Anti-RBD antibody and application thereof

Abstract

The invention provides an anti-RBD antibody and application thereof. The antibody provided by the invention comprises a monospecific antibody and a bispecific antibody, can specifically bind to RBD protein of SARS-CoV-2, can effectively inhibit virus invasion, has excellent binding activity with variant viruses such as Armitron and the like, and has good broad-spectrum neutralizing activity for broad-spectrum variant viruses.

Description

An anti-RBD antibody and its use Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-RBD antibody and uses thereof. Background Art

[0002] Since the discovery of SARS-CoV-2, there have been more than 20 variants worldwide. As of January 7, 2022, the WHO has defined five variants of concern (VOC), namely Alpha (α), Beta (β), Gamma (γ), Delta (δ) and Omicron (ο). In May 2022, the WHO stated that 128 countries and regions have reported the discovery of the Omicron variant. The number of mutated amino acid residues in the Delta variant is 18, while in the new strain Omicron there are 43. These mutations are diverse, and most of them are located in areas that interact with human cells. Due to the high mutation rate of the virus and its wide range of transmission, a large number of vaccines and drugs are also being developed. The risk of drug resistance and off-target effects of vaccines and antibody drugs is increasing, which also puts higher demands on the development of new antibody drugs and vaccines, and provides more opportunities.

[0003] The novel coronavirus has four structural proteins that mediate the assembly and infection of viral particles. The N protein forms the nucleocapsid outside the genome, followed by the outer layer of the viral envelope structure composed of the membrane protein (M), spike protein (S), and envelope protein (E).

[0004] The SARS-CoV-2 S protein is a hallmark transmembrane protein on the surface of the virus. It is a homotrimer composed of three identical subunits non-covalently bound together. Its molecular weight is 141,178 and contains 1,273 amino acids. The S protein is primarily composed of a signal peptide, an N-terminal domain, a receptor-binding domain (RBD), a fusion peptide, heptad repeats (HR) 1 and HR2, a transmembrane domain, and a cytoplasmic domain. It also possesses two cleavage sites, S1 / S2 and S2. Related studies have shown that HR1 and HR2 form a fusion core (6-HB) after the S1 RBD binds to ACE2 on target cells, enhancing the efficiency of viral fusion and infection.

[0005] Angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, is the primary active peptide of the renin-angiotensin system. It is currently believed that ACE2 is the primary receptor for viral entry in SARS-CoV-2 infection. ACE2, a type I transmembrane protein containing a zinc carboxypeptidase, is primarily expressed in the kidneys, heart, and male reproductive system, as well as in tissues such as the lungs, small intestine, and liver. The spike protein plays a central role in viral invasion, with the S protein serving as the primary immunogen. This protein stimulates the production of large quantities of antibodies, which bind to the S protein surface, blocking viral-host recognition and inhibiting conformational splicing, thereby suppressing viral invasion. The S protein often aggregates as a trimer on the viral surface. The S protein is a homotrimer, anchored at its C-terminus to the viral membrane. The S protein is crucial for coronavirus entry into cells, making it an attractive antiviral target. Antibodies against the RBD can inhibit the formation of S protein trimers.

[0006] Summary of the Invention

[0007] In order to solve the problem that there are no antibodies with good broad-spectrum neutralizing activity against Omicron virus on the market, the present invention provides an anti-RBD antibody, including monospecific antibodies and bispecific antibodies, which can effectively inhibit viral invasion, have good virus binding activity, long half-life, and good broad-spectrum neutralizing activity against a wide spectrum of mutant viruses.

[0008] The present invention provides isolated monoclonal human antibodies or antigen-binding portions thereof, including monospecific antibodies and bispecific antibodies, which specifically bind to coronaviruses such as SARS-CoV-2 at the spike protein or fragment thereof, such as the extracellular domain, or more specifically, the receptor binding domain of the spike protein, (i) prevent or block coronaviruses such as SARS-CoV-2 from entering host cells, and / or (ii) induce complement-mediated inactivation and / or phagocytosis of coronaviruses such as SARS-CoV-2. The antibodies of the present invention can also bind to the spike protein of coronaviruses such as SARS-CoV-2 displayed on infected host cells, inducing complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated viral inhibition (ADCVI) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) to eliminate infected cells. The antibodies of the present invention can be coated on coronaviruses such as SARS-CoV-2 or infected cells displaying coronavirus spike proteins to form immune complexes, which can be recognized by antigen-presenting cells such as dendritic cells, inducing humoral and cellular antiviral immune responses.

[0009] The antibody or its antigen-binding portion can be used for in vitro detection of coronaviruses such as SARS-CoV-2, and for treating or preventing diseases caused by coronaviruses.

[0010] Therefore, the present invention provides an isolated monoclonal antibody or an antigen-binding portion thereof that specifically binds to a coronavirus spike protein or a fragment thereof, wherein the antibody or the antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, (a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof comprising up to three amino acid mutations; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof comprising up to three amino acid mutations; the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof comprising up to three amino acid mutations; and / or

[0011] (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4 or 10 or a variant thereof comprising up to three amino acid mutations; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof comprising up to three amino acid mutations; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof comprising up to three amino acid mutations.

[0012] In certain embodiments, HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, respectively, or variants thereof comprising up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 4 or 10, 5, 6, respectively, or variants thereof comprising up to three amino acid mutations.

[0013] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7, 13, 14, or 42.

[0014] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 8, 11, or 47.

[0015] In certain embodiments, the heavy chain variable region and the light chain variable region comprise amino acid sequences that are at least 80% identical to the amino acid sequences shown in SEQ ID NOs: 7, 13, 14, 42 and 8, 11, 47, respectively.

[0016] In certain embodiments, it is a full length antibody, Fab, Fab', (Fab')2, Fd, Fv, dAb, scFv, or scFv-scFv.

[0017] In certain embodiments, it comprises an antibody or antigen binding portion thereof linked to a second antibody or antigen binding portion thereof, wherein the second antibody or antigen binding portion thereof has a different binding specificity than the antibody or antigen binding portion thereof described in any of the foregoing, and the second antibody or antigen binding portion thereof is capable of binding to another epitope of the coronavirus spike protein.

[0018] In certain embodiments, the second antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein

[0019] (a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof comprising at most three amino acid mutations; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising at most three amino acid mutations; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising at most three amino acid mutations; and / or

[0020] (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20 or 45 or a variant thereof comprising up to three amino acid mutations; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21 or a variant thereof comprising up to three amino acid mutations; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22 or a variant thereof comprising up to three amino acid mutations.

[0021] In certain embodiments, the heavy chain variable region and light chain variable region of the second antibody, or antigen-binding portion thereof, comprise an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 23 or 45 and SEQ ID NO: 24 or 46, respectively.

[0022] In certain embodiments, the bispecific antibody comprises two single-chain antibody fragments scFv, wherein the sequence of the heavy chain variable region VH of the scFv is shown in SEQ ID NO: 27 or 14, and the sequence of the light chain variable region VL of the scFv is shown in SEQ ID NO: 28 or 47.

[0023] In certain embodiments, the scFv is respectively connected to the N-terminus or C-terminus of the two heavy chains or two light chains of the second antibody or its antigen-binding portion. In certain embodiments, the scFv is respectively connected to the N-terminus of the two light chains of the second antibody or its antigen-binding portion.

[0024] In certain embodiments, it comprises a heavy chain constant region and / or a light chain constant region. In certain embodiments, the heavy chain constant region is an IgG, IgA, IgD, IgE, and IgM constant region. In certain embodiments, the heavy chain constant region is an IgG1, IgG2, IgG3, or IgG4 constant region. In certain embodiments, the light chain constant region is a kappa or lambda constant region.

