CN113354729A

CN113354729A - Monoclonal antibody for resisting novel coronavirus and application thereof

Info

Publication number: CN113354729A
Application number: CN202010151348.6A
Authority: CN
Inventors: 刘映霞; 吴燕; 沈晨光; 刘磊; 谭曙光; 李德林; 王非然; 高福
Original assignee: Third Peoples Hospital of Shenzhen; Institute of Microbiology of CAS
Current assignee: Third Peoples Hospital of Shenzhen; Institute of Microbiology of CAS
Priority date: 2020-03-06
Filing date: 2020-03-06
Publication date: 2021-09-07
Anticipated expiration: 2040-03-06
Also published as: CN113354729B; WO2021174595A1

Abstract

The present invention relates to the fields of immunology and molecular virology, in particular to the fields of diagnosis, prevention and treatment of novel coronaviruses. In particular, the invention relates to monoclonal antibodies against novel coronaviruses, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the invention relates to the use of said antibodies. The antibodies of the invention are useful for diagnosing, preventing and/or treating infections with the novel coronavirus and/or diseases caused by said infections (e.g., novel coronavirus pneumonia).

Description

Monoclonal antibody for resisting novel coronavirus and application thereof

Technical Field

The present invention relates to the fields of immunology and molecular virology, in particular to the fields of diagnosis, prevention and treatment of novel coronaviruses. In particular, the invention relates to monoclonal antibodies against novel coronaviruses, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the invention relates to the use of said antibodies. The antibodies of the invention are useful for diagnosing, preventing and/or treating infections by novel coronaviruses and/or diseases caused by said infections (e.g., novel coronavirus pneumonitis).

Background

By 3 months and 3 days in 2020, the pneumonia caused by the novel coronavirus 2019-nCoV is diagnosed in 80303 cases, the accumulated death cases are 2947 cases, the accumulated diagnosis cases in 9742 cases and the accumulated death cases in 171 cases in countries (54 countries such as Tai nations, America, Germany, Australia and Iran) outside China, and the lives and the health of the public are seriously threatened. At present, no specific medicine exists for treating the novel coronavirus.

The novel coronavirus 2019-nCoV is a pathogen causing novel coronavirus pneumonia (COVID-19), is a single-stranded RNA virus, and belongs to the family of coronaviridae, namely severe acute respiratory syndrome coronavirus (SARS-CoV) causing epidemic situations in 2002-2003 and middle east respiratory syndrome coronavirus (MERS-CoV) causing epidemic situations in 2012. The Spike protein (Spike, S protein) on the surface of the virus is combined with angiotensin converting enzyme 2 (ACE2) molecule of a host cell receptor in the process of infecting a host, so that fusion of a virus membrane and a host cell membrane is started, and the host cell is infected with the virus. The S protein is divided into two parts of S1 and S2, and the Receptor Binding Domain (RBD) of C Terminal (CTD) of S1 is proved to be combined with ACE2 to mediate the process of membrane fusion.

To date, neutralizing antibodies have proven to be an effective method of treating viral diseases. Currently marketed drugs for the treatment and prevention of viral infections are palivizumab (Synagis) for the prevention of Respiratory Syncytial Virus (RSV) infection in children, abalizumab (Trogarzo) for the treatment of HIV infection, and Rabishield for the prevention after rabies virus exposure. Furthermore, there are also a number of monoclonal antibodies against different viruses at different stages of clinical research (https:// clinicaltralials. gov /). Antibodies act primarily through two aspects. In one aspect, an antibody having neutralizing activity can block viral infection by binding to viral envelope proteins, blocking binding of the virus to cellular receptors. On the other hand, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) recruit immune cells and immune molecules, such as macrophages or complement, to eliminate free virus and infected cells.

Therefore, there is a need to develop neutralizing antibodies against the novel coronavirus 2019-nCoV to provide a means to effectively prevent and treat the novel coronavirus infection.

Disclosure of Invention

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified into kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; chothia et al (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, ch.7(Paul, w., ed., 2 nd edition, Raven Press, n.y. (1989), which is incorporated herein by reference in its entirety for all purposes.

In some cases, the antigen-binding fragment of the antibody is a single chain antibody (e.g., scFv), in which the VL and VH domains pair to form a single valent molecule through a linker that enables it to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 242: 423426 (1988) and Huston et al, proc.natl.acad.sci.usa 85: 58795883 (1988)). Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-. Other linkers useful in the present invention are described by Alfthan et al (1995), Protein Eng.8: 725-.

In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a diabody in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, e.g., Holliger p. et al, proc.natl.acad.sci.usa 90: 64446448 (1993), and Poljak r.j. et al, Structure 2: 11211123 (1994)).

Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibody H4 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of antibodies are specifically screened for specificity in the same manner as for intact antibodies.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained by the hybridoma technique first reported by Kohler et al (Nature, 256:495,1975), but can also be obtained by recombinant DNA techniques (see, for example, Journal of biological methods,2009,158(1-2): 171-.

As used herein, "neutralizing antibody" refers to an antibody or antibody fragment that eliminates or significantly reduces the virulence (e.g., the ability to infect cells) of a target virus.

As used herein, the term "e.coli expression system" refers to an expression system consisting of e.coli (strain) derived from commercially available strains such as, but not limited to: GI698, ER2566, BL21(DE3), B834(DE3), BLR (DE 3).

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector is referred to as an expression vector when it enables expression of a protein encoded by the inserted polynucleotide. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) of P1 origin; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody has less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less binds to the antigen with an affinity (KD).

As used herein, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody (e.g., monoclonal antibody H4 of the invention) is present at less than about 10^-5M, e.g. less than about 10^-6 M、10^-7M、10^-8M、10^-9M or 10^-10M or less dissociation equilibrium constant (KD) binding antigens (e.g., RBD of novel coronavirus S proteins), e.g., as using surface plasmonsResonance Spectroscopy (SPR) was performed in a BIACORE instrument.

In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "neutralizing activity" means that the antibody or antibody fragment has a functional activity of binding to an antigenic protein on the virus, thereby preventing maturation of virus-infected cells and/or virus progeny and/or release of virus progeny, and the antibody or antibody fragment having neutralizing activity can prevent amplification of the virus, thereby inhibiting or eliminating infection by the virus.

As used herein, the terms "novel coronavirus" and "2019-nCoV" refer to a class of coronaviruses found at the end of 2019, both having the same meaning and being used interchangeably.

As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia resulting from infection with a novel coronavirus, which have the same meaning and are used interchangeably.

After extensive experimental research, the inventors of the present application discovered an antibody which can specifically recognize and target the S protein of a novel coronavirus, particularly the Receptor Binding Domain (RBD) of the S protein, and can block the binding of the RBD of the S protein to the cellular receptor angiotensin converting enzyme 2 (ACE2), showing a high virus-neutralizing ability. Accordingly, the antibodies of the invention are particularly useful for the diagnosis, prevention and treatment of novel coronavirus infections or diseases associated with novel coronavirus infections (e.g., novel coronavirus pneumonia).

In a first aspect of the present application, there is provided a monoclonal antibody or antigen-binding fragment thereof comprising heavy chain variable region (VH) complementarity determining regions 1-3(CDR1-3) having amino acid sequences shown in SEQ ID NOS: 1-3, respectively; and/or light chain variable region (VL) complementarity determining regions 1-3(CDR1-3) having amino acid sequences shown in SEQ ID NOS: 4-6, respectively.

In certain preferred embodiments, the monoclonal antibody comprises the heavy chain variable region (VH) shown as SEQ ID NO: 7.

In certain preferred embodiments, the monoclonal antibody comprises the light chain variable region (VL) as set forth in SEQ ID NO: 8.

In certain preferred embodiments, the monoclonal antibody comprises: VH CDR1-3 with amino acid sequences shown as SEQ ID NO. 1-3, and VL CDR1-3 with amino acid sequences shown as SEQ ID NO. 4-6, respectively.

In certain preferred embodiments, the monoclonal antibodies comprise: VH shown as SEQ ID NO. 7 and VL shown as SEQ ID NO. 8.

In certain preferred embodiments, the monoclonal antibody further has a leader sequence at the N-terminus of the heavy chain variable region. In certain preferred embodiments, the leader sequence has the amino acid sequence shown as SEQ ID NO 11.

In certain preferred embodiments, the monoclonal antibody further has a leader sequence at the N-terminus of the light chain variable region. In certain preferred embodiments, the leader sequence has the amino acid sequence shown as SEQ ID NO 11.

In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (e.g., scFv), human antibody, chimeric antibody, or bispecific or multispecific antibody.

In certain preferred embodiments, the monoclonal antibody further comprises a heavy chain constant region. In certain preferred embodiments, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO 9.

In certain preferred embodiments, the monoclonal antibody further comprises a light chain constant region. In certain preferred embodiments, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO 10.

In certain preferred embodiments, the light chain of the monoclonal antibody is of the kappa type.

In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to the spike protein (S protein) of a novel coronavirus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of targeting the Receptor Binding Domain (RBD) of the spike protein (S protein) of a novel coronavirus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of inhibiting Receptor Binding Domain (RBD) -mediated receptor binding and/or membrane fusion processes of the S protein, inhibiting infection of a cell by a virus.

