An anti-RBD antibody and its application

Abstract

The present invention provides an anti-RBD antibody and its application. The anti-RBD antibody provided by the present invention can specifically bind to the RBD protein of SARS-CoV-2, effectively inhibiting viral invasion, with excellent binding activity against variant viruses such as Omicron, and has good blocking and neutralizing activity against a wide spectrum of variant viruses.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-RBD antibody and applications 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 bound non-covalently. 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. The amino acid sequence of the S protein. 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 in 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 being the primary immunogen. This protein stimulates the production of antibodies, which bind to the S protein surface, blocking viral recognition and inhibiting conformational changes in the viral protein, thereby suppressing viral invasion.

[0006] Therefore, developing antibodies that inhibit the novel coronavirus S protein from recognizing its host (blocking the binding of RBD to ACE2 protein) can effectively inhibit viral invasion. The present invention provides a new anti-RBD antibody that can effectively inhibit viral invasion, has good virus binding activity, and has good blocking and neutralizing activity against a wide spectrum of mutant viruses. Summary of the Invention

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

[0008] The present invention provides isolated monoclonal human antibodies or antigen-binding portions thereof that 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) preventing or blocking coronaviruses such as SARS-CoV-2 from entering host cells, and / or (ii) inducing 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 that 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 to detect coronaviruses such as SARS-CoV-2 in vitro, and to treat or prevent diseases caused by coronaviruses.

[0010] Therefore, the present invention provides an anti-RBD monoclonal antibody or an antigen-binding portion thereof, which specifically binds to the spike protein or a fragment thereof of a coronavirus, wherein the antibody or the antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein

[0011] (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

[0012] (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 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.

[0013] 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, 5, 6, respectively, or variants thereof comprising up to three amino acid mutations.

[0014] 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, or 16.

[0015] 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 or 14.

[0016] In certain embodiments, the heavy chain variable region and the light chain variable region have amino acid sequences that are at least 80% identical to the amino acid sequences shown in SEQ ID NOs: 7, 13, 16 and 8, 14, respectively.

[0017] In certain embodiments, the monoclonal antibody, or antigen-binding portion thereof, comprises a heavy chain constant region and / or a light chain constant region.

[0018] In certain embodiments, the heavy chain constant region is an IgG, IgA, IgD, IgE, and IgM constant region.

[0019] In certain embodiments, the heavy chain constant region is an IgG1, IgG2 (eg, IgG2a), IgG3, or IgG4 constant region.

[0020] In certain embodiments, an antibody or antigen-binding portion of the invention may be an antibody fragment, such as Fab, F(ab')2, Fv, scFv, or (scFv)2.

[0021] In another aspect, the present invention provides a bispecific molecule, which may comprise an antibody or antigen binding portion thereof of the present invention connected to a second functional portion (e.g., a second antibody or antigen binding portion thereof), the second functional portion having a different binding specificity than the antibody or antigen binding portion thereof of the present invention, such as binding to another epitope of the coronavirus spike protein. The present invention also provides an immunoconjugate, such as an antibody-drug conjugate, which may comprise an antibody or antigen binding portion thereof of the present invention connected to a therapeutic agent such as a cytotoxin. In another aspect, the antibody or antigen binding portion thereof of the present invention can be made into part of a chimeric antigen receptor (CAR). Also provided are immune cells, such as T cells, that may comprise antigen chimeric receptors.

[0022] 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. Also provided is a method for preparing an antibody or antigen-binding portion thereof of the present invention using a host cell, which method can include the following steps: (i) expressing the antibody or antigen-binding portion thereof in a host cell and (ii) isolating the antibody or antigen-binding portion thereof from the host cell or cell culture thereof.

[0023] In another aspect, the present invention provides a composition that 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 some embodiments, the composition may include more than one antibody or antigen-binding portion thereof of the present invention. In some embodiments, the composition may include a vector expressing more than one antibody or antigen-binding portion thereof of the present invention. In some embodiments, the composition may include an immunoconjugate produced with more than one antibody or antigen-binding portion thereof of the present invention. In some embodiments, the composition may include a bispecific molecule with more than one antibody or antigen-binding portion thereof of the present invention. In some embodiments, the composition may include an immune cell with more than one antibody or antigen-binding portion thereof of the present invention in CAR.

[0024] 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 some embodiments, more than one antibody or its antigen-binding portion is administered. In some 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 some embodiments, the coronavirus is SARS-CoV-2.

[0025] 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. The coronavirus can be SARS-CoV, MERS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. In certain embodiments, the subject is a human. The subject may have been exposed to a disease caused by a coronavirus or to a coronavirus (e.g., SARS-CoV-2) but cannot be protected by vaccination.