[0025] In certain embodiments, the heavy chain constant region optionally contains a mutation. In certain embodiments, the heavy chain constant region may contain a mutation, such as an M252Y / S254T / T256E (YTE) mutation.

[0026] In certain embodiments, it binds to the spike protein or a fragment thereof of a coronavirus selected from SARS-CoV, SARS-CoV-2, and MERS-CoV.

[0027] In certain embodiments, it binds to the receptor binding domain or extracellular domain of the spike protein of the coronavirus. In certain embodiments, it (a) binds to the spike protein of SARS-CoV-2 or a fragment thereof, (b) inhibits the binding of the spike protein of SARS-CoV-2 or a fragment thereof to human ACE2, (c) prevents SARS-CoV-2 from entering host cells, (d) induces complement-mediated inactivation of SARS-CoV-2, (e) induces phagocytosis of SARS-CoV-2, (f) induces complement-dependent cytotoxicity against SARS-CoV-2 infected cells, (g) induces antibody-dependent cellular phagocytosis against SARS-CoV-2 infected cells, (h) induces antibody-dependent cell-mediated viral inhibition against SARS-CoV-2 infected cells, (i) induces antibody-dependent cell-mediated cytotoxicity against SARS-CoV-2 infected cells, (j) induces humoral and cellular antiviral immune responses, and / or (k) blocks the trimerization of the spike protein.

[0028] In certain embodiments, it binds to the extracellular domain of the spike protein of SARS-CoV-2 and mediates antibody neutralization.

[0029] In another aspect, the present invention also provides an immunoconjugate, such as an antibody-drug conjugate, which may comprise an antibody of the present invention or an antigen-binding portion thereof linked to a therapeutic agent such as a cytotoxin.

[0030] In another aspect, the antibodies of the present invention or their antigen-binding portions can be made into part of a chimeric antigen receptor (CAR). The present invention also provides immune cells, such as T cells, that can contain antigen chimeric receptors.

[0031] In another aspect, the present invention provides a nucleic acid molecule encoding an antibody or antigen-binding portion thereof of the present invention, as well as an expression vector that can contain such a nucleic acid molecule and a host cell that can contain the expression vector.

[0032] In another aspect, the present invention provides a method for preparing an antibody or antigen-binding portion thereof of the present invention using a host cell, which method may comprise the steps of: (i) expressing the antibody or antigen-binding portion thereof in the host cell and (ii) isolating the antibody or antigen-binding portion thereof from the host cell or its cell culture.

[0033] In another aspect, the present invention provides a composition, which may include an antibody or antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, an immune cell or expression vector with CAR, and a pharmaceutically acceptable carrier. In certain embodiments, the composition may include more than one antibody or antigen-binding portion thereof of the present invention. In certain embodiments, the composition may include a vector expressing more than one antibody or antigen-binding portion thereof of the present invention. In certain embodiments, the composition may include an immunoconjugate produced with more than one antibody or antigen-binding portion thereof of the present invention. In certain embodiments, the composition may include a bispecific molecule with more than one antibody or antigen-binding portion thereof of the present invention. In certain embodiments, the composition may include an immune cell with more than one antibody or antigen-binding portion thereof of the present invention in CAR.

[0034] In another aspect, the present invention provides a method for treating a disease caused by a coronavirus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention or its antigen-binding portion thereof. In certain embodiments, more than one antibody or its antigen-binding portion thereof is administered. In certain embodiments, the method may comprise administering a bispecific molecule, immunoconjugate, or immune cell with a CAR of the present invention. The coronavirus may be SARS-CoV, MERS-CoV, or SARS-CoV-2. In certain embodiments, the coronavirus is SARS-CoV-2.

[0035] In another aspect, the present invention provides a method for preventing a disease caused by a coronavirus infection in a subject in need thereof, comprising administering an antibody or antigen-binding portion thereof of the present invention to the subject. In certain embodiments, the coronavirus may be SARS-CoV, MERS-CoV, or SARS-CoV-2. In certain embodiments, the coronavirus is SARS-CoV-2. In certain embodiments, the subject is a human. In certain embodiments, the subject may be exposed to a disease caused by a coronavirus or to a coronavirus (e.g., SARS-CoV-2), but cannot be protected by vaccination.

[0036] Other features and advantages of the present invention will be apparent from the following detailed description and examples which should not be construed as limiting.The contents of all references, GenBank entries and patents cited throughout this application are expressly incorporated herein by reference.

[0037] Definition of terms

[0038] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for the sake of clarity and / or for ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not be construed as representing a difference from what is generally understood in the art.

[0039] The term "SARS-CoV-2" refers to severe acute respiratory syndrome coronavirus 2, which belongs to the Coronaviridae family, Betacoronavirus genus (which includes SARS-CoV and MERS-CoV).

[0040] The term "spike protein" refers to the transmembrane protein that coronaviruses use to invade host cells. The SARS-CoV-2 spike protein contains a first subunit (S1 subunit) that binds to host cell receptors and a second subunit (S2 subunit) that facilitates fusion of the viral and cell membranes. The S1 subunit contains a receptor binding domain (RBD) that binds to host cell receptors.

[0041] The term "antibody" encompasses immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each L chain is linked to an H chain by a single covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each heavy chain has a variable region at its N-terminus, followed by a constant region. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. This heavy chain constant region comprises three regions (domains): CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one region (domain, CL1). The VH and VL regions are further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies can be of different subclasses.

[0042] The term "full-length antibody" refers to an antibody in its substantially intact form, as compared to an antibody fragment. In particular, a full-length antibody includes a 4-chain antibody having heavy and light chains including an Fc region.

[0043] The term "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., SARS-CoV-2 spike protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) Fab' and F(ab')2 fragments, wherein F(ab')2 is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of the VH domain; (vi) scFv; (viii) scFv-scFv.

[0044] The term "Fc region" refers to the tail region of an antibody, which interacts with Fc receptors and some proteins of the complement system to activate the immune system. The IgG, IgA, and IgG Fc regions are composed of two identical fragments of the second and third constant domains (CH2 and CH3) from the antibody heavy chain, while the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4). The Fc region can bind to the complement component C1q to activate the classical complement cascade reaction, can bind to Fc receptors on phagocytes (i.e., macrophages, granulocytes, and dendritic cells) to induce phagocytosis of cells bound by antibodies, can bind to Fc receptors on immune effector cells (mainly natural killer cells) to induce immune effector cells to release cytotoxic granules, leading to the death of antibody-coated cells, and can bind to Fc receptors on antigen-presenting cells such as dendritic cells to induce humoral and cellular antiviral immune responses.

[0045] The term "chimeric antibody" is an antibody molecule (or antigen-binding portion thereof) in which (1) the constant region or a portion thereof is altered, substituted or replaced so that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function and / or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (2) the variable region or a portion thereof is altered, substituted or replaced with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement of the human constant region, the chimeric antibody can retain its specificity for recognizing the antigen while having reduced antigenicity in humans compared to the original mouse antibody.

[0046] The term "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from human antibody sequences. A humanized antibody may contain some or all of the CDRs from non-human animals or synthetic antibodies, while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of heterologous protein components they carry. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. In order to avoid a decrease in immunogenicity and a resulting decrease in activity, the human antibody variable region framework sequences can be subjected to minimal reverse mutations or back mutations to maintain activity.

