected from 2 or more kinds of monoclonal antibodies.

The immunoassay reagent used in the competitive binding assay method may be prepared, for example, as a predetermined amount of a viral antigen labeled with an enzyme, colloidal metal particles, colored latex particles, a luminescent substance, a fluorescent substance, a radioactive substance, or the like. The reagent can be used to conduct a competitive reaction with a sample containing a certain amount of the monoclonal antibody of the present invention, the labeled viral antigen and the antigen to be measured, and the amount of the antigen in the sample to be measured can be quantified from the amount of the labeled viral antigen bound or unbound to the antibody, thereby conducting immunoassay.

In the present invention, the antibody or antigen can be bound to a solid phase or a labeled substance by a physical adsorption method, a chemical binding method, or the like (see "protein nucleic acid enzyme", Japanese patent application No.31, 37-45 (1987)).

The labeled anti-SARS-CoV-2 virus monoclonal antibody can be prepared by binding the anti-SARS-CoV-2 virus monoclonal antibody to a labeling substance. The label may be an enzyme, a colloidal metal particle, a colored latex particle, a fluorescent latex particle, a luminescent substance, a fluorescent substance, or the like. The Enzyme may be various enzymes used in Enzyme-linked immunoassays (EIA), such as alkaline phosphatase, peroxidase, β -D-galactosidase, and the like; as the colloidal metal particles, for example, colloidal gold particles, colloidal selenium particles, and the like can be used.

The method of binding the marker to the monoclonal antibody against SARS-CoV-2 virus can be carried out by a known method of generating a covalent bond or a non-covalent bond. Examples of the bonding method include: glutaraldehyde method, periodic acid method, maleimide method, dithiodipyridine method, method using various crosslinking agents, etc. (for example, "protein nucleic acid enzyme", Japanese patent application No.31, 37-45 (1985)). In the binding method using a crosslinking agent, for example, N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidocaproic acid, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, and the like can be used as the crosslinking agent. In the method of covalent bonding, depending on the use of a functional group present in the antibody, a labeled monoclonal antibody against SARS-CoV-2 virus can be produced by introducing a functional group such as thiol, amino, carboxyl, hydroxyl or the like into the antibody by a conventional method, and then binding the functional group to the label by the above-mentioned binding method. The non-covalent bonding method may be a physical adsorption method.

As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates, etc., which correspond to the enzyme of the label and are represented as follows, can be used.

(a) Chromogenic substrate: 2, 2 ' -azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS), 3 ', 5, 5 ' -Tetramethylbenzidine (TMB), Diaminobenzidine (DAB) in combination with hydrogen peroxide for peroxidases; 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), sodium 5-bromo-4-chloro-3-indolyl phosphate (BCIP. Na) are used for alkaline phosphatase.

(b) Fluorescent substrate: 4-methylumbelliferyl phosphate (4-MUP) for alkaline phosphatase; 4-Methylumbelliferyl phenyl-beta-D-galactoside (4MUG) was used for beta-D-galactosidase.

(c) Luminescent substrate: 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxetane.2sodium salt (AMPPD) for alkaline phosphatase; 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-beta-D-galactopyranosyl) phenyl-1, 2-dioxetane (AMGPD) for beta-D-galactosidase; luminol, isoluminol obtained in combination with hydrogen peroxide is used in peroxidases.

By assaying various biological samples derived from a human or an animal using the monoclonal antibody against the S protein of SARS-CoV-2 virus of the present invention, diagnosis of SARS-CoV-2 virus infection can be carried out.

Drawings

FIG. 1, SARS-CoV-2S1 antigen immune mouse serum titer.

FIG. 2, serum neutralizing antibody titer of SARS-CoV-2S1 antigen-immunized mice.

FIG. 3, determination of the binding ability of purified murine antibody S1B-30-3 to SARS-CoV-2S 1.

FIG. 4, determination of the cross-reactivity of the purified murine antibody S1B-30-3 with SARS-CoV S.

FIG. 5, murine mAb S1B-30-3 competes with ACE2-HRP for the ability to bind to SARS-CoV-2S 1.

FIG. 6, murine antibody S1B-30-3 blocks binding of the spike S protein to 293T-ACE2 cells.

FIG. 7, the molecular docking model of murine antibody S1B-30-3/RBD, ACE2/RBD structure (PDB 6M0J) and antibody CR3022/RBD structure (PDB6W 41).

FIG. 8 shows the alignment of the amino acid sequences of the heavy chain variable region of the humanized antibody hS1B-30-3 with that of the parent murine antibody.

FIG. 9 shows the alignment of the amino acid sequences of the light chain variable regions of the humanized antibody hS1B-30-3 and the parent murine antibody.

FIG. 10, indirect ELISA method for determination of humanized antibody hS1B-30-3 and SARS-CoV-2S trimer antigen binding ability.

FIG. 11, competition ELISA assay humanized antibody hS1B-30-3 blocks the ability of SARS-CoV-2S trimer to bind to human ACE 2.

FIG. 12, humanized antibody hS1B-30-3 blocks binding of spike S protein to 293T-ACE2 cells.

FIG. 13, assay of the in vitro pseudovirus inhibitory activity of humanized antibody hS 1B-30-3.

Detailed Description

EXAMPLE 1 preparation of murine monoclonal antibody against SARS-CoV-2S1 protein

Antigen preparation: SARS-CoV-2S1 antigen preparing process: the 326-685aa segment (labeled S1B) was selected from the full-length amino acid sequence of the novel coronavirus S protein disclosed in Uniprot (Uniprot Entry P0DTC2) and used as an antigen for screening antibodies in this example. In order to obtain the target protein with high expression efficiency, the coding gene of S1B is artificially modified and optimized, the eukaryotic expression vector pcDNA3.1-S1B of the target gene is constructed according to a conventional molecular biology method, the recombinant expression plasmid with correct sequencing is transfected into CHO cells, and the expression and purification are carried out according to a conventional method, so as to obtain the purified antigen for immunization.