[0026] Specifically, the present invention includes the following technical solutions:

[0027] 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,

[0028] (a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3,

[0029] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof comprising at most three amino acid mutations; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof comprising at most three amino acid mutations; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof comprising at most three amino acid mutations; and / or

[0030] (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3,

[0031] The LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4 or a variant of the amino acid sequence comprising at most three amino acid mutations; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 or a variant of the amino acid sequence comprising at most three amino acid mutations; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6 or a variant of the amino acid sequence comprising at most three amino acid mutations.

[0032] 2. The antibody or antigen-binding portion thereof according to claim 1, wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, and 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, 5, and 6, respectively, or variants thereof comprising up to three amino acid mutations.

[0033] 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, or 16.

[0034] 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 or 14.

[0035] 5. The antibody or antigen-binding portion thereof according to any one of schemes 1 to 4, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7, 13, 16 and SEQ ID NO: 8, 14, respectively.

[0036] 6. The antibody or antigen-binding portion thereof according to any one of schemes 1 to 5, which comprises a heavy chain constant region and / or a light chain constant region.

[0037] 7. The antibody or antigen-binding portion thereof according to claim 6, wherein the heavy chain constant region is an IgG, IgA, IgD, IgE, and IgM constant region.

[0038] 8. The antibody or antigen-binding portion thereof according to claim 6 or 7, wherein the heavy chain constant region is an IgG1, IgG2, IgG3 or IgG4 constant region.

[0039] 9. The antibody or antigen-binding portion thereof according to claim 6, wherein the heavy chain constant region is a sequence having amino acid mutations as shown in SEQ ID NOs: 15 and 17.

[0040] 10. The antibody or antigen-binding portion thereof according to claim 6, wherein the light chain constant region is a kappa or lambda constant region.

[0041] 11. The antibody or antigen-binding portion thereof according to claim 10, wherein the light chain constant region is a human kappa constant region having the amino acid sequence of SEQ ID NO: 12.

[0042] 12. The antibody or antigen-binding portion thereof according to any one of schemes 1 to 11, which is a human antibody or antigen-binding portion thereof.

[0043] 13. The antibody or antigen-binding portion thereof according to any one of schemes 1 to 12, which is a full-length IgG antibody or a Fab fragment.

[0044] 14. The antibody or antigen-binding portion thereof according to any one of schemes 1 to 13, which binds to the spike protein or a fragment thereof of a coronavirus selected from SARS-CoV, SARS-CoV-2 and MERS-CoV.

[0045] 15. The antibody or antigen-binding portion thereof according to any one of schemes 1-14, which binds to the receptor binding domain or extracellular domain of the spike protein of a coronavirus.

[0046] 16. An antibody or antigen-binding portion thereof according to any one of schemes 1-15, which (a) binds to the spike protein or fragment thereof of SARS-CoV-2, (b) inhibits the binding of the spike protein or fragment 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, and / or (j) induces humoral and cellular antiviral immune responses.

[0047] 17. The antibody or antigen-binding portion thereof according to Scheme 16, which binds to the extracellular domain of the spike protein of SARS-CoV-2 and blocks the binding of the extracellular domain of the spike protein of SARS-CoV-2 to human ACE2.

[0048] 18. The antibody or antigen-binding portion thereof according to Scheme 17, which binds to the receptor binding domain of the spike protein of SARS-CoV-2 and blocks the binding of the SARS-CoV-2 spike receptor binding domain to human ACE2.

[0049] 19. A bispecific molecule, immunoconjugate, or chimeric antigen receptor comprising the antibody or antigen-binding portion thereof of any one of Schemes 1-18.

[0050] 20. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof according to any one of Schemes 1 to 18.

[0051] 21. An expression vector comprising the nucleic acid molecule of Scheme 20.

[0052] 22. A host cell comprising the expression vector of Scheme 21.

[0053] 23. A composition comprising the antibody or antigen-binding portion thereof of any one of Schemes 1-18, the bispecific molecule, immunoconjugate or chimeric antigen receptor of Scheme 19, the nucleic acid molecule of Scheme 20, the expression vector of Scheme 21, and / or the host cell of Scheme 22.

[0054] 24. A method for treating a disease caused by a coronavirus infection in a subject in need thereof, comprising administering the composition of Scheme 23 to the subject.

[0055] 25. The method of claim 24, wherein the disease is caused by SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0056] 26. The method of claim 25, wherein the disease is caused by SARS-CoV-2.

[0057] 27. The method of claim 24-26, wherein the subject is a human.

[0058] 28. A method for 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 Schemes 1-18.

[0059] 29. The method according to claim 28, for preventing a disease caused by SARS-CoV, MERS-CoV or SARS-CoV-2.

[0060] 30. The method according to claim 29, for preventing disease caused by SARS-CoV-2.