In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof has neutralizing capacity (e.g., is capable of neutralizing a novel coronavirus). In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of inhibiting infection by a novel coronavirus or entry into a host cell. Thus, the monoclonal antibody or antigen-binding fragment thereof is capable of neutralizing the novel coronavirus, and thereby preventing and treating infection by the novel coronavirus.

The present application also provides isolated nucleic acid molecules encoding the monoclonal antibodies or antigen binding fragments thereof of the present invention. Such nucleic acid molecules are not limited to the method by which they are produced, and can be obtained using genetic engineering recombinant techniques or chemical synthetic methods.

Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises: the amino acid sequence is VH CDR1-3 of SEQ ID NO. 1-3.

In certain preferred embodiments, the antibody heavy chain variable region has the amino acid sequence shown as SEQ ID NO 7.

In certain preferred embodiments, the nucleic acid molecule has the nucleotide sequence set forth as SEQ ID NO 12.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding an antibody light chain variable region, wherein the antibody light chain variable region comprises: the amino acid sequence is VL CDR1-3 of SEQ ID NO. 4-6.

In certain preferred embodiments, the antibody light chain variable region has the amino acid sequence shown as SEQ ID NO 8.

In certain preferred embodiments, the nucleic acid molecule has the nucleotide sequence set forth as SEQ ID NO 13.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding an antibody heavy chain variable region as defined above, and a nucleotide sequence capable of encoding an antibody light chain variable region as defined above.

In certain preferred embodiments, the antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO 7. In certain preferred embodiments, the nucleotide sequence capable of encoding the antibody heavy chain variable region has the nucleotide sequence shown in SEQ ID NO. 12.

In certain preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a leader sequence located 5' to the nucleotide sequence capable of encoding the variable region of the antibody heavy chain. In certain preferred embodiments, the leader sequence has the amino acid sequence shown as SEQ ID NO. 11. In certain preferred embodiments, the nucleotide sequence encoding the leader sequence has the nucleotide sequence set forth in SEQ ID NO 16.

In certain preferred embodiments, the antibody light chain variable region comprises the amino acid sequence set forth in SEQ ID NO 8. In certain preferred embodiments, the nucleotide sequence capable of encoding the variable region of an antibody light chain has the nucleotide sequence set forth in SEQ ID NO 13.

In certain preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a leader sequence located 5' to the nucleotide sequence capable of encoding the variable region of the antibody light chain. In certain preferred embodiments, the leader sequence has the amino acid sequence shown as SEQ ID NO. 11. In certain preferred embodiments, the nucleotide sequence encoding the leader sequence has the nucleotide sequence set forth in SEQ ID NO 16.

In certain preferred embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO. 12 and the nucleotide sequence set forth as SEQ ID NO. 13.

In certain preferred embodiments, the isolated nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence encoding a leader sequence and a nucleotide sequence capable of encoding an antibody heavy chain variable region; and, a second polynucleotide comprising a nucleotide sequence encoding a leader sequence and a nucleotide sequence capable of encoding an antibody light chain variable region.

In certain preferred embodiments, the isolated nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO. 16 and a nucleotide sequence set forth as SEQ ID NO. 12; and a second polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO. 16 and a nucleotide sequence set forth as SEQ ID NO. 13.

In certain preferred embodiments, the isolated nucleic acid molecule further comprises a nucleotide sequence capable of encoding an antibody heavy chain constant region. In certain preferred embodiments, the heavy chain constant region has the amino acid sequence shown as SEQ ID NO 9. In certain preferred embodiments, the nucleotide sequence capable of encoding an antibody heavy chain constant region has the nucleotide sequence set forth in SEQ ID NO. 14.

In certain preferred embodiments, the isolated nucleic acid molecule further comprises a nucleotide sequence capable of encoding an antibody light chain constant region. In certain preferred embodiments, the light chain constant region has the amino acid sequence shown in SEQ ID NO 10. In certain preferred embodiments, the nucleotide sequence capable of encoding an antibody light chain constant region has the nucleotide sequence set forth in SEQ ID NO. 15.

In certain preferred embodiments, the isolated nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence encoding a leader sequence, a nucleotide sequence capable of encoding an antibody heavy chain variable region, and a nucleotide sequence capable of encoding an antibody heavy chain constant region; and, a second polynucleotide comprising a nucleotide sequence encoding a leader sequence, a nucleotide sequence capable of encoding an antibody light chain variable region, and a nucleotide sequence capable of encoding an antibody light chain constant region.