Animal immunization: the SARS-CoV-2S1 protein antigen is emulsified fully with Freund' S adjuvant, and then the male Balb/C mouse (Shanghai Si Laike laboratory animal, Inc.) is immunized by multipoint immunization, 50 μ g/mouse, and the immunization period is once in three weeks. On day 10 after the 3rd immunization, blood was drawn from the orbit, and the degree of immune response of the mice was monitored by testing the antibody titer against SARS-CoV-2S1 in serum by indirect ELISA as described in example 2.1, and the results are shown in FIG. 1, and those in which the neutralizing antibody level against SARS-CoV-2S1 in serum by competitive ELISA are shown in FIG. 2. Mice that produced the highest anti-SARS-CoV-2S 1 antibody titers and the highest levels of neutralizing antibodies were then boosted once 3 days prior to fusion. After 3 days, the mice were sacrificed and their spleens were removed and fused with a mouse myeloma Sp2/0 cell line.

Cell fusion and antibody screening: mixing 2X 10⁸Sp2/0 cells and 2X 10 cells⁸Splenocytes were fused in 50% polyethylene glycol (molecular weight 1450) and 5% Dimethylsulfoxide (DMSO) solution. Using Iscove's medium (containing 10% fetal calf serum, 100 units/mL penicillin, 100. mu.g/mL streptomycin, 0.1)mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.M thymidine) to adjust the number of spleen cells to 5X 10⁵0.3 mL/mL, added to wells of a 96-well plate and placed at 37 ℃ in 5% CO₂In the incubator. After 10 days of culture, positive wells that compete with hACE2-Fc were selected by detecting the ability of the antibody in the supernatant to compete with HRP-labeled hACE2-Fc (ACE2(18-740) -Fc) (ACRO Biosystems, same source as below) for binding to SARS-CoV-2S1 by high-throughput ELISA, respectively (see example 2.3 for the method). The above-mentioned well fused cells containing the monoclonal antibody capable of inhibiting the binding of hACE2-Fc to SARS-CoV-2S1 were subcloned, and hybridoma cell line # S1B-30-3 expressing a high affinity murine monoclonal antibody was obtained by screening by the competitive ELISA method in the same manner. Clones producing specific antibodies were cultured in RPMI 1640 medium supplemented with 10% FCS. When the cell density reaches about 5X 10⁵At individual cells/mL, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged, and the culture supernatant was collected. The antibody was purified using a protein A column and the monoclonal antibody eluate was dialyzed against 150mM NaCl. The dialyzed solution was filter-sterilized through a 0.2 μm filter to obtain the purified murine monoclonal antibody S1B-30-3 to be tested.

Example 2 functional characterization of anti-SARS-CoV-2S 1 murine monoclonal antibody

2.1 Indirect ELISA method for determining the binding ability of murine antibody to SARS-CoV-2S1 antigen

The microplate was coated with SARS-CoV-2S1(ACRO Biosystems) overnight at room temperature. The coating solution was discarded, blocked with skim milk powder dissolved in PBS buffer for 1h, and the plates were washed 3-4 times with PBST (PBS containing 0.05% Tween 20(Tween-20), pH 7.4). Then 100. mu.L of purified anti-SARS-CoV-2S 1 murine antibody S1B-30-3 and human antibody CR3022 against SARS-CoV and SARS-CoV-2S1 (the heavy chain variable region and the light chain variable region of which are published in GenBank (Access numbers: DQ168569 and DQ 168570)) positive control were added to each well, incubated at room temperature for 1h, washed with PBS containing 0.05% Tween 20, then 100. mu.L of HRP-labeled goat anti-mouse IgG polyclonal antibody (Jackson Laboratory) was added to each well as a detection antibody, then the plates were washed 3-4 times with PBST, the substrate TMB was added for 10 minutes of color development, and then 0.2M H was added₂SO₄The reaction was terminated and the absorbance value (OD value) was read after that, and the result is shown in FIG. 3.

2.2 anti-SARS-CoV-2S 1 murine antibody Cross-reactivity assay

The binding ability of the obtained murine monoclonal antibody S1B-30-3 to SARS-CoV S was determined.

SARS-CoV S1 protein (ACRO Biosystems) was diluted to 0.1. mu.g/mL with PBS buffer, added to a 96-well plate at a volume of 100. mu.L/well, and left at 4 ℃ for 16 to 20 hours. After aspirating the supernatant and washing the plate 1 time with PBST buffer, 200. mu.L of PBST (PBST/1% skim milk) containing 1% skim milk was added to each well and blocked by incubation at room temperature for 1 h. After removing the blocking solution and washing the plate 3 times with PBST buffer, the above murine antibody S1B-30-3 was added at 100. mu.L/well and incubated at room temperature for 1.5 h. The reaction was removed and after 3 washes of the plates with PBST, 50. mu.L/well 1: a4000-diluted HRP-labeled goat anti-mouse IgG secondary antibody (The Jackson Laboratory) was incubated at room temperature for 1 h. After washing the plate 3 times with PBST, 100. mu.L of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50. mu.L of 0.2M H was added to each well₂SO₄The reaction was stopped and the OD read with a microplate reader at a dual wavelength of 450/620 nm.

As shown in FIG. 4, murine antibody S1B-30-3 did not cross SARS-CoV S.

2.3 competitive ELISA method for determining the ability of murine antibody to block the binding of SARS-CoV-2S1 with ACE2

SARS-CoV-2S1 protein (ACRO Biosystems) was diluted to 0.1. mu.g/mL with PBS buffer and added to a 96-well plate at 100. mu.L/well overnight at room temperature. The coating solution was discarded and 200. mu.L of PBST/1% skim milk powder was added to each well and incubated at room temperature for 1h for blocking. The blocking solution was removed and after washing the plate 3 times with PBST buffer, 100. mu.L of a mixture of hACE2-Fc labeled with horseradish peroxidase (HRP) and antibody S1B-30-3 was added per well. PBST was used as a blank control. After sufficient incubation, unbound HRP-labeled hACE2-Fc was washed off with PBS and incubated at room temperature for 1 h. After washing the plate 3 times with PBST, 100. mu.L of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50. mu.L of 0.2M H was added to each well₂SO₄The reaction was stopped and the OD read with a microplate reader at a dual wavelength of 450/620 nm.