[0047] The term "monoclonal antibody" refers to an antibody that is homogeneous and directed only against a specific antigenic epitope. Compared to conventional polyclonal antibody preparations that typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.

[0048] The term "isolated monoclonal antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to the SARS-CoV-2 spike protein is substantially free of antibodies that specifically bind to antigens other than the SARS-CoV-2 spike protein). However, an isolated antibody that specifically binds to the SARS-CoV-2 spike protein may have cross-reactivity with other antigens, for example, spike proteins from other viruses (such as coronaviruses). Furthermore, the isolated antibody can be substantially free of other cellular material and / or chemicals.

[0049] The term "bispecific antibody" refers to a protein molecule that can specifically bind to two target antigens or target antigen epitopes.

[0050] The term "scFv" refers to a molecule comprising an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, using 1-6 repeated GGGGS amino acid sequences or variants thereof.

[0051] The term "affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., the antigen binding moiety of MIAC) and its binding partner (e.g., antigen). Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple amino acid sites through weak non-covalent forces; The greater the interaction, the stronger the affinity. Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, for example, by using surface plasmon resonance (SPR) technology (e.g., instrument) or biolayer interferometry (e.g., instrument).

[0052] The term “chimeric antigen receptor” or “CAR” refers to an engineered receptor that transfers a defined specificity onto immune effector cells, typically T cells, and enhances T cell function. Newer-generation CARs consist of an extracellular binding domain, including a scFv, a hinge region, a transmembrane domain, and an intracellular signaling domain (primarily the cytoplasmic domain of CD3-zeta, the primary transmitter of T cell activation signals), plus one or more co-stimulatory domains. CARs may further harbor factors that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines and co-stimulatory ligands.

[0053] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.

[0054] The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer nucleotide coding information into a host cell. The term "vector" encompasses all types of vectors, regardless of their function. Vectors that can direct the expression of expressible nucleic acids to which they are operatively linked are generally referred to as "expression vectors." In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vectors.

[0055] The term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence to express a selected gene of interest. The term includes the progeny of a parent cell, regardless of whether the progeny is identical in morphology or genetic makeup to the original parent cell, as long as the gene of interest is present in the progeny. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0056] The term "treat" refers to inhibiting, eliminating, reducing and / or ameliorating the symptoms, severity of symptoms and / or frequency of symptoms associated with the disease or condition being treated, such as disease caused by coronavirus.

[0057] The term "prophylaxis" refers to actions taken to prevent a specific disease, such as a disease caused by a coronavirus. Prophylaxis can include administering antibodies to a subject who has been exposed to the disease or the virus that causes the disease and who is not protected by vaccination.

[0058] The term "administering" refers to the act of delivering or causing delivery of a therapeutic or pharmaceutical composition to a subject's body by the methods described herein or known in the art. Administering a therapeutic or pharmaceutical composition includes prescribing a therapeutic or pharmaceutical composition to be delivered to a patient. Exemplary administration forms include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0059] The term "effective amount" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-associated conditions of the invention or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-associated condition of the invention in a patient.

[0060] The term "pharmaceutically acceptable carrier" includes any or all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic and sustained-release agents, and the like that are compatible with drug administration. This includes various excipients, diluents, and buffers, etc., which are suitable for administration to humans and / or animals without excessive adverse side effects and are suitable for maintaining the activity of the drug or active agent located therein. Suitable carriers are described in the standard reference text in the latest edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference in its entirety. Examples of suitable carriers or diluents include, but are not limited to, water, saline solution, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and hydrophobic media such as fixed oils may also be used. The use of media and agents for pharmaceutically active substances is well known in the art. Except for conventional media or agents that are incompatible with the active ingredient, their use in the composition can achieve the desired effect.

[0061] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent sequence identity. Comparisons for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST software or the FASTA program package. The term "at least 80% identity" means that the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence is greater than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0062] The term "complement-dependent cytotoxicity" or "CDC" refers to an antibody-mediated immune mechanism in which antibodies bind to the complement component C1q and activate the classical complement cascade, leading to the formation of the membrane attack complex (MAC) on the cell surface and subsequent cell lysis.

[0063] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to a cell-mediated immune mechanism in which the Fc portion of an antibody binds to Fc receptors on phagocytes (i.e., macrophages, granulocytes, and dendritic cells) to induce phagocytosis of the antibody-bound cell.

[0064] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated immune mechanism in which the Fc portion of an antibody binds to Fc receptors on immune effector cells (primarily natural killer cells), resulting in the release of cytotoxic granules from the immune effector cells, leading to the death of the antibody-coated cells.

[0065] The antibodies or antigen-binding portions thereof of the present invention specifically bind to the spike protein or fragments thereof of coronaviruses, such as SARS-CoV-2, more specifically to the receptor binding domain of the spike protein, and inhibit the binding of the spike protein to its entry receptor ACE2. The antibodies or antigen-binding portions thereof of the present disclosure may also bind to the spike protein or fragments thereof, such as the receptor binding domain, displayed on virus-infected host cells.

[0066] The antibodies of the invention, or antigen-binding portions thereof, can induce (i) complement-mediated inactivation of coronaviruses, such as SARS-CoV-2, (ii) phagocytosis of coronaviruses, (iii) complement-dependent cytotoxicity (CDC) against coronavirus-infected host cells, (iv) antibody-dependent cellular phagocytosis (ADCP) against infected host cells, (v) antibody-dependent cell-mediated viral inhibition (ADCVI) against coronavirus-infected host cells, (vi) antibody-dependent cell-mediated cytotoxicity (ADCC) against coronavirus-infected host cells, and / or (vii) humoral and cellular antiviral immune responses.

[0067] The amino acid sequences and ID numbers of the heavy / light chain CDRs and variable regions of the antibodies or antigen-binding portions thereof of the present invention are listed in Table 1 below. The heavy chain variable region CDRs and light chain variable region CDRs have been defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined based on the heavy chain / light chain variable region sequences by other systems such as Chothia, AbM or Contact numbering systems / methods.

[0068] The antibodies of the present invention may contain a heavy chain constant region, such as a human IgG1 heavy chain constant region having the amino acid sequence set forth in, for example, SEQ ID NO: 9, or a variant thereof, and / or a light chain constant region having the amino acid sequence set forth in, for example, SEQ ID NO: 12, or a variant thereof. The antibodies of the present invention may also contain other suitable heavy chain constant regions and / or light chain constant regions, or variants thereof.

[0069] Table 1. Amino acid sequences of antibodies or antigen-binding portions BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 shows the Omicron RBD binding activity of mouse antibodies.

[0071] Figure 2 shows the binding activity of mouse antibodies to RBD of various mutant virus strains.

[0072] FIG3 shows the neutralizing activity of mouse antibodies against Omicron pseudovirus.

[0073] FIG4 shows the Omicron virus binding activity of humanized antibody 1.

[0074] FIG5 shows the Omicron virus binding activity of humanized antibody 2.

[0075] FIG6 shows the Omicron virus binding activity of humanized antibody 3.

[0076] FIG7 shows the pseudovirus neutralizing activities of murine antibody, chimeric antibody, humanized antibody 1, humanized antibody 2, and humanized antibody 3.

[0077] FIG8 is a schematic diagram of the structure of antibody a.

[0078] FIG9 is a schematic diagram of the structure of antibody b.

[0079] FIG10 is a schematic diagram of the structure of antibody c.

[0080] FIG11 is a schematic diagram of the structure of antibody d.

[0081] FIG12 shows the Omicron virus binding activity of antibodies a1, b1, and c1.