In certain preferred embodiments, the isolated nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO 16, SEQ ID NO 12, and SEQ ID NO 14; and a second polynucleotide comprising the nucleotide sequence set forth as SEQ ID NO 16, SEQ ID NO 13 and SEQ ID NO 15.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the invention as defined above.

In another aspect, the invention provides a vector comprising an isolated nucleic acid molecule as defined above. The vector of the present invention may be a cloning vector or an expression vector. In certain preferred embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like.

In another aspect, host cells comprising the isolated nucleic acid molecules or vectors of the invention are also provided. Such host cells include, but are not limited to, prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). The cell of the invention may also be a cell line, such as 293T cells.

In another aspect, there is also provided a method of making a monoclonal antibody of the invention, or an antigen-binding fragment thereof, comprising culturing a host cell of the invention under suitable conditions, and recovering the monoclonal antibody of the invention, or an antigen-binding fragment thereof, from the cell culture.

In another aspect, the invention provides a composition comprising a monoclonal antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector or a host cell as described above.

In another aspect, the invention provides a kit comprising a monoclonal antibody of the invention, or an antigen-binding fragment thereof. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a detectable label. In certain preferred embodiments, the kit further comprises a second antibody that specifically recognizes the monoclonal antibody of the invention, or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable label. Such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances and enzymes (e.g., horseradish peroxidase), and the like.

In another aspect, the present invention provides a method of detecting the presence or level of RBD of a novel coronavirus, or S protein thereof, in a sample, comprising using a monoclonal antibody, or antigen binding fragment thereof, of the invention. In certain preferred embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label. In another preferred embodiment, the method further comprises detecting the monoclonal antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label. The methods can be used for diagnostic purposes (e.g., the sample is a sample from a patient), or for non-diagnostic purposes (e.g., the sample is a cellular sample, not a sample from a patient).

In another aspect, the present invention provides a method of diagnosing whether a subject is infected with a novel coronavirus, comprising: detecting the presence of a novel coronavirus, or S protein thereof, or RBD of S protein in a sample from said subject using a monoclonal antibody or antigen binding fragment thereof of the invention. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a detectable label. In another preferred embodiment, the method further comprises detecting the monoclonal antibody or antigen-binding fragment thereof or the anti-idiotype antibody of the invention using a second antibody carrying a detectable label.

In another aspect, there is provided the use of a monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody of the invention in the manufacture of a kit for detecting the presence or level of a novel coronavirus, or its S protein or RBD of the S protein, in a sample, or for diagnosing whether a subject is infected with a novel coronavirus.

In certain preferred embodiments, the sample includes, but is not limited to, fecal matter from a subject (e.g., a mammal, preferably a human), oral or nasal secretions, alveolar lavage fluid, and the like.

In certain preferred embodiments, the monoclonal antibody is an antibody comprising: VH CDR1-3 with amino acid sequences shown as SEQ ID NO. 1-3, and/or VL CDR1-3 with amino acid sequences shown as SEQ ID NO. 4-6; preferably, it comprises: VH shown as SEQ ID NO. 7 and/or VL shown as SEQ ID NO. 8.

General methods for using antibodies or antigen-binding fragments thereof to detect the presence or level of a virus or antigen of interest (e.g., a novel coronavirus or its S protein or RBD of S protein) in a sample are well known to those skilled in the art. In certain preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition, and the like.

In another aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody of the invention, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and/or excipient. In certain preferred embodiments, the monoclonal antibodies comprise: VH CDR1-3 with amino acid sequences shown as SEQ ID NO. 1-3, and/or VL CDR1-3 with amino acid sequences shown as SEQ ID NO. 4-6; preferably, the monoclonal antibody comprises: VH shown as SEQ ID NO. 7 and/or VL shown as SEQ ID NO. 8.

In another aspect, the invention provides a method for neutralizing the virulence of a novel coronavirus in a sample, comprising contacting a sample comprising the novel coronavirus with a monoclonal antibody, or antigen-binding fragment thereof, of the invention. Such methods may be used for therapeutic purposes, or for non-therapeutic purposes (e.g., the sample is a cell sample, not a patient or a sample from a patient).

In another aspect, there is provided the use of a monoclonal antibody of the invention, or an antigen-binding fragment thereof, for the preparation of a medicament for neutralizing the virulence of a novel coronavirus in a sample. In another aspect, the present invention provides a monoclonal antibody or antigen binding fragment thereof as described above for use in neutralizing the virulence of a novel coronavirus in a sample.