FIG. 5 shows that murine mAb S1B-30-3 competes with ACE2 for binding to SARS-CoV-2S1, i.e., functions by blocking the binding of SARS-CoV-2S1 and hACE 2.

2.4 determination of kinetic constants and affinities of anti-SARS-CoV-2S 1 murine antibodies by biofilm interferometry

The binding affinity constants of the purified murine antibody S1B-30-3, the control antibodies CR3022 and hACE2-Fc and SARS-CoV-2S1(ACRO Biosystems) were determined by the biofilm interference technique (BLI). In the measurement, biotinylated SARS-CoV-2S1 to be measured is immobilized on the surface of SA (Streptavidin) sensor, and an anti-SARS-CoV-2S 1 murine antibody is used as an analyte. Processing the data, fitting with a model combined with analytical software 1:1, the fitting data being substantially overlapped with the experimental data to obtain a combination and dissociation rate constant k_aAnd k_dBy k_dDivided by k_aObtaining the equilibrium dissociation constant K_D(see Table 1). The results showed that the murine antibody S1B-30-3 binds to the K of SARS-CoV-2S1_DThe value is 2 orders of magnitude lower than human ACE 2.

TABLE 1 kinetic constants and affinity measurements for murine antibodies

Example 3 anti-SARS-CoV-2S 1 murine antibody blocks the binding of the spike S protein to 293T-ACE2 cells

At the cellular level, FACS method is used for determining the competitive blocking of the combination of the spike S protein-mouse Fc fusion protein (S-mFc) and 293T-ACE2 cells by the antibody to be detected, fluorescence labeled goat anti-mouse secondary antibody is used for detecting the average fluorescence intensity of the S-mFc combined on the cells, and IC of the antibody to be detected for blocking the combination of the S protein and ACE2 on the cell surface is calculated₅₀The blocking effect of the antibody to be detected is evaluated.

The preparation process of the S-mFc antigen comprises the following steps: based on the full-length amino acid sequence of the novel coronavirus S protein disclosed in Uniprot (Uniprot Entry P0DTC2), a full-length segment of the S protein was selected, and a murine IgG2a Fc fragment (Uniprot Entry P01863 (107-. In order to obtain the target protein with high-efficiency expression, the coding gene of the S-mFc is artificially modified and optimized, the eukaryotic expression vector pcDNA3.1-S-mFc of the target gene is constructed according to a conventional molecular biology method, the recombinant expression plasmid with correct sequencing is transfected into CHO cells, and the expression and purification are carried out according to a conventional method.

The blocking experiment of the murine antibody in vitro comprises the following specific steps. The 293T-ACE2 cells were trypsinized and added to a 96-well U-bottom plate at a cell density of 1X 106 cells/mL, 100. mu.L/well and incubated at 4 ℃ for 30 minutes; diluting S-mFc with 1% PBSB to a certain concentration; diluting a murine antibody sample S1B-30-3 to be detected to 8 mu g/mL, diluting the murine antibody sample by a 4-fold ratio, carrying out 9 gradients, and then mixing the diluted S-mFc and the antibodies 1 with different concentration gradients: 1, mixing, pre-incubating for 30 minutes at room temperature, adding into the U-shaped bottom plate with 96 holes, and incubating for 1 hour at 4 ℃; the supernatant was centrifuged off and the cells were washed 3 times with 1% PBSB; AF 647-goat anti-mouse IgG Fc (Jackson Immuno) was purified using 1% PBSB at 1: 400 dilution, 100 u L/hole, 4 degrees C were incubated for 1 hours; the supernatant was centrifuged off and the cells were washed 3 times with 1% PBSB; resuspend cells with 1% PBSB, 150. mu.L/well; the flow cytometer detects the signal intensity. And analyzing by using the average fluorescence intensity as an axis Y and the antibody concentration as an axis X through a GraphPad Prism 6 software, and calculating the IC of the anti-murine antibody S1B-30-3 for blocking the combination of the S-mFc protein and 293T-ACE2 cells₅₀The value is obtained.

As shown in FIG. 6, at cellular level, the anti-SARS-CoV-2S 1 murine antibody S1B-30-3 can compete well for blocking the binding of S protein and ACE2, IC₅₀The concentration was 11.93 ng/mL.

Example 4 evaluation of the Effect of murine antibody S1 protein on the binding Capacity between RBD/ACE2 Using computer molecular docking technology

The structural modeling of the murine antibody S1B-30-3 is carried out by adopting computer software Discovery Studio, the molecular docking space conformation of the murine antibody and an antigen RBD structural domain is simulated, the binding site of the S protein murine antibody on the RBD structural domain is predicted, and the influence of the antibody on the binding capacity between RBD/ACE2 is evaluated.

A three-dimensional structure model of the murine antibody S1B-30-3 is constructed by using Discovery Studio software. The modeling takes 3 steps to perform: 1. search and mouse sourceThe antibody light chain and heavy chain variable regions are respectively provided with three-dimensional structure templates with high similarity of amino acid sequences. Searching a three-dimensional structural template with high amino acid sequence similarity with the whole murine antibody variable region (light and heavy chains together) so as to determine the relative orientation of the light and heavy chains in the murine antibody variable region; 2. constructing a framework region structure model of the murine antibody by using the 3 structure model templates obtained in the step 1 and the light and heavy chain amino acid sequences of the variable region of the murine antibody; 3. and (3) constructing a structural model of six CDR circular regions on the basis of the step 2. The RBD structure model in the molecular docking simulation calculation adopts protein database