[0082] FIG13 shows the Omicron virus binding activity of antibodies d1, a2, and b2.

[0083] FIG14 shows the Omicron virus binding activity of antibodies C2 and D2.

[0084] FIG15 shows the Omicron pseudovirus neutralizing activity of antibodies a1, a2, b1, and b2.

[0085] FIG16 shows the neutralization activity of antibodies c1, c2, d1, and d2 against Omicron pseudovirus.

[0086] FIG17 shows the broad-spectrum neutralization activity of antibody d2 against pseudoviruses.

[0087] FIG18 shows the neutralizing activity of humanized bispecific antibodies against Delta and Omicron BA.2 pseudoviruses.

[0088] FIG19 shows the neutralizing activity of batch 1 and batch 2 of the humanized bispecific antibody against Omicron BA.1 pseudovirus.

[0089] Figure 20 shows the neutralizing activity of batch 1 and batch 2 of humanized bispecific antibodies against Omicron BA.4 & BA.5 pseudoviruses.

[0090] Figures 21(a)-(f) are the fitting curves of the competitive activities of humanized bispecific antibodies against L452R, T478K / Delta, B.1.1.529 / Omicron, BA.1.1 / Omicron, BA.2 / Omicron, BA.4&BA.5 / Omicron, and BF7&BA.4.6 / Omicron proteins, respectively.

[0091] Figure 22 shows the BA.2 lung viral load curves of each group after administration of humanized bispecific antibodies. DETAILED DESCRIPTION

[0092] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The scope of this application is not limited by these embodiments and is subject to the scope of the patent application.

[0093] Example 1 Mouse immunization

[0094] Three to five six-week-old female Balb / c mice were intraperitoneally injected with the SARS-CoV-2 Spike RBD, His Tag (B.1.1.529 / Omicron) (Acro: SPD-C522E) as the immunogen. Three days prior to vaccination, negative serum was collected. For the first immunization, 50 μg of the immunogen emulsified in Freund's complete adjuvant was injected. On days 14 and 35, 25 μg of the immunogen emulsified in Freund's incomplete adjuvant were injected intraperitoneally. Seven days later, tail blood was collected, serum was titered, and serum titers were determined by ELISA. Coat an ELISA plate overnight with SARS-CoV-2 Spike RBD, His Tag (B.1.1.529 / Omicron) (Acro: SPD-C522E). Discard the coating solution and block with 2% BSA. Add serum diluent and allow the reaction to proceed. After the reaction is complete, wash with PBST, add horseradish peroxidase-conjugated goat anti-mouse secondary antibody, and wash the plate with PBST. Develop with TMB colorimetric solution, terminate the reaction with 1M hydrochloric acid, and measure optical density at 450nm. When titer results meet the requirements, harvest mouse spleens and lymph nodes for cell fusion.

[0095] Example 2 Hybridoma Cell Fusion

[0096] The mice were sacrificed, and the spleen and lymph nodes were removed. The cells were ground through a cell sieve to obtain lymphocytes. The total cell count was calculated, and the obtained lymphocytes were mixed with myeloma SP2 / 0 cells at a ratio of 2:1. The cell suspension was centrifuged at 1000 rpm for 8 minutes, and the cells were washed twice with electrofusion solution and then electrofusion solution was used at a total cell density of 1×10 7 Resuspend the cells at 400 μg / mL and place the cell suspension in a fusion cell. Perform cell fusion using conventional electroporation. Culture the fusions in complete DMEM medium containing feeder cells and HAT at 37°C, 5% CO2. The feeder cells used in the fusion selection culture are macrophages from the peritoneal cavity of unimmunized animals to support the growth of new hybrid B lymphocyte hybridomas.

[0097] Example 3 Screening of positive clones of the present invention

[0098] ELISA was used to screen clones that bind to SARS-COV-2 Spike RBD, His Tag (B.1.1.529 / Omicron) (Acro: SPD-C522E), SARS-COV-2 Spike RBD (L4S2R T478K), His Tag (Acro: SPD-C52Hh), and SARS-COV-2 S1 Protein RBD, His Tag (MALS verifiled) (Acro: SPD-C52H3). All of the above proteins are commercially available.

[0099] The target protein was coated onto an ELISA plate at 4°C overnight, then blocked with 2% BSA and incubated at 37°C for 1 hour. Hybridoma cell culture supernatant was added to an ELISA plate, with SP2 / 0 cell supernatant used as a negative control and pre-fusion mouse serum diluted 100-fold as a positive control (100 μL / well) incubated at 37°C for 1 hour. The plates were washed three times with PBST, and HRP-labeled goat anti-mouse IgG-Fc antibody (100 μL / well) was added incubated at 37°C for 45 minutes. After washing, TMB was used for color development for 10 minutes, and the reaction was terminated with 1M hydrochloric acid. OD450 readings were measured on a microplate reader. Positive clones were identified if OD450 > 1.0 and < 0.1 for the negative control. Positive clones were counted by blowing air into the wells, and cells were plated at 1 cell / well in a 96-well culture plate. After 7-10 days at 5% CO2 and 37°C, the supernatant was collected and analyzed by ELISA. The single antibody of the present invention binds to the RBD of three viral strains and can be used as a candidate molecule.

[0100] Example 4 Production of murine antibodies

[0101] After obtaining a stable hybridoma cell line selected according to the present invention, monoclonal antibodies were obtained using in vitro culture. The cell line was expanded in a T75 culture flask and cultured until the cell confluence reached 40-50%. The cell supernatant was discarded and 50 mL of hybridoma-SFM medium was added and cultured at 37°C in 5% CO2. After 6-7 days of culture, when the cell viability fell below 20%, the culture supernatant was collected after low-speed centrifugation and purified using a Protein A affinity chromatography column to obtain purified mouse antibodies.

[0102] Example 5 Obtaining the variable region sequence of mouse antibody

[0103] The DNA sequence of the variable region of a murine antibody was determined using a degenerate primer-based PCR method. Hybridoma cell lines were cultured separately, harvested by centrifugation at 1000 rpm, and total RNA was extracted using Trizol. First-strand cDNA was synthesized using this as a template. The corresponding variable region DNA sequence was then amplified using PCR using the first-strand cDNA as a subsequent template. The PCR products were recovered, subjected to TA cloning, and sequenced to obtain candidate hybridoma heavy and light chain variable region sequences. The heavy chain variable region sequence comprised the amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region sequence comprised the amino acid sequence set forth in SEQ ID NO:8.