In another aspect, there is provided the use of a monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody of the invention in the preparation of a pharmaceutical composition for the prevention or treatment of a novel coronavirus infection or a disease associated with a novel coronavirus infection (e.g., novel coronavirus pneumonia) in a subject. In another aspect, the invention provides a monoclonal antibody or antigen-binding fragment thereof as described above for use in preventing or treating a novel coronavirus infection or a disease associated with a novel coronavirus infection (e.g., novel coronavirus pneumonia) in a subject.

In another aspect, the present invention provides a method for preventing or treating a novel coronavirus infection or a disease associated with a novel coronavirus infection (e.g., novel coronavirus pneumonia) in a subject, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition of the present invention.

In certain preferred embodiments, the subject is a mammal, e.g., a human.

The monoclonal antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention can be administered to a subject by any suitable route of administration. Such routes of administration include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

The drugs and pharmaceutical compositions provided by the present invention may be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., antiviral drugs such as faviravir, ridciclovir, and interferon). In certain preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

Sequence information

The information of the partial sequence referred to in the present application is shown in table 1 below.

TABLE 1 partial sequence information

Advantageous effects

The monoclonal antibody of the present application (e.g., H4 antibody) is capable of binding to the novel coronavirus S protein RBD with high affinity and has strong neutralizing activity against the novel coronavirus. For example, the H4 antibody of the invention has an affinity for RBD of 4.48nM and a neutralizing titer (semi-inhibitory concentration, IC) against the novel coronavirus₅₀) It was 0.896. mu.g/mL. Thus, the monoclonal antibodies of the present application (e.g., the H4 antibody) have clinical utility in the prevention and treatment of novel coronavirus infections.

Drawings

FIG. 1 shows the results of molecular sieve chromatography and SDS-PAGE detection of the novel coronavirus S protein RBD.

FIG. 2 shows the results of molecular sieve chromatography and SDS-PAGE detection of the recombinantly expressed H4 antibody, wherein "-" on the gel indicates no addition of DTT (non-reducing SDS-PAGE); "+" indicates addition of DTT (reducing SDS-PAGE).

FIG. 3 shows the results of the kinetic curves of the H4 antibody binding to RBD protein.

FIG. 4 shows the cell surface fluorescence detected with BD FACSCAnto in example 6.

Figure 5 shows the neutralizing activity of H4 antibody against 2019-nCoV live virus at different concentrations.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassays used in the present invention are essentially described in reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available on the market. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

In order to obtain a neutralizing antibody having a protective effect, the present inventors first screened memory B cells capable of specifically binding to an S protein RBD from Peripheral Blood Mononuclear Cells (PBMCs) of persons infected with 2019-nCoV and cured at discharge by flow sorting using the S protein RBD of 2019-nCoV expressed in escherichia coli as an antigen, and then subjected the single B cells obtained by screening to RT-PCR to obtain a sequence encoding an antibody variable region. Further, a sequence encoding a variable region of the antibody and a constant region gene were ligated into an expression vector, and expression and purification were performed in mammalian cells, thereby obtaining antibody H4. A series of functional tests are carried out on the antibody H4, and the results show that the antibody H4 can specifically bind to the S protein RBD, block the binding of the S protein RBD and ACE2, inhibit the infection of 2019-nCoV on human cells, and has the neutralization activity of resisting the infection of 2019-nCoV.

Example 1: expression and purification of 2019-nCoV virus S protein RBD

A DNA fragment encoding the 2019-nCoV/2019 strain spike protein S protein RBD (the amino acid sequence of which is shown in SEQ ID NO: 17) was ligated to pET21a vector using NdeI and XhoI enzymes, and the nucleotide sequence encoding the 6 His tag (6 His tag) and a stop codon were ligated to the 3' end of the coding region. The ligation product was transformed into BL21 E.coli competent cells. Then, selecting a single clone, inoculating the single clone into 40mL of LB culture medium, and culturing for 6-8 hours; then, the cells were inoculated into 4L of LB medium and cultured at 37 ℃ until OD600 became 0.4-0.6. Subsequently, IPTG was added to the culture to a final concentration of 1mM and the culture was continued at 37 degrees Celsius for 4-6 hours. After the culture, the inclusion bodies were harvested and renatured. The renatured protein solution was concentrated and dialyzed into 20mM Tris, 150mM NaCl, pH9.0 buffer. Subsequently, the protein in the solution was purified by molecular sieve chromatography using AKTA-purifier (GE) and superdex200 Hiload 16/60 column (GE) and buffer A (20 mM Tris, 150mM NaCl, pH9.0), and during the purification, UV absorbance at 280nm was monitored simultaneously, and the fractions containing the protein of interest were recovered. After the purification, the purity of the objective protein (RBD of S protein) was confirmed by SDS-PAGE. The results are shown in FIG. 1. The results in FIG. 1 show that a highly pure RBD protein having a size of 32kDa is obtained.