High resolution RBD structure (PDB ID 6M 0J). 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 identical to that in 6W41, i.e., the rotamer1 conformation. This conformation is the highest occupancy, and the side chain of F486 does not exist in this conformation due to binding of the RBD in 6M0J to ACE 2. The software for molecular docking adopts ZDCK software in the Discovery Studio software package. The parameters used in the molecular docking simulation experiment are all default values. The murine antibody is used as a molecular docking receptor, and the RBD is used as a molecular docking ligand. Acceptor blocked amino acids (Receptor blocked residues) are selected from the variable region amino acids which are located away from the CDR regions in the opposite spatial positions to those of the CDR regions. Receptor binding site amino acids (Receptor binding site residues) the top amino acid of HCDR3 loop exposed on the surface of the protein was selected. The results of molecular docking of murine antibody S1B-30-3 with RBD are shown in FIG. 7. ACE2(PDB 6M0J, Lan J et al,2020, Nature,581: 215-. As can be seen from the figure, there is a large overlap region between the murine antibody S1B-30-3 and the binding site of ACE2 on the RBD domain, indicating that S1B-30-3 competes directly with ACE2 for binding to RBD.

The molecular docking result shows that the murine antibody S1B-30-3 competes with ACE2 for binding to RBD, can block the binding between RBD and ACE2, and provides a reasonable explanation on the molecular level for the antibody to inhibit SARS-CoV-2 virus infection of host cells.

Example 5 humanization of the SARS-CoV-2 coronavirus S1 protein murine antibody

We used CDR grafting (CDR grafting) to humanize murine antibodies. The basic principle of CDR grafting is to transplant the CDR area of the mouse antibody to the template of the human antibody, and to introduce the stable CDR conformation and several or some key residues of the mouse anti-FR area important for antigen-antibody combination into the template of the human antibody (backbmutions), so as to achieve the aim of reducing the immunogenicity of the mouse antibody and maintaining the affinity of the mouse antibody. In addition to the above CDR-grafting procedures, we further calculated the isoelectric Point (PI), hydrophobic aggregation (aggregation), post-translational modification (PTM, such as glycosylation, fragmentation, isomerization site, etc.) and immunogenicity (immunogenicity) of the humanized antibody after CDR-grafting, and mutated the amino acids that cause the problems in this respect, so that the humanized antibody can sufficiently exert its pharmacological effects in clinical use.

The specific procedure for antibody humanization is as follows. Human antibody germline databases of the IMGT website (IMGT human antibody germline database, http:// www.imgt.org/3D structure-DB/cgi/DomainGapAlign. cgi) were searched for human antibody templates with high similarity to murine antibodies (VH IGHV3-7 x 01, VL IGKV1-39 x 01). CDR region annotation was performed on murine and humanized antibody templates using Discovery Studio, and CDR regions were defined according to the Kabat or IMGT protocol (Table 2). Six CDR regions of the humanized antibody template were replaced with six CDR regions of a murine antibody, respectively. Each individual CDR region of the grafted 6 CDR regions may be an amino acid region as defined by Kabat, or an amino acid region as defined by IMGT. After CDR-grafting, back-mutation from the murine antibody to the humanized template FR region was performed. The key murine anti-FR region amino acids that stabilize antibody CDR region conformation and are important for antigen-antibody binding include the 4 classes of amino acid residues: 1) CDR region

Amino acids buried beneath the surface of the antibody; 2) CDR region

Amino acids that are internally exposed at the surface of the antibody; 3) interfacial amino acids between antibody light and heavy chain domains; and 4) Vernier zone resins that stabilize the conformation of the CDR regions of the antibody (Foote J and Winter G,1992, J Mol Biol,224: 487-499). The above 4 types of key murine anti-FR region residues were determined by modeling the murine anti-FR three-dimensional structure. For these 4 types of amino acids of the human-derived template that do not correspond to the sequence of the mouse antibody, amino acids important for maintaining the CDR conformation and antigen-antibody binding are selected by three-dimensional structural analysis, and amino acid transplantation or substitution from the mouse antibody to the human-derived template is performed. Then, the humanized antibody generated after the 4-type amino acid transplantation is further subjected to isoelectric point calculation, hydrophobic aggregation, post-translational modification and immunogenicity calculation, and the problem amino acid is mutated, so as to obtain the final humanized antibody sequence (table 3), and fig. 8 shows the comparison result of the humanized antibody hS1B-30-3 and the heavy chain variable region amino acid sequence of the parent murine antibody; FIG. 9 shows the alignment of the amino acid sequences of the light chain variable regions of the humanized antibody hS1B-30-3 with that of its parent murine antibody.

TABLE 2 CDR regions in murine antibody variable regions

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 were spliced or assembled using conventional techniques with the sequences of the antibody heavy chain constant region (preferably from human IgG1, IgG2 or IgG4) and the light chain constant region (preferably from human kappa light chain, with the amino acid sequence shown in SEQ ID NO: 17). For example, in one embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG1 (amino acid sequence 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 shown in SEQ ID NO: 19). Or a modified human IgG2 constant region sequence; in one embodiment, the humanized antibody molecule comprises human IgG2 modified at the hinge region according to EU numbering (e.g., deletion of ERKCC, amino acid sequence shown in SEQ ID NO: 20). In another embodiment, the humanized antibody molecule comprises human IgG4 (amino acid sequence shown in SEQ ID NO: 21) mutated at position 228 (e.g., S to P) according to EU numbering.

TABLE 3 humanized antibodies corresponding to murine antibodies and their variable region amino acid sequences

Example 6 construction of humanized antibody expression vector and protein expression

The cDNA coding for the heavy chain and light chain of the humanized antibody obtained in the above method is inserted into PcDNA3.1 or its derived plasmid, or other eukaryotic expression vector 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. Meanwhile, the vector plasmid contains a selectable marker gene to confer ampicillin resistance in bacteria and G418 resistance in mammalian cells. In addition, the vector plasmid contains the DHFR gene, and in a suitable host cell, the humanized antibody gene and the DHFR gene can be co-amplified with Methotrexate (MTX, Sigma) (see, for example, patent CN 103333917B).