[0104] Example 6 Binding activity of mouse antibodies to Omicron RBD

[0105] Dilute SARS-CoV-2 Spike RBD, His Tag (B.1.1.529 / Omicron) (Manufacturer: Acro, Cat: SPD-C522e, Lot: 5716-21CCF2-11B) to 1 μg / mL and coat a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating solution and wash the plate with 300 μL per well of 1× PBST. Wash three times with a plate washer and pat dry on a lint-free tissue. Prepare 3% skim milk powder at 300 μL / well and incubate at 37°C for 1 hour. Discard the blocking solution and wash the plate with 300 μL per well of 1× PBST. Wash three times with a plate washer and pat dry on a lint-free tissue to block the protein. The mouse antibody and positive control antibody were diluted to 20 μg / mL with 3% skim milk powder. Three-fold dilutions were performed using this initial concentration, resulting in 11 dilution steps. A blank well was added with only the diluent. 100 μL / well was added, and the plate was incubated at 37°C for 1 hour. Discard any remaining liquid, wash the plate three times with 1× PBST, and pat dry on a lint-free tissue paper. Goat anti-mouse IgG Fc (HRP) (abcam, Cat#ab97265) was diluted 1:10,000 with 3% skim milk powder, adding 100 μL / well, and incubate at 37°C for 45 minutes. Discard the secondary antibody solution, wash the plate six times with 1× PBST, and pat dry on a lint-free tissue paper. Add 100 μL / well of one-component TMB colorimetric mix (Solarion, Cat#PR1200), and develop at 37°C in the dark for 7 minutes. The color reaction was terminated by adding 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water) as a stop solution, 100 μL / well. The plate was read at 450 nm on a microplate reader. Binding curves of the mouse antibody, the positive control antibody (heavy chain variable region sequence as shown in SEQ ID NO: 15, light chain variable region sequence as shown in SEQ ID NO: 16) and the Omicron RBD were obtained, as shown in Figure 1. 50 The values ​​were 0.95 and 3.60 μg / mL, respectively. It can be seen that the mouse antibody is more effective than the positive control antibody.

[0106] Example 7 Binding experiment of mouse antibodies to RBD of various mutant virus strains

[0107] SARS-CoV-2 Spike RBD, His tag. (BA.1 / Omicron) (Manufacturer: Acro, Cat: SPD-c522e, Lot: 5716-21CCF2-Z4), SARS-CoV-2 Spike RBD, His tag. (BA.1.1 / Omicron) (Manufacturer: Acro, Cat: SPD-c522j, Lot: 6159-2248F1-12M), SARS-CoV-2 Spike RBD, His tag. (BA.2 / Omicron) (Manufacturer: Acro, Cat: SPD-c522g, Lot: 5911-223BF1-11Z), SARS-CoV-2 Spike RBD, His tag. (Delta) (Manufacturer: Acro), SARS-CoV-2 Spike RBD, His tag. Tag. (wild type) (manufacturer: Acro) was diluted to 0.8 μg / mL and coated in a 96-well ELISA plate at 100 μL / well at 4°C overnight. Discard the coating solution, wash each well with 1×PBST at 300 μL, wash three times with a plate washer, and pat dry on a paper towel. Block with 3% skim milk powder at 300 μL / well, incubate at 37°C for 1h20min, discard the blocking solution, wash each well with 1×PBST at 300 μL, wash three times with a plate washer, and pat dry on a lint-free paper. The positive control antibody (heavy chain variable region sequence as shown in SEQ ID NO: 15, light chain variable region sequence as shown in SEQ ID NO: 16) and the mouse antibody were diluted to 10 μg / mL with 3% skim milk powder, 100 μL / well, and incubated at 37°C for 1h. Discard the liquid in the wells, wash three times with a plate washer, and pat dry on a paper towel. Dilute Goat pAb to Ms IgG (HRP) (Manufacturer: abcam, Lot: GR3393964-2) 1:10,000 with 3% skim milk powder, add 100 μL / well, and incubate at 37°C for 45 min. Discard any liquid from the wells, wash the plate with 1× PBST (300 μL per well), wash six times on a microplate washer, and pat dry on lint-free paper. Add 100 μL / well of the one-component TMB colorimetric mixture, wrap with aluminum foil, and develop at 37°C in the dark for 3 min. Terminate the color reaction by adding 100 μL / well of the stop solution (83 mL of 37% concentrated hydrochloric acid + 917 mL of pure water). Read at 450 nm on a microplate reader. As shown in Figure 2, both mouse antibodies and positive control antibodies can cross-bind to SARS-CoV-2 wild type, Delta, Omicron virus strain BA.1, and Omicron virus strain BA.2, and their binding activity to Omicron virus strain BA.1.1 is slightly weaker.

[0108] Example 8 Single-site neutralization of mouse antibodies and Omicron pseudovirus

[0109] The mouse antibody and the positive control antibody (the heavy chain variable region sequence is shown in SEQ ID NO: 15, and the light chain variable region sequence is shown in SEQ ID NO: 16) were melted at 4°C and diluted with DMEM complete medium to 13.3 μg / mL (final concentration 10 μg / mL). The diluted antibody was added to a 96-well white plate at 75 μL / well. The pseudovirus SARS-Cov-2 spike (Omikron) Fluc-GFP Pseudovirus (manufacturer: Acro, Cat: PSSO-HLGB003) was re-dissolved at room temperature and diluted according to the pseudovirus: diluent = 1:125. The diluted virus was added to a 96-well white plate at 25 μL / well and incubated with the antibody diluent at room temperature for 1 hour. HEK293-ACE2 hu cells were cultured at 5×10 4 Add 100 μL of luciferase developer solution to the culture plate and incubate in an incubator for 48 hours. Remove the luciferase developer solution and equilibrate the 96-well plate to room temperature. Discard 100 μL of culture medium from the sample wells and immediately add 100 μL of luciferase developer solution. Incubate at room temperature for 3-5 minutes. Read the chemiluminescent signal with a microplate reader. The results of a single-point neutralization test of the mouse antibody against the Omicron pseudovirus are shown in Table 2. The mouse antibody exhibited over 99.81% inhibitory activity against the Omicron pseudovirus, comparable to the positive control antibody.

[0110] Table 2. Results of single-site neutralization assays of mouse antibodies and Omicron pseudovirus

[0111] Example 9 Neutralization experiment of mouse antibodies and Omicron pseudovirus

[0112] Melt the mouse antibody, positive control antibody, and irrelevant antibody at 4°C and dilute with DMEM complete medium to 13.3μg / ml (final concentration 10μg / mL). Take 200μL of the first column of antibody diluent, take 30μL to 150μL of diluent and dilute 6 times, and so on, for a total of 7 gradients. Add the diluted antibody to a 96-well white plate at 75μg / well. Redissolve the pseudovirus SARS-Cov-2 spike (Omikron) Fluc-GFP Pseudovirus (manufacturer: Acro, Cat: PSSO-HLGB003) at room temperature and dilute it according to the pseudovirus: diluent = 1:125. Add the diluted virus to a 96-well white plate at 25μL / well and incubate with the antibody diluent at room temperature for 1h. Incubate HEK293-ACE2hu cells at 5×10 4Add 100 μL / well to the culture plate and culture in the incubator for 48 hours. Remove the luciferase colorimetric solution and the 96-well white plate in advance and equilibrate to room temperature. Discard 100 μL of culture medium in the sample well and immediately add 100 μL of luciferase colorimetric solution. Incubate at room temperature for 3-5 minutes and read the chemical fluorescence signal with a microplate reader. As shown in Figure 3, the mouse antibody and the positive control antibody have obvious neutralizing activity against the Omicron pseudovirus, IC 50 The values ​​were 0.0021μg / mL and 0.0097μg / mL respectively. The activity of mouse antibody was higher than that of positive control antibody.