[0061] 31. The method of any one of schemes 28-30, wherein the subject is human.

[0062] 32. The method of any one of schemes 28-31, wherein the subject is not protected by vaccination.

[0063] 33. A method of diagnosing a coronavirus infection in a subject, comprising contacting the antibody or antigen-binding portion thereof of any one of schemes 1-18 with a tissue sample from the subject.

[0064] 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, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0065] Definition of terms

[0066] 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.

[0067] 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).

[0068] The term "spike protein" refers to the transmembrane protein used by coronaviruses to invade host cells. The spike protein of SARS-CoV-2 contains a first subunit (S1 subunit) that binds to the host cell receptor and a second subunit (S2 subunit) for fusion of the viral and cell membranes. The S1 subunit contains a receptor binding domain (RBD) that binds to the host cell receptor. For detailed information about the RBD protein and the SARS-CoV-2 spike protein, please refer to Meng Yuan et al., 2020, A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV, Science / j

[0069] 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.

[0070] The term "antigen-binding portion" or "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the 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) Fab fragments, consisting of V L 、V H 、C L and CH1 (ii) F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region; (iii) H and C H1 (iv) an Fd fragment consisting of a V domain of an antibody L and V H (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; (vi) isolated complementarity determining regions (CDRs); and (viii) nanobodies, heavy chain variable regions, comprising one variable domain and two constant domains. L and V H Encoded by different genes, they can be joined using recombinant methods with synthetic linkers to make them into a single protein chain, where V L and V H The domains pair to form monovalent molecules. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0071] The term "Fc region" refers to the tail region of an antibody that interacts with Fc receptors and some proteins of the complement system to activate the immune system. The IgG, IgA, and IgG Fc regions consist of the second and third constant domains (C H2 and C H3 ), while the IgM and IgE Fc regions contain three heavy chain constant domains (C H The Fc region can bind to the complement component C1q to activate the classical complement cascade, bind to Fc receptors on phagocytes (i.e., macrophages, granulocytes, and dendritic cells) to induce phagocytosis of antibody-bound cells, bind to Fc receptors on immune effector cells (primarily natural killer cells) to induce the release of cytotoxic granules from immune effector cells, leading to the death of antibody-coated cells, and bind to Fc receptors on antigen-presenting cells such as dendritic cells to induce humoral and cellular antiviral immune responses.

[0072] The term "chimeric antibody" is an antibody molecule (or antigen-binding fragment 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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 conventional methods known in the art, for example, by using surface plasmon resonance (SPR) technology (e.g., instrument) or biolayer interferometry (e.g., instrument) to measure.

[0077] 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.

[0078] 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.

[0079] 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 vector form.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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%.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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: 11, and / or a light chain constant region, such as a human kappa constant region having the amino acid sequence set forth in, for example, SEQ ID NO: 12. The antibodies of the present invention may also contain other suitable heavy chain constant regions and / or light chain constant regions.

[0094] Table 1. Amino acid or nucleotide sequences of the heavy and light chains of the antibodies of the present invention

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The binding activity of the murine antibody of the present invention to Omicron RBD.

[0105] Figure 2 It is the binding activity of the mouse-derived antibody of the present invention with the RBD of various mutant virus strains.

[0106] Figure 3 This is an experiment on binding of the mouse-derived antibody of the present invention to the RBD of various mutant virus strains.

[0107] Figure 4 The mouse antibody of the present invention single-point blocks the binding of Omicron RBD to hACE2.

[0108] Figure 5 The mouse-derived antibody of the present invention blocks the binding of Omicron RBD to hACE2 at multiple points.

[0109] Figure 6 The mouse-derived antibody of the present invention neutralizes the Omicron pseudovirus at multiple points.

[0110] Figure 7 This is a binding experiment between the humanized antibody of the present invention and Omicron RBD.

[0111] Figure 8 This is a binding experiment between the humanized antibody of the present invention (containing Fc mutation and FR back mutation) and Omicron RBD.

[0112] Figure 9 This is a blocking experiment of the humanized antibody and the humanized antibody (containing Fc mutation and FR back mutation) of the present invention.

[0113] Figure 10 This is a pseudovirus neutralization experiment of the humanized antibody and the humanized antibody (containing Fc mutation and FR reversion mutation) of the present invention.

[0114] Figure 11 This is a blocking experiment of the humanized antibody (containing Fc mutation) of the present invention.

[0115] Figure 12 This is a pseudovirus neutralization experiment of the humanized antibody (containing Fc mutation) of the present invention.

[0116] Figure 13This is a pseudovirus broad-spectrum neutralization experiment of the humanized antibody (containing Fc mutation and FR reversion mutation) of the present invention.