Example 2: isolation of memory B cells that specifically recognize RBD proteins

With the informed consent of the person who infected 2019-nCoV virus and recovered from the discharge, 10mL of blood was collected and PBMCs were isolated. Isolating the PBMCs at 10⁷density/mL combined with incubation on ice for half an hour of RBD protein (prepared as in example 1) at a final concentration of 400 nM; then washed 2 times with PBS and incubated with the following antibodies (all from BD): anti-human CD3/PE-Cy5, anti-human CD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC, and anti-His/PE. After half an hour incubation on ice, PBMCs were washed 2 times with PBS. Subsequently, PBMCs are sorted by FACSAria III and PE is collected^-Cy5^-APC^- APC-Cy7⁺Pacific Blue⁺FITC⁺PE⁺The cells of (i.e., B cells) were collected directly into a 96-well plate at 1 cell/well.

Example 3: isolation and characterization of H4 antibody and construction of recombinant expression vector

The B cells obtained in example 2 were subjected to reverse transcription (at 55 ℃ C. for 60 minutes) using Superscript III reverse transcriptase (Invitrogen), wherein the reverse transcription primers used were as shown in Table 2.

TABLE 2 sequence information of the reverse transcription primers used

Performing a first round of PCR (PCRa) with HotStar Tap Plus enzyme (QIAgen) using the reverse transcription product as a template to amplify the sequence of the antibody variable region; wherein, the primers used are shown in table 3; the reaction conditions used were as follows: 95 ℃ for 5 min; 35 cycles (95 ℃ 30s, 55 ℃ (heavy chain/kappa chain) 30s, 72 ℃ 90 s); 72 ℃ for 7 min. Then, using the amplification product as a template to perform a second round of PCR (PCRb); wherein, the primers used are shown in table 4; the reaction conditions used were as follows: 95 ℃ for 5 min; 35 cycles (95 ℃ 30s, 58 ℃ (heavy chain)/60 ℃ (κ chain)/64 ℃ (λ chain) 30s, 72 ℃ 90 s); 72 ℃ for 7 min.

The PCR products were separated by electrophoresis on a 1% agarose gel. The PCR product with the band size of 400-500 bp was recovered and sent to the sequencing company for sequencing. The sequencing results were analyzed using NCBI online software.

Through sequence determination, the sequence of one antibody is obtained and named as H4. The amino acid sequence of the heavy chain variable region of the H4 antibody is shown as SEQ ID NO. 7 (the coding gene is shown as SEQ ID NO. 12), and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8 (the coding gene is shown as SEQ ID NO. 13). The sequence identity of the H4 antibody to germline genes is shown below in tables 5-6.

TABLE 3 primers used in the first round of PCR (PCRa)

TABLE 4 primers used in the second round of PCR (PCRb)

TABLE 5 comparison of H4 antibody heavy chain and germline genes

TABLE 6 comparison of H4 antibody light chain and germline genes

The nucleotide sequences encoding the heavy chain/light chain variable regions obtained by analysis were linked to the corresponding nucleotide sequences encoding the heavy chain/kappa chain constant regions by bridge-PCR, and then cloned into expression vectors pCAGGS (purchased from Addgene), thereby obtaining recombinant expression vectors encoding the antibody heavy and light chains, respectively. The construction scheme of the constructs for expressing the heavy and light chains is as follows:

heavy chain coding sequence (5 '-3'): CMV promoter-EcoR I restriction enzyme site-leader sequence gene-VH gene-CH gene-Xho I restriction enzyme site;

light chain (κ) coding sequence (5 '-3'): CMV promoter-Sac I restriction enzyme site-leader sequence gene-VL gene-CL (kappa) gene-Xho I restriction enzyme site;

wherein, the amino acid sequence of the leader sequence is shown as SED ID NO. 11 (the coding gene is shown as SEQ ID NO. 16), the amino acid sequence of the CH is shown as SED ID NO. 9 (the coding gene is shown as SEQ ID NO. 14), and the amino acid sequence of the CL is shown as SED ID NO. 10 (the coding gene is shown as SEQ ID NO. 15).

Example 4: expression of H4 antibody

293T cells were cultured in DMEM containing 10% FBS. 293T cells were co-transfected with the recombinant expression vectors encoding the heavy and light chains of the antibody, respectively, obtained in example 3. 4-6 hours after transfection, the cell culture medium was changed to serum-free DMEM, and culture was continued for 3 days. The supernatant was collected, then DMEM was added, the culture was continued for 4 days, and then the supernatant was collected again.