[0113] Example 10 Humanized Antibody Binding Experiment with Omicron Virus

[0114] SARS-CoV-2 Spike RBD, His tag (Manufacturer: Acro, Cat: SPD-c522e Lot: 5716-21CCF2-Z4) was diluted to 0.8 μg / mL and coated into a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating solution, wash the plate with 300 μL per well of 1× PBST, wash three times with a plate washer, and pat dry on paper. Block the plate with 3% skim milk powder at 300 μL / well and incubate at 37°C for 1 hour 20 minutes. Discard the blocking solution, wash the plate with 300 μL per well of 1× PBST, wash three times with a plate washer, and pat dry on clean paper. A chimeric antibody was used as a positive control antibody. Chimeric antibody, humanized antibody 1, humanized antibody 2, and humanized antibody 3 were diluted to 10 μg / mL with 3% skim milk powder. Eleven dilutions were added in a three-fold dilution series at 100 μL / well and incubated at 37°C for 1 hour. Discard the liquid from the wells, wash three times with a plate washer, and pat dry on paper. Goat anti-human IgG (HRP) (Manufacturer: Jackson, Lot: 109-035-098) was diluted 1:10,000 with 3% skim milk powder at 100 μL / well and incubate at 37°C for 45 minutes. Discard the liquid from the wells, wash the plate six times with 1× PBST, and pat dry on lint-free paper. Add 100 μL / well of the one-component TMB colorimetric mixture, wrap with aluminum foil, and develop at 37°C in the dark for 3 minutes. Stop the color reaction by adding 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water) at 100 μL / well. Read the plate at 450 nm on a microplate reader. As shown in Figures 4-6, there is little difference in activity between humanized antibody 1, humanized antibody 2, and humanized antibody 3, with humanized antibody 3 exhibiting relatively higher activity.

[0115] Example 11 Pseudovirus Neutralization Experiments with Murine Antibodies, Chimeric Antibodies, Humanized Antibody 1, Humanized Antibody 2, and Humanized Antibody 3

[0116] Melt the mouse antibody, chimeric antibody, humanized antibody 1, humanized antibody 2, humanized antibody 3, and irrelevant antibody at 4°C and dilute to 13.3 μg / mL (final concentration 10 nM) with DMEM complete medium. Take 200 μL of the first column of antibody diluent, take 30 μL to 150 μL of diluent and dilute 6 times, and so on, for a total of 7 gradients. Add the diluted antibody to a 96-well white plate at 75 μL / well. Redissolve the pseudovirus SARS-Cov-2 spike (Omikron) Fluc-GFP Pseudovirus (manufacturer: Acro, Cat: PSSO-HLGB003) at room temperature and dilute it according to the pseudovirus: diluent = 1:125. Add the diluted virus to a 96-well white plate at 25 μL / well and incubate with the antibody diluent at room temperature for 1 hour. HEK293-ACE2 hu cells were cultured at 5×10 4 Add 100 μL / well to the culture plate and culture in the incubator for 48 hours. Take out the luciferase colorimetric solution and the 96-well white plate in advance and equilibrate to room temperature. Discard 100 μL of culture medium in the sample well and immediately add 100 μL of luciferase colorimetric solution. Incubate at room temperature for 3-5 minutes and read the chemical fluorescence signal with a microplate reader. As shown in Figure 7, humanized antibody 1, humanized antibody 2, and humanized antibody 3 have significant neutralizing activity against Omicron pseudovirus, IC 50 The values ​​were 0.023 nM, 0.022 nM and 0.021 nM, respectively.

[0117] Example 12 Construction of the bispecific antibody of the present invention

[0118] Eight bispecific antibodies were constructed based on murine antibodies and the murine antibodies described in patent CN202210892796.0, namely antibodies a1, a2, b1, b2, c1, c2, d1, and d2. A1, b1, c1, and d1 are antibody structures without mutations in the Fc region, and a2, b2, c2, and d2 are antibody structures with mutations in the Fc region. Exogenously expressed bispecific antibody suspensions, in which antibodies a1 and a2 have the same structure (abbreviated as antibody a structure), b1 and b2 have the same structure (abbreviated as antibody b structure), c1 and c2 have the same structure (abbreviated as antibody c structure), and d1 and d2 have the same structure (abbreviated as antibody d structure). The structures of antibodies ad are shown in Figures 8-11, respectively.

[0119] Example 13 Binding experiment of the bispecific antibody of the present invention and Omicron virus

[0120] SARS-CoV-2 Spike RBD, His tag (Manufacturer: Acro, Cat: SPD-c522e Lot: 5716-21CCF2-Z4) was diluted to 0.8 μg / mL and coated into a 96-well microtiter plate at 100 μL / well at 4°C overnight. Discard the coating solution, wash the plate with 300 μL / well of 1× PBST, wash three times with a plate washer, and pat dry on paper. Block the plate with 300 μL / well of 3% skim milk powder, incubate at 37°C for 1 hour 20 minutes, discard the blocking solution, wash the plate with 300 μL / well of 1× PBST, wash three times with a plate washer, and pat dry on clean tissue paper. The humanized antibody in patent CN202210892796.0 (the full-length sequence of the heavy chain is shown in SEQ ID NO: 40, and the full-length sequence of the light chain is shown in SEQ ID NO: 41) was used as a positive control antibody. The positive control antibody, antibody a1, a2, b1, b2, c1, c2, d1, and d2 were diluted to 5 μg / mL (final concentration 2.5 μg / mL) with 3% skim milk powder, 11 gradients of 3-fold dilution, 100 μL / well, and incubated at 37°C for 1 hour. Discard the liquid in the well, wash the plate three times with a plate washer, and pat dry on a flat paper. Goat anti-human IgG (HRP) (manufacturer: Jackson, Lot: 109-035-098) was diluted 1:10000 with 3% skim milk powder, 100 μL / well, and incubated at 37°C for 45 minutes. Discard the liquid in the wells and wash the plate with 300 μL per well of 1× PBST. Wash six times in a microplate washer and pat dry on lint-free paper. Add 100 μL / well of the single-component TMB colorimetric mixture, wrap with aluminum foil, and incubate at 37°C in the dark for 3 min. Terminate the colorimetric reaction by adding 100 μL / well of the stop solution (83 mL of 37% concentrated hydrochloric acid + 917 mL of pure water). Read the plate at 450 nm on a microplate reader. As shown in Figures 12-14, there is little difference in the activity of antibodies a1, a2, b1, b2, c1, c2, d1, and d2. Antibodies a1, b1, b2, c1, c2, d1, and d2 all exhibit stronger activity than the positive control antibody.

[0121] Example 14 Omicron pseudovirus neutralization experiment with the bispecific antibody of the present invention

[0122] The humanized antibody described in patent CN202210892796.0 (full-length heavy chain sequence as shown in SEQ ID NO:40, full-length light chain sequence as shown in SEQ ID NO:41) was used as a positive control antibody. The positive control antibody, antibodies a1, a2, b1, b2, c1, c2, d1, and d2 were thawed at 4°C and diluted with complete DMEM medium to 13.3 μg / mL (final concentration 10 nM). From 200 μL of the first column of antibody diluent, 30 μL was taken to 150 μL of the diluent and diluted 6-fold, and so on, for a total of 7 steps. The diluted antibody was added to a 96-well white plate at 75 μL / well. The pseudovirus SARS-Cov-2 spike (Omicron) Fluc-GFP pseudovirus (manufacturer: Acro, Cat: PSSO-HLGB003) was reconstituted at room temperature and diluted to a ratio of 1:125. The diluted virus was added to a 96-well white plate at 25 μL / well and incubated with the antibody dilution at room temperature for 1 hour. HEK293-ACE2 hu cells were cultured at 5×10 4 Add 100 μL / well of the sample wells to the culture plate and incubate in an incubator for 48 hours. Remove the luciferase colorimetric solution and equilibrate the 96-well white plate to room temperature in advance. Discard 100 μL of culture medium from the sample wells and immediately add 100 μL of luciferase colorimetric solution. Incubate at room temperature for 3-5 minutes and read the chemiluminescent signal with a microplate reader. As shown in Figures 15 and 16, antibodies a1, a2, b1, b2, c1, c2, d1, and d2 all showed significant neutralizing activity against Omicron pseudovirus, with greater activity than the positive control antibody. Antibody d2 showed the highest activity.