[0117] Figure 14 Figure 2 is the binding and dissociation curve of the humanized antibody of the present invention binding to Omicron RBD.

[0118] Figure 15 : The binding and dissociation curves of the humanized antibody (containing Fc mutation) of the present invention binding to Omicron RBD.

[0119] Figure 16 The binding and dissociation curves of the humanized antibody of the present invention (containing Fc mutations and FR back mutations) binding to Omicron RBD. DETAILED DESCRIPTION

[0120] 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.

[0121] Example 1 Mouse immunization

[0122] Three to five six-week-old female Balb / c mice were intraperitoneally injected with SARS-CoV-2 Spike S1 (Acro: S1N-C52Hu) as the immunogen. Three days prior to immunization, negative serum was collected. For the first immunization, 50 μg of the immunogen, fully emulsified in Freund's complete adjuvant, was injected intraperitoneally. On days 14 and 35, 25 μg of the immunogen, fully emulsified in Freund's incomplete adjuvant, were injected intraperitoneally for the second and third immunizations. Seven days later, tail blood was collected, serum was titered and diluted, and serum titers were determined by ELISA. ELISA plates were coated overnight with SARS-CoV-2 Spike RBD, His Tag (B.1.1.529 / Omicron) (Acro: SPD-C522E). The coating solution was discarded and the plates were blocked with 2% BSA. After blocking, serum diluent was added and the reaction was allowed to proceed. After the reaction, the plates were washed with PBST, and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody was added. The plates were then washed with PBST. TMB colorimetric solution was added for color development, and the reaction was terminated with 1M hydrochloric acid. The optical density was measured at 450 nm. When the titer results met the requirements, the mouse spleen and lymph nodes were harvested and cell fusion was performed.

[0123] Example 2 Hybridoma Cell Fusion

[0124] 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.

[0125] Example 3 Screening of positive clones of the present invention

[0126] 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.

[0127] 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 using 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 monoclonal antibodies of the present invention bind to the RBDs of three viral strains and can be used as candidate molecules.

[0128] Example 4 Production of murine antibodies of the present invention

[0129] After obtaining a stable hybridoma cell line selected according to the present invention, monoclonal antibodies were obtained by 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 the purified murine antibody of the present invention.

[0130] Example 5 Obtaining the variable region sequence of the mouse antibody of the present invention

[0131] The DNA sequences of the variable regions of the murine antibodies of the present invention were 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 sequences were 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 the candidate hybridoma heavy and light chain variable region sequences. The amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0132] Example 6 Binding activity of the murine antibody of the present invention to Omicron RBD

[0133] SARS-CoV-2Spike RBD, His Tag (B.1.1.529 / Omicron) (Manufacturer: Acro, Cat: SPD-C522e, Lot: 5716-21CCF2-11B) was diluted to 1 μg / mL and coated in a 96-well ELISA plate at 100 μL / well at 4°C overnight. Pour out the coating solution, wash the plate with 300 μL per well of 1×PBST, wash 3 times with a plate washer, and pat dry on a dust-free paper. Prepare 3% skim milk powder, 300 μL / well, incubate at 37°C for 1 hour, pour out the blocking solution, wash the plate with 300 μL per well of 1×PBST, wash 3 times with a plate washer, pat dry on a dust-free paper to block the protein. The antibody of the present invention was diluted to 20 μg / mL with 3% skim milk powder, and diluted 3 times with this as the initial concentration, with a total of 11 dilution gradients. Set up another blank well and add only the diluent. Discard the liquid from the wells and wash the plate with 300 μL per well of 1× PBST, wash three times on a microplate washer, and pat dry on a lint-free tissue. Dilute goat anti-mouse IgG Fc (HRP) (abcam, Cat# ab97265) 1:10,000 with 3% nonfat dry milk, add 100 μL per well, and incubate at 37°C for 45 min. Discard the secondary antibody solution and wash the plate six times with 300 μL per well of 1× PBST on a microplate washer, and pat dry on a lint-free tissue. Add 100 μL per well of one-component TMB colorimetric mix (Solarion, Cat# PR1200) and develop at 37°C in the dark for 7 min. Stop the color reaction by adding 100 μL per well of 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water). Read at 450 nm on a microplate reader. The binding curve of the mouse antibody of the present invention to Omicron RBD was obtained, and its EC 50 The value is 0.057 μg / mL, such as Figure 1 shown.

[0134] Example 7 Affinity Detection of the Mouse Antibody of the Present Invention and RBD

[0135] HISIK Biosensors (manufacturer: ForteBio. Inc., Cat: 18-5120) were pre-wetted with PBST buffer for 20 minutes and added to the Omicron RBD (manufacturer: Acro, Cat: SPD-C522e) protein diluted to 20 μg / ml with PBST, so that the target protein loading