The collected supernatant was centrifuged at 5000rpm for 30min, then mixed with an equal volume of buffer containing 20mM sodium phosphate (pH 7.0), followed by filtration through a 0.22 μm filter, and then loaded onto a protein A pre-column (5mL, GE Healthcare). Proteins bound to the pre-packed column were eluted with 10mM glycine (pH 3.0). The eluted fractions were concentrated and then purified by molecular sieve chromatography. Subsequently, the purified protein of interest was detected by SDS-PAGE (reducing and non-reducing). The results are shown in FIG. 2. The results in fig. 2 show that purified H4 antibody was obtained.

Example 5: evaluation of binding ability of H4 antibody to S protein RBD

In this example, surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The method comprises the following specific steps:

first, an antibody against anti-human IgG was immobilized in an amino-coupled manner on a channel (flow cell, Fc) of a CM5 chip. The fixed amount is controlled around 8,000 Response Units (RU). The purified H4 antibody was then bound by means of antibody capture. In addition, RBD protein was serially diluted in multiple ratios with 20mM HEPES, 150mM NaCl, pH 7.4 solution. Then, serial dilutions of RBD protein were passed sequentially through each channel (loading one by one starting from low concentration). Kinetic curves of H4 antibody binding to RBD protein were recorded (fig. 3) and kinetic constants were calculated using BIAevaluation software 8K (Biacore, Inc.) software (as shown in table 4). The results in fig. 3 and table 4 show that the H4 antibody is able to bind with higher affinity the RBD of the S protein of 2019-nCoV.

TABLE 4 binding kinetics constants of antibodies to RBD proteins

	ka(1/Ms)	kd(1/s)	KD(M)
				H4	8.47E+04	3.79E-04	4.48E-09

Example 6: assessment of the ability of H4 to block RBD binding to ACE2

The gene encoding hACE2 protein (Genbank accession: NP-068576.1) was cloned into pEGFP-N1 vector (purchased from Addgene) using XhoI and BamHI, and the gene was expressed in fusion with the gene encoding GFP, thereby constructing plasmid pEGFP-hACE 2. Plasmid pEGFP-hACE2 was transfected into HEK293T cells. After 24h, GFP expression was observed under a fluorescent microscope. HEK293T-hACE2 cells were collected. Irrelevant antibodies were incubated with 200ng/mL RBD protein at a molar ratio of 10:1 for 1h at room temperature. Then, HEK293T-hACE2 cells (2X 10)⁵Reaction) RBD protein (200ng/ml, carrying 6 × His tag) after incubation with irrelevant antibody was incubated for 30min at room temperature. After centrifugation at 500Xg for 5min, the supernatant was removed and the cells were washed 2 times with PBS. Subsequently, the cells were incubated with anti-His/APC (gentle, 130-119-820) for 30min at room temperature, then washed 2 times with PBS, and then the fluorescence on the cell surface was detected with BD FACSCAnto.

To examine the blocking effect of the H4 antibody, the H4 antibody purified in example 4 was incubated with 200ng/mL RBD protein at a molar ratio of 10:1 at room temperature for 1H, followed by incubation with HEK293T-hACE2 cells. Then, the binding of RBD protein to the cells was detected by anti-His/APC as described above. The results are shown in FIG. 4. FIG. 4 shows the cell surface fluorescence profile detected with BD FACSCAnto. The results show that the number of hACE2 positive and RBD positive cells (i.e., cells carrying bifluorescence) is significantly less in the right panel than in the left panel; this indicates that the H4 antibody was effective in blocking the binding of the S protein RBD of 2019-nCoV to HEK293T-hACE2 cells.

Example 7: assessment of the ability of the H4 antibody to neutralize 2019-nCoV live Virus

The purified H4 antibody of example 4 was diluted from 200. mu.g/mL to a 12 th gradient (0.098. mu.g/mL) and then separately dosed with half the Tissue Culture Infectious Dose (TCID)₅₀) The BetacoV/Shenzhen/SZTH-003/2020 virus (obtained from the third national Hospital of Shenzhen, GISAID No.: EPI _ ISL _406594) was incubated at 37 ℃ for 2 hours with mixing. After incubation, the virus was added to 96-well plates pre-seeded with Vero cells and incubated at 37 ℃ in 5% CO₂After 4 days in the incubator, cytopathic effect (CPE) was observed and the neutralizing titer of the H4 antibody was calculated. The results are shown in FIG. 5. Figure 5 shows the neutralizing activity of H4 antibody against 2019-nCoV live virus at different concentrations. The results show that the H4 antibody neutralizes the live 2019-nCoV virus (half inhibitory concentration, IC)₅₀) 0.896. mu.g/mL, and has excellent neutralizing activity.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof, comprising heavy chain variable region (VH) complementarity determining regions 1-3(CDR1-3) having amino acid sequences shown in SEQ ID NOS: 1-3, respectively; and/or, light chain variable region (VL) complementarity determining regions 1-3(CDR1-3) having amino acid sequences shown in SEQ ID NOS: 4-6, respectively;