[0123] Example 15: Broad-spectrum neutralization experiment of pseudovirus by antibody d2

[0124] Thaw antibody d2 at 4°C and dilute to 1.5 μg / mL (final concentration 10 nM) in complete DMEM medium. Prepare a 6-fold dilution series of 200 μL of antibody dilution buffer, then add 30 μL to 150 μL of dilution buffer, and repeat this process for a total of 7 dilutions. Add 75 μL / well of the diluted antibody to a 96-well plate. Reconstitute the pseudoviruses SARS-CoV-2 spike (Acro) Fluc-GFP pseudovirus (Manufacturer: Acro, Cat: PSSO-HLGB003), SARS-CoV-2 Spike (Delta) Fluc-GFP pseudovirus (Manufacturer: Acro, Cat: PSSD-HLGB002), and SARS-CoV-2 Spike (Acro BA.2) Fluc-GFP pseudovirus (Manufacturer: Acro, Cat: PSSO-HLGB011) at room temperature and dilute at a ratio of 1:125 between pseudovirus and diluent. The diluted virus was added to a 96-well white plate at 25 μL / well and incubated with the antibody dilution at room temperature for 1 hour. HEK293-ACE2 hu cells were cultured at 5×10 4 100 μL / well was added to the culture plate and cultured in the incubator for 48 hours. The luciferase colorimetric solution and the 96-well white plate were taken out in advance and equilibrated to room temperature. 100 μL of culture medium in the sample well was discarded and 100 μL of luciferase colorimetric solution was immediately added. The cells were incubated at room temperature for 3-5 minutes and the chemiluminescence signal was read by the enzyme reader. As shown in Figure 17, antibody d2 had obvious broad-spectrum neutralizing activity against the new coronavirus pseudovirus, with an IC of 0.05 for Delta virus, BA.1, BA.2, and BA.4 / BA.5 strains. 50 The values ​​are 0.235nM, 0.028nM, 0.086nM, and 0.089nM, respectively.

[0125] Example 16 Humanization of Antibody D2

[0126] Antibody d2 was humanized to obtain a humanized bispecific antibody, wherein the full-length amino acid sequence of the heavy chain was SEQ ID NO: 43, and the full-length amino acid sequence of the scFv + second antibody light chain was SEQ ID NO: 44.

[0127] Example 17 Neutralizing activity of humanized bispecific antibodies against wild-type and different mutant strains

[0128] The neutralizing activity of the humanized bispecific antibody was studied using a pseudovirus assay with different subtype mutants. The pseudovirus recognizes the ACE2 receptor on the surface of HEK293-ACE2 cells through its surface RBD protein, leading to viral invasion and intracellular expression of luciferase. The humanized bispecific antibody effectively inhibited infection of host cells by wild-type Delta and the various mutant strains of Omicron BA.1, Omicron BA.2, and Omicron BA.4&5, and suppressed luciferase gene expression. The neutralizing activity of batches 1 and 2 was compared using pseudoviruses of the BA.1 / Omicron and BA.4&BA.5 / Omicron strains. The data demonstrated that batches 1 and 2 exhibited identical neutralizing activity against the BA.1 and BA.4&5 mutants. Specific test data are shown in Table 3 and Figures 18-20.

[0129] Table 3. Neutralizing activity of humanized bispecific antibodies against wild-type and different mutant strains (EC50 values, ng / mL)

[0130] Example 18 In vitro neutralization activity of humanized bispecific antibodies against live viruses

[0131] We commissioned the Kunming Institute of Zoology to further validate the cellular neutralizing activity of humanized bispecific antibodies using live virus strains to investigate the binding and competitive activity of humanized bispecific antibodies against RBD proteins of various subtypes of the virus. The in vitro activity of the humanized bispecific antibodies was evaluated using the COVID-19 Omicron variant (BA.2) and the recently circulating BA.5 strain. The inhibition of viral infection of Vero E6 cells by the humanized bispecific antibodies was evaluated by measuring the concentration at which the humanized bispecific antibodies completely inhibited cytopathic effect (CPE). First, the antibody drug was diluted in culture medium according to the concentration series in a well plate. An equal volume of 100 TCID50 of virus was added to each diluted sample, mixed, and incubated at 37°C, 5% CO2 for 1 hour. The sample-virus mixture, along with positive and negative controls, was then added to plates seeded with Vero-E6 cells and incubated at 37°C, 5% CO2 for 4-6 days. Observe and record the cytopathic effect (CPE) in each well under a microscope. Based on the cytopathic effect, calculate the dilution multiple of the sample with a neutralization effect of more than 50%, and use this as the neutralizing antibody titer of the sample. Table 4 shows the statistical results of CPE of samples at various concentrations of the neutralizing activity of the humanized bispecific antibody against the BA.2 strain. The test results show that the humanized bispecific antibody has 100% live virus neutralization ability against the BA.2 strain at an extremely low concentration of 2 ng / mL. Table 5 shows the statistical results of CPE of samples at various concentrations of the neutralizing activity of the humanized bispecific antibody against the BA.5 strain.

[0132] Table 4. CPE statistical results of samples at different concentrations for neutralization activity of humanized bispecific antibodies against BA.2 strain Note: The denominator represents the number of replicates, the numerator represents the number of wells where no CPE was observed, and the percentage represents the neutralizing antibody protection rate of the sample at that concentration.

[0133] Table 5. CPE statistical results of samples at different concentrations for neutralization activity of humanized bispecific antibodies against BA.5 strain

[0134] Example 19 Competitive Activity Test of Humanized Bispecific Antibodies Against Proteins of Different Mutant Strains

[0135] Flow cytometry was used to test the competitive activity of the humanized bispecific antibodies against proteins from different mutant strains: L452R, T478K Delta, B.1.1.529 / Omicron, BA.1.1 / Omicron, BA.2 / Omicron, BA.4 & BA.5 / Omicron, and BF7 & BA.4.6 / Omicron. Flow cytometry results showed that the RBD proteins from different mutant strains had strong binding activity to HEK293 cells expressing ACE2. The humanized bispecific antibodies competed for the binding of the RBD proteins from different mutant strains to HEK293 cells in a concentration-dependent manner. The competitive activity results of batches 1 and 2 against proteins from different mutant strains were consistent. Specific test data are shown in Table 6 and Figures 21(a)-(f).

[0136] Table 6. Competitive activity of humanized bispecific antibodies against proteins of different mutant strains (EC50 values, μg / mL)

[0137] Example 20 In vivo pharmacodynamics study of humanized bispecific antibodies in animal infection models

[0138] The in vivo efficacy of the humanized bispecific antibody in an animal infection model was tested at the Kunming Institute of Zoology, Chinese Academy of Sciences. Existing data show that a single intraperitoneal injection of the humanized bispecific antibody in hamsters infected with the BA.2 mutant strain of Omicron effectively inhibited lung infection in these hamsters. High-dose administration virtually eliminated lung infection in all hamsters. The specific experimental plan and results are as follows.

[0139] After the Syrian golden hamsters aged 5-7 weeks were raised, 1*10 4Hamsters were infected with the TICD50 COVID-19 Omicron mutant strain BA.2 (isolated and cultured at the Kunming Institute of Zoology, Chinese Academy of Sciences). Two hours after infection, the hamsters received a single intraperitoneal injection of different doses of the humanized bispecific antibody: a low-dose group received 25 mg / kg, and a high-dose group received 50 mg / kg. Three days after dosing, the hamsters were dissected, and the viral load in their lung tissues was measured. Lung tissue sections were also collected for pathological examination. The specific dosing protocol is shown in Table 7.