The above-constructed recombinant expression vector plasmid is transfected into a mammalian host cell line to express a humanized antibody. For stable high level expression, a preferred host cell line is a dihydrofolate reductase (DHFR) -deficient Chinese Hamster Ovary (CHO) cell (see, e.g., Chasin, l. et al, U.S. patent No. 4818679). The preferred method of transfection is electroporation, although other methods may be used, including calcium phosphate co-precipitation, lipofection, and protoplast fusion, among others. In electroporation, 2X 10 cells were placed in a cuvette using a Gene Pulser (Bio-Rad Laboratories) set at a 250V electric field and a 960. mu. Fd capacitance⁷The cells were suspended in 0.8ml of PBS and contained 10. mu.g of expression vector plasmid DNA linearized with PvuI (Takara). 2 days after transfection, the cells were transfected with 0.2mg/mL G418 and 200nM methotrexate (methotrexate or MTX). To achieve higher levels of expression, the transfected humanized antibody gene was co-amplified with the DHFR gene inhibited by the MTX drug. The secretion rate of each cell line was measured by limiting dilution of the subcloned transfectants and ELISA, and cell lines expressing the humanized antibody at a high level were selected. Conditioned media of humanized antibodies were collected for determination of their biological activity in vitro and in vivo.

For example, the nucleotide sequences encoding the heavy and light chains of humanized antibody hS1B-30-3 shown in Table 4 were inserted into the expression vectors constructed above, and cell lines stably and highly expressing the target antibody were selected by pressure, subcloned, cultured and purified to obtain each target antibody.

TABLE 4 amino acid sequences of the heavy and light chains of humanized antibodies and the nucleotide sequences encoding them

Antibody numbering	HC amino acid sequence	LC amino acid sequence	HC nucleotide sequence	LC nucleotide sequence
					hS1B-30-3	SEQ ID NO：22	SEQ ID NO：23	SEQ ID NO：24	SEQ ID NO：25

Example 7 functional characterization of humanized antibodies

7.1 Indirect ELISA method for determining the binding Capacity of humanized antibody to SARS-CoV-2S trimer antigen

The plate was coated with SARS-CoV-2S trimer (ACRO BioSystems) overnight at room temperature. The coating solution was discarded, blocked with skim milk powder dissolved in PBS buffer for 1h, and the plates were washed 3-4 times with PBST (pH7.4, PBS containing 0.05% Tween-20). Then, 100. mu.l of purified humanized antibody hS1B-30-3 against SARS-CoV-2S1 RBD and receptor hACE2-Fc against SARS-CoV-2S1 RBD were added to each well, incubated at room temperature for 1 hour, the wells were washed with PBS containing 0.05% Tween (Tween)20, then 100. mu.l of HRP-labeled goat anti-human IgG polyclonal antibody (Jackson Laboratory) was added to each well as a detection antibody, the plates were washed with PBST 3 to 4 times, substrate TMB was added thereto, developed for 10 minutes, and then 0.2M H was added₂SO₄The reaction was terminated and the absorbance value (OD value) was read thereafter, and the result is shown in FIG. 10.

7.2 competitive ELISA assay of the ability of the humanized antibodies to block binding of SARS-CoV-2S trimer to ACE2

SARS-CoV-2S trimer protein (ACRO BioSystems) was diluted to 0.1. mu.g/ml with PBS buffer, and added to a 96-well plate at 100. mu.l/well overnight at room temperature. The coating solution was discarded and 200. mu.l PBST/1% skim milk powder was added to each well and incubated at room temperature for 1h for blocking. After removing the blocking solution and washing the plate 3 times with PBST buffer, 100. mu.l of a mixture of hACE2-Fc labeled with horseradish peroxidase (HRP) and humanized antibody hS1B-30-3 and receptor hACE2-Fc against SARS-CoV-2S1 RBD, respectively, was added to each well. PBST was used as a blank control. After sufficient incubation, unbound HRP-labeled hACE2-Fc was washed off with PBS and incubated at room temperature for 1 h. After washing the plate 3 times with PBST, 100. mu.l of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50. mu.l of 0.2M H was added to each well₂SO₄The reaction was stopped and the OD read with a microplate reader at a dual wavelength of 450/620 nm. The results are shown in FIG. 11.

7.3 determination of the ability of the humanized antibody to block the binding of SARS-CoV-2S1 to ACE2 by competitive ELISA

The competition ELISA method for determining the binding capacity of the humanized antibody hS1B-30-3 for blocking SARS-CoV-2S1 and ACE2 is shown in example 2.3.

As shown in Table 5, the humanized antibody hS1B-30-3 competed with hACE2 for binding to SARS-CoV-2S1 by blocking the binding of SARS-CoV-2S1 and hACE2, and EC was determined₅₀The value was 0.03307. mu.g/ml.

TABLE 5 ability of humanized antibodies to compete for binding to hACE2

	EC₅₀(μg/ml)
		hS1B-30-3	0.03307
hACE2-Fc	1.31470

7.4 determination of kinetic constants and affinity of humanized antibody against SARS-CoV-2S1 by biofilm interferometry

Experimental methods for the determination of the kinetic constants and affinity equilibrium dissociation constants of humanized antibodies are described in example 2.4. The results are shown in Table 6. Compared with the mouse antibody, the humanized antibody hS1B-30-3 has an affinity equilibrium dissociation constant reaching pM level, which is superior to the parent mouse antibody S1B-30-3.

TABLE 6 kinetic constants and affinity assay results for humanized antibodies

7.5 anti-SARS-CoV-2S 1 humanized antibody blocks the binding of spike S protein to 293T-ACE2 cell

See example 3 for experimental methods for blocking binding of the spike S protein to 293T-ACE2 cells. The results of the experiment are shown in FIG. 12. On the cellular level, the humanized antibody hS1B-30-3 for resisting SARS-CoV-2S1 can compete well to block the combination of S protein and ACE2, IC₅₀It was 6.91 ng/mL. The blocking effect of the antibody after humanization did not change significantly compared to the murine antibody.