preferably, the monoclonal antibody comprises a heavy chain variable region (VH) as shown in SEQ ID NO:7, and/or a light chain variable region (VL) as shown in SEQ ID NO: 8;

preferably, the monoclonal antibody comprises: VH CDR1-3 with amino acid sequences shown as SEQ ID NO. 1-3, and VL CDR1-3 with amino acid sequences shown as SEQ ID NO. 4-6;

preferably, the monoclonal antibodies include: VH shown as SEQ ID NO. 7 and VL shown as SEQ ID NO. 8;

preferably, the monoclonal antibody further has a leader sequence at the N-terminus of the heavy chain variable region, and/or the monoclonal antibody further has a leader sequence at the N-terminus of the light chain variable region;

preferably, the leader sequence has an amino acid sequence shown as SEQ ID NO. 11;

preferably, the monoclonal antibody or antigen binding fragment thereof is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (e.g., scFv), human antibody, chimeric antibody, or bispecific or multispecific antibody;

preferably, the monoclonal antibody further comprises a heavy chain constant region; preferably, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO 9;

preferably, the monoclonal antibody further comprises a light chain constant region; preferably, the amino acid sequence of the light chain constant region is shown in SEQ ID NO 10.

2. An isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises: VH CDR1-3 with amino acid sequence of SEQ ID NO 1-3;

for example, the variable region of the antibody heavy chain has an amino acid sequence shown as SEQ ID NO. 7;

for example, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO. 12.

3. An isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody light chain variable region, wherein the antibody light chain variable region comprises: VL CDR1-3 having an amino acid sequence of SEQ ID NO. 4-6;

for example, the variable region of the antibody light chain has an amino acid sequence shown as SEQ ID NO. 8;

for example, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO. 13.

4. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of claim 1.

5. A vector comprising the isolated nucleic acid molecule of any one of claims 2-4.

6. A host cell comprising the isolated nucleic acid molecule of any one of claims 2-4 or the vector of claim 5.

7. A method of making the monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising culturing the host cell of claim 6 under suitable conditions, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cell culture.

8. A composition comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1, the isolated nucleic acid molecule of any one of claims 2-4, the vector of claim 5, the host cell of claim 6.

9. A kit comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1;

for example, the monoclonal antibody or antigen binding fragment thereof or anti-idiotype antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, and an enzyme;

for example, the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody; optionally, the second antibody further comprises a detectable label such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance and an enzyme.

10. A method for detecting the presence or level of a RBD of a novel coronavirus, or S protein thereof, in a sample, comprising administering the monoclonal antibody or antigen-binding fragment thereof of claim 1;

for example, the monoclonal antibody or antigen binding fragment thereof or anti-idiotype antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, and an enzyme;

for example, the method further comprises detecting the monoclonal antibody or antigen-binding fragment thereof or the anti-idiotype antibody using a second antibody that carries a detectable label (e.g., a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, and an enzyme).

11. Use of the monoclonal antibody or antigen binding fragment thereof of claim 1 in the preparation of a kit for detecting the presence or level of a novel coronavirus, or its S protein or the RBD of the S protein, in a sample, or for diagnosing whether a subject is infected with a novel coronavirus;

preferably, the sample is an excretion, oral or nasal secretion, or alveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

12. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1, and a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition further comprises other pharmaceutically active agents, such as favipiravir, ridciclovir, interferon and the like.

13. A method for neutralizing the virulence of a novel coronavirus in a sample comprising contacting a sample comprising a novel coronavirus with the monoclonal antibody or antigen-binding fragment thereof of claim 1.

14. Use of the monoclonal antibody or antigen-binding fragment thereof of claim 1 for the preparation of a medicament for neutralizing the virulence of a novel coronavirus in a sample, or for preventing or treating a novel coronavirus infection or a disease associated with a novel coronavirus infection (e.g. novel coronavirus pneumonia) in a subject;

preferably, the subject is a mammal, e.g., a human;

preferably, the medicament is used alone or in combination with other pharmaceutically active agents (e.g., favipiravir, ridciclovir, interferon, etc.).