[0140] Table 7. In vivo efficacy test plan for humanized bispecific antibody BA.2 infection model

[0141] The viral loads in the lungs of the hamster control group and the treatment group 3 days after the challenge and administration are shown in Figure 22. The experimental data showed that the humanized bispecific antibody has very significant neutralizing activity against the BA.2 strain, effectively inhibiting the virus's lung infection in hamsters and effectively reducing the viral load of hamster lung infection, with statistical significance. In the low-dose administration group, the lung viral load of 8 hamsters decreased significantly, and in the high-dose group, the lung viral load of almost all hamsters decreased completely. The decrease in lung viral load was dose-dependent, with an average lung viral load decrease of 2 lg values ​​in the low-dose group and an average lung viral load decrease of 3.8 lg values ​​in the high-dose group.

[0142] Table 8. BA.2 lung viral load in each group after administration of humanized bispecific antibodies (viral load copies / 1 μg RNA)

[0143] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. An isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to a coronavirus spike protein or a fragment thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, characterized in that: (a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising at most three amino acid mutations; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising at most three amino acid mutations; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising at most three amino acid mutations; and / or (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4 or 10 or a variant thereof comprising up to three amino acid mutations; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof comprising up to three amino acid mutations; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof comprising up to three amino acid mutations.

2. The antibody or antigen-binding portion thereof according to claim 1, wherein HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 1, 2, 3, or variants thereof comprising up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 4 or 10, 5, 6, or variants thereof comprising up to three amino acid mutations.

3. The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein The heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7, 13, 14, or 42.

4. The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein The light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 8, 11, or 47.

5. The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein The heavy chain variable region and the light chain variable region comprise amino acid sequences that are at least 80% identical to the amino acid sequences shown in SEQ ID NOs: 7, 13, 14, 42 and 8, 11, 47, respectively.

6. The antibody or antigen-binding portion thereof according to any one of claims 1 to 5, characterized in that It is a full-length antibody, Fab, Fab', (Fab')2, Fd, Fv, dAb, scFv or scFv-scFv.

7. A bispecific antibody or antigen-binding portion thereof, comprising the antibody or antigen-binding portion thereof of any one of claims 1-6 connected to a second antibody or antigen-binding portion thereof, wherein the second antibody or antigen-binding portion thereof has a different binding specificity than the antibody or antigen-binding portion thereof of any one of claims 1-6, and the second antibody or antigen-binding portion thereof is capable of binding to another epitope of the coronavirus spike protein.

8. The bispecific antibody or antigen-binding portion thereof according to claim 7, wherein: The second antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein (a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof comprising at most three amino acid mutations; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising at most three amino acid mutations; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising at most three amino acid mutations; and / or (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20 or a variant thereof comprising up to three amino acid mutations; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21 or a variant thereof comprising up to three amino acid mutations; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22 or a variant thereof comprising up to three amino acid mutations.

9. The bispecific antibody or antigen-binding portion thereof according to claim 8, wherein The heavy chain variable region and light chain variable region of the second antibody or antigen-binding portion thereof comprise an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 23 or 45 and SEQ ID NO: 24 or 46, respectively.

10. The bispecific antibody or antigen-binding portion thereof according to claim 9, wherein: The bispecific antibody comprises two single-chain antibody fragments scFv, wherein the sequence of the heavy chain variable region VH of the scFv is shown in SEQ ID NO. 27 or 14, and the sequence of the light chain variable region VL of the scFv is shown in SEQ ID NO. 28 or 47.

11. The bispecific antibody or antigen-binding portion thereof according to claim 10, wherein: The scFv is respectively linked to the N-terminus or C-terminus of the two heavy chains or two light chains of the second antibody or its antigen-binding portion; preferably, the scFv is respectively linked to the N-terminus of the two light chains of the second antibody or its antigen-binding portion.

12. The antibody or antigen-binding portion thereof according to any one of claims 1 to 11, wherein It comprises a heavy chain constant region and / or a light chain constant region.

13. The antibody or antigen-binding portion thereof according to claim 12, wherein The heavy chain constant region is an IgG, IgA, IgD, IgE, or IgM constant region.

14. The antibody or antigen-binding portion thereof according to claim 12, wherein wherein the heavy chain constant region is an IgG1, IgG2, IgG3 or IgG4 constant region.

15. The antibody or antigen-binding portion thereof according to claim 12, wherein wherein the light chain constant region is a kappa or lambda constant region.

16. The antibody or antigen-binding portion thereof according to any one of claims 1 to 15, wherein wherein the heavy chain constant region optionally contains a mutation.

17. The antibody or antigen-binding portion thereof according to any one of claims 1 to 15, wherein It binds to the spike protein or a fragment thereof of a coronavirus selected from SARS-CoV, SARS-CoV-2 and MERS-CoV.

18. The antibody or antigen-binding portion thereof according to any one of claims 1 to 15, wherein It binds to the receptor binding domain or extracellular domain of the coronavirus's spike protein.

19. The antibody or antigen-binding portion thereof according to any one of claims 1 to 18, wherein It (a) binds to the spike protein or fragments thereof of SARS-CoV-2, (b) inhibits the binding of the spike protein or fragments thereof of SARS-CoV-2 to human ACE2, (c) prevents SARS-CoV-2 from entering host cells, (d) induces complement-mediated inactivation of SARS-CoV-2, (e) induces phagocytosis of SARS-CoV-2, (f) induces complement-dependent cytotoxicity against SARS-CoV-2-infected cells, (g) induces antibody-dependent cellular phagocytosis against SARS-CoV-2-infected cells, (h) induces antibody-dependent cell-mediated viral inhibition against SARS-CoV-2-infected cells, (i) induces antibody-dependent cell-mediated cytotoxicity against SARS-CoV-2-infected cells, (j) induces humoral and cellular antiviral immune responses, and / or (k) blocks trimer formation of the spike protein.

20. The antibody or antigen-binding portion thereof according to any one of claims 1 to 19, wherein It binds to the extracellular domain of the spike protein of SARS-CoV-2 and mediates antibody neutralization.

21. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-20. An expression vector comprising the nucleic acid molecule according to claim 21 . A host cell comprising the expression vector according to claim 22.

24. A composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-20, the nucleic acid molecule of claim 21, the expression vector of claim 22, and / or the host cell of claim 23.

25. A method of treating a disease caused by a coronavirus infection in a subject in need thereof, comprising administering the composition of claim 24 to the subject.

26. The method of claim 25, wherein the disease is caused by SARS-CoV, MERS-CoV, or SARS-CoV-2.

27. The method of claim 26, wherein the disease is caused by SARS-CoV-2.

28. The method of any one of claims 25-27, wherein the subject is a human.

29. A method of preventing a disease caused by a coronavirus infection in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding portion thereof of any one of claims 1-20.

30. The method of claim 29, for preventing a disease caused by SARS-CoV, MERS-CoV or SARS-CoV-2.

31. The method of claim 30, for preventing a disease caused by SARS-CoV-2.

32. The method of any one of claims 29-31, wherein the subject is a human.

33. The method of any one of claims 29-32, wherein the subject is not protected by vaccination.

34. A method of diagnosing a coronavirus infection in a subject comprising contacting the antibody, or antigen binding portion thereof, of any one of claims 1-20 with a tissue sample from the subject.

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