7.6 anti-SARS-CoV-2S 1 humanized monoclonal antibody in vitro pseudovirus inhibition experiment

The method comprises the steps of infecting 293T-ACE2 cells after incubation of new crown S protein pseudoviruses and antibodies to be detected, detecting the luminescence value RLU of Luciferase by a chemiluminescence method, calculating the pseudovirus inhibition rate of the antibodies to be detected according to the reading value of the RLU, and evaluating the neutralization effect of the antibodies to be detected. The new corona S protein pseudoviral genome encodes firefly luciferase, and when the viral genome enters the cell for integration, the expression and activity of the firefly luciferase are directly proportional to the number of transduced cells. Pseudoviruses infect cells only once compared to euviruses.

The humanized antibody is used for inhibiting pseudovirus in the following steps. Diluting an antibody sample to be detected to 20 mu g/mL by using DMEM, diluting the antibody sample by 3 times, and carrying out 8 gradients; the pseudovirus was removed from-80 ℃ and reconstituted at 4 ℃ and the reconstituted pseudovirus (assist in san Jose Biotech Co., Ltd.) was diluted 50-fold as a working solution; adding the diluted working solution of the antibody to be detected and the pseudovirus into a 96-hole white board at a rate of 25 mu L/hole respectively, uniformly mixing, repeating the holes for 2 times, and incubating for 1 hour at room temperature; 293T-ACE2 cells (Shanghai assist saint Biotech Co., Ltd.) were cultured to logarithmic growth phase with medium DMEM + 10% FBS + 0.75. mu.g/mL Puromycin (Puromycin), trypsinized, resuspended in medium DMEM + 10% FBS, homogenized and cell counted at a cell density of 2X 10⁵Spreading the cells/mL, 50 μ L/well on a white board, and culturing in a 5% CO2 incubator at 37 deg.C for 24 hr; after 24 hours, adding 50 mu L of a culture medium DMEM + 10% FBS into each hole, and continuously putting the mixture into a cell culture box for culturing for 24 hours; carefully pipette off the supernatant, add 50. mu.L of newly formulated luciferase developing solution immediately, incubate for 5 minutes at room temperatureThe 96-well plate is placed in a microplate reader, and the chemiluminescence signal of each well is read. Positive control group: pseudovirus and DMEM medium were added to a 96-well white plate at 25. mu.L/well, mixed well and incubated at 37 ℃ for 1 hour. Negative control group: DMEM medium was added to a 96-well white plate and incubated at 37 ℃ for 1 hour. The inhibition ratio (%) <1 — (sample RLU signal value-negative control RLU signal value)/(positive control RLU signal value-negative control RLU signal value). The inhibition ratio was set as the Y-axis and the antibody concentration was set as the X-axis, and the quantitative response curve of the antibody was obtained by analyzing the results with the software GraphPad Prism 6 (fig. 13). Table 7 shows the inhibition rate of the humanized antibody hS1B-30-3 against the novel coronavirus protein pseudovirus at various concentrations. As shown in FIG. 13 and Table 7, the humanized monoclonal antibody hS1B-30-3 can remarkably and dose-dependently inhibit the infection of 293T-ACE2 cells by new coronavirus, and the hS1B-30-3 can block the early invasion of host cells by SARS-CoV-2 infection and play a role in protection.

TABLE 7 anti-SARS-CoV-2S 1 humanized monoclonal antibody pseudovirus inhibition results

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Anyuan pharmaceutical technology (Shanghai) Co., Ltd

<120> SARS-CoV-2 virus neutralizing antibody and use thereof

<130> S1B-30-3

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> murine antibody S1B-30-3 HCDR1-1 amino acid sequence (as defined by Kabat)

<400> 1

Asp Ala Trp Met Asp

1 5

<210> 2

<211> 8

<212> PRT

<213> murine antibody S1B-30-3 HCDR1-2 amino acid sequence (defined by IMGT)

<400> 2

Gly Phe Thr Phe Ser Asp Ala Trp

1 5

<210> 3

<211> 19

<212> PRT

<213> murine antibody S1B-30-3 HCDR2-1 amino acid sequence (as defined by Kabat)

<400> 3

Gln Ile Arg Asn Lys Ala Asn Ser His Ala Thr Asn Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210> 4

<211> 10

<212> PRT

<213> murine antibody S1B-30-3 HCDR2-2 amino acid sequence (defined by IMGT)

<400> 4

Ile Arg Asn Lys Ala Asn Ser His Ala Thr

1 5 10

<210> 5

<211> 9

<212> PRT

<213> murine antibody S1B-30-3 HCDR3-1 amino acid sequence (as defined by Kabat)

<400> 5

Gly Leu Thr Gly Tyr Val Leu Asp Tyr

1 5

<210> 6

<211> 11

<212> PRT

<213> murine antibody S1B-30-3 HCDR3-2 amino acid sequence (defined by IMGT)

<400> 6

Ile Arg Gly Leu Thr Gly Tyr Val Leu Asp Tyr

1 5 10

<210> 7

<211> 11

<212> PRT

<213> murine antibody S1B-30-3 LCDR1-1 amino acid sequence (as defined by Kabat)

<400> 7

Lys Ala Ser Gln Asn Val Arg Thr Ala Val Ala

1 5 10

<210> 8

<211> 6

<212> PRT

<213> murine antibody S1B-30-3 LCDR1-2 amino acid sequence (defined by IMGT)

<400> 8

Gln Asn Val Arg Thr Ala

1 5

<210> 9

<211> 7

<212> PRT

<213> murine antibody S1B-30-3 LCDR2-1 amino acid sequence (as defined by Kabat)

<400> 9

Leu Ala Ser Asn Arg His Thr

1 5

<210> 10

<211> 3

<212> PRT

<213> murine antibody S1B-30-3 LCDR2-2 amino acid sequence (defined by IMGT)

<400> 10

Leu Ala Ser

1

<210> 11

<211> 9

<212> PRT

<213> murine antibody S1B-30-3 LCDR3-1 amino acid sequence (as defined by Kabat)

<400> 11

Leu Gln His Trp Asn Tyr Pro Leu Thr

1 5

<210> 12

<211> 9

<212> PRT

<213> murine antibody S1B-30-3 LCDR3-2 amino acid sequence (defined by IMGT)

<400> 12

Leu Gln His Trp Asn Tyr Pro Leu Thr

1 5

<210> 13

<211> 120

<212> PRT

<213> murine antibody S1B-30-3 heavy chain variable region amino acid sequence ()

<400> 13

Glu Val Lys Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Asp Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ala Asn Ser His Ala Thr Asn Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Phe Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Ile Arg Gly Leu Thr Gly Tyr Val Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 14

<211> 107

<212> PRT

<213> murine antibody S1B-30-3 light chain variable region amino acid sequence ()

<400> 14

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Ser Lys Ala Ser Gln Asn Val Arg Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Phe Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 15

<211> 120

<212> PRT

<213> humanized antibody hS1B-30-3 heavy chain variable region amino acid sequence ()

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ala Asn Ser His Ala Thr Asn Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ile Arg Gly Leu Thr Gly Tyr Val Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 107

<212> PRT

<213> humanized antibody hS1B-30-3 light chain variable region amino acid sequence ()

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Phe Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 17

<211> 107

<212> PRT

<213> amino acid sequence of human kappa constant region ()

<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> wild-type constant region amino acid sequence of human IgG1 ()

<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> wild-type constant region amino acid sequence of human IgG2 ()

<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> 21

<211> 327

<212> PRT

<213> human IgG4(S228P mutant constant region amino acid sequence)

<400> 21

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> 441

<212> PRT

<213> humanized antibody hS1B-30-3 heavy chain amino acid sequence ()

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ala Asn Ser His Ala Thr Asn Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ile Arg Gly Leu Thr Gly Tyr Val Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Thr Val Val Glu Cys Pro Pro Cys

210 215 220

Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

245 250 255

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

260 265 270

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

275 280 285

Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

305 310 315 320

Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

325 330 335

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

340 345 350

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

355 360 365

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

370 375 380

Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

385 390 395 400

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

405 410 415

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

420 425 430

Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 23

<211> 214

<212> PRT

<213> humanized antibody hS1B-30-3 light chain amino acid sequence ()

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Phe Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 24

<211> 1323

<212> DNA

<213> humanized antibody hS1B-30-3 heavy chain nucleic acid sequence ()

<400> 24

gaggtccagc tggtcgagtc cggcggcggc ctggtgcagc ctggcggctc cctgcggctg 60

agctgcgctg cttctggctt cacattctcc gatgcctgga tggattgggt gcgacaggct 120

cctggcaaag gcttggactg ggttgcccag atccggaaca aggccaactc ccacgccacc 180

aactacgccg actccgtcaa gggcagattt accatctctc gcgacgattc taagaactcc 240

gtgtatctgc agatgaactc tctgaagacc gaggataccg ccgtctacta ctgcatcaga 300

ggcctgacag gctacgtgct ggactactgg ggccagggca ccaccgtgac cgtgtcctcc 360

gcaagcacca agggaccttc tgtgtttcct ctggccccat gtagtcgctc aacttccgag 420

agcaccgcag cactgggatg cctggtgaag gattacttcc cagaacccgt cacagtgtct 480

tggaacagtg gggccctgac aagcggtgtc cacacttttc cagctgtgct gcagtcatcc 540

ggactgtact ctctgagctc tgtggtcact gtgcccagtt caaatttcgg gacccagaca 600

tatacttgta acgtggacca taagccttcc aataccaagg tcgataaaac agtggtggaa 660

tgtccacctt gcccagctcc accagtcgca ggacctagcg tgttcctgtt tcctccaaag 720

cccaaagaca cactgatgat ctcacgcaca cctgaggtca cttgcgtggt cgtggacgtg 780

tcccacgagg accctgaagt ccagtttaac tggtacgtgg atggcgtcga agtgcataat 840

gccaagacca aaccaagaga ggaacagttc aactccacat ttcgcgtcgt gagcgtgctg 900

actgtcgtgc accaggactg gctgaacggc aaggagtata agtgtaaagt gagcaataag 960

ggcctgcctg ctccaatcga gaaaaccatt tctaagacaa aaggccagcc cagagaacct 1020

caggtgtaca cactgccccc ttcccgcgag gaaatgacta agaaccaggt cagcctgacc 1080

tgcctggtga aaggttttta tcccagtgac atcgccgtgg agtgggaatc aaatggccag 1140

cctgagaaca attacaagac taccccaccc atgctggact cagatggttc cttctttctg 1200

tattcaaagc tgaccgtgga taaatccagg tggcagcagg gcaacgtctt ctcttgtagt 1260

gtgatgcatg aggctctgca caatcattac acacagaagt cactgtccct gagcccaggc 1320

aag 1323

<210> 25

<211> 642

<212> DNA

<213> light chain nucleic acid sequence of humanized antibody hS1B-30-3 ()

<400> 25

gatatccaga tgacccagtc tccatcttcc ctctccgcat ctgtgggcga tagagtgacg 60

atcacctgca aggcttccca gaacgtgcgg acagccgtgg cttggtacca acaaaagcct 120

ggccagtctc ccaaggctct gatcttcctg gccagcaacc ggcacaccgg cgtccctgac 180

agattctccg ggtccggctc tggcaccgac ttcaccctca ccatcagtag cctgcagcct 240

gaagacttcg ccacctactt ctgcctgcag cactggaact accctctgac cttcggcgga 300

ggcaccaaag tggagatcaa acgtactgtc gctgcaccaa gcgtgttcat ttttcctcca 360

tctgacgaac agctgaagtc tggaaccgct agtgtcgtgt gcctgctgaa caatttttac 420

cccagggagg caaaggtcca gtggaaagtg gataacgccc tgcagagcgg caattctcag 480

gagagtgtga ccgaacagga ctcaaaggat tccacatata gcctgtcatc cactctgacc 540

ctgagcaaag ctgactacga gaagcacaaa gtctatgcat gcgaagtgac ccatcaggga 600

ctgagctctc ctgtgacaaa gtctttcaac cggggggagt gc 642

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to a SARS-CoV-2 coronavirus S protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising at least one, two, or three Complementarity Determining Regions (CDRs) selected from the group consisting of:

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 3 VH variable region CDRs and 3 VL variable region CDRs selected from the group consisting of:

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is murine or chimeric and the heavy chain variable region comprises the heavy chain FR region of murine IgG1, IgG2, IgG3 or variants thereof; and a light chain variable region thereof comprising the light chain FR region of a murine kappa, lambda chain or variant thereof.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the VH domain of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 13, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO: 14, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.

5. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is humanized.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the VH domain of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 15, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO: 16, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.

7. The antibody of claim 6, wherein said antibody comprises a heavy chain constant region and a light chain constant region from a human immunoglobulin; more preferably, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE heavy chain constant regions; more preferably, the heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, and IgG 4; and, the heavy chain constant region has a native sequence or a sequence having substitution, deletion or addition of one or more amino acids compared to the native sequence from which it is derived; and the light chain constant region is preferably as set forth in SEQ ID NO: 17 constant region of human kappa appa chain.

8. The antibody of claim 7, wherein said antibody comprises a heavy chain constant region selected from the group consisting of:

(i) as shown in SEQ ID NO: 18, the heavy chain constant region of wild-type human IgG 1;

(ii) as shown in SEQ ID NO: 19, the heavy chain constant region of wild-type human IgG 2;

(iii) as shown in SEQ ID NO: 20, the heavy chain constant region of a hinge region modified human IgG 2;

(iv) as shown in SEQ ID NO: 21, heavy chain constant region of human IgG4 containing the Ser228Pro mutation.

9. The antibody of claim 7 or 8, wherein the heavy chain of said antibody has the amino acid sequence set forth in SEQ ID NO: 22; or a sequence having one or several 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 more identity compared to any of the above sequences; and/or the light chain of the antibody has the amino acid sequence shown as SEQ ID NO: 23; or a sequence having one or several 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 more identity compared to any of the above sequences.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof has a K of 10nM or less_DBinds to the S protein, more preferably with a K of 1nM or less_D(ii) a binding S protein; more preferably, at a K of 100pM or less_D(ii) a binding S protein; more preferably, at a K of 10pM or less_D(ii) a binding S protein; most preferably, at a K of 1pM or less_DBinds to the S protein.

11. A DNA molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-10.

12. The DNA molecule of claim 11, wherein the DNA molecule encoding the heavy chain of said antibody has the amino acid sequence set forth in SEQ ID NO: 24; and/or, the DNA molecule encoding the light chain of said antibody has the amino acid sequence as set forth in SEQ ID NO: 25.

13. A vector comprising a DNA molecule according to claim 11 or 12.

14. A host cell comprising the vector of claim 13; the host cell comprises a prokaryotic, yeast or mammalian cell, preferably a CHO cell.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and a pharmaceutically acceptable excipient, carrier or diluent.

16. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-10, comprising: (a) obtaining the gene of the antibody or the antigen binding fragment thereof, and constructing an expression vector of the antibody or the antigen binding fragment thereof; (b) transfecting the expression vector into a host cell by a genetic engineering method; (c) culturing the above host cell under conditions that allow production of the antibody or antigen-binding fragment thereof; (d) isolating, purifying the antibody or antigen-binding fragment thereof produced;

wherein, the expression vector in the step (a) is selected from one or more of plasmids, bacteria and viruses, preferably, the expression vector is a PXY1A1M vector;

wherein, the constructed vector is transfected into a host cell by a genetic engineering method in the step (b), and the host cell comprises prokaryotic cells, yeast or mammalian cells, such as CHO cells, NS0 cells or other mammalian cells, preferably CHO cells;

17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 in the manufacture of a medicament for the treatment and prevention of a disease caused by SARS-CoV-2 coronavirus; preferably, the disease is novel coronavirus pneumonia (COVID-19).

18. A method for detecting the presence of SARS-CoV-2 virus or its corresponding antigen in a sample, comprising the steps of:

(1) incubating a biological sample to be tested with at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 under suitable conditions;

(2) detecting the presence of the bound complex in the step;

wherein the biological sample is selected from the group consisting of plasma, whole blood, mouthwash, throat swab, urine, stool, and bronchial perfusate;

wherein said antigen binding fragment is selected from the group consisting of F (ab')₂Fab', Fab and Fv.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 in the preparation of a SARS-CoV-2 virus detection kit.

20. A test kit comprising at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-10.

21. The test kit of claim 20, comprising:

(1) selected from any one of:

a. a solid support and a first antibody;

b. a solid support coated with a first antibody;

the first antibody is selected from any one of the monoclonal antibodies or antigen binding fragments thereof of any one of claims 1-10;

(2) a second antibody;

said second antibody is optionally suitably labeled and is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-10 that is capable of being used in conjunction with the first antibody of (1).

22. The test kit according to claim 21, wherein the solid support is selected from nitrocellulose membranes, latex particles, magnetic particles, colloidal gold, beads or optical sensors such as glass, fibreglass or polymers (e.g. polystyrene or polyvinyl chloride) or fibre optic sensors.

23. The test kit of claim 21, wherein the label is a radioisotope (e.g., a radioisotope)¹²⁵I) Enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin and coloring substances; preferably, the enzymes include, for example, alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease, and glucose oxidase; the fluorescent substance includes, for example, fluorescein derivatives and rhodamine derivatives, and rare earth elements or rare earth element complexes, such as europium or europium complexes; the phosphorescent substances include such as acridine ester and isoluminol; the radioactive isotopes include¹²⁵I、³H、¹⁴C and³²p; the coloring matter includes, for example, latex particles and colloidal gold.

24. Use of the detection kit according to any one of claims 20 to 23 for the diagnosis of a disease caused by infection with the SARS-CoV-2 virus; preferably, the disease is novel coronavirus pneumonia (COVID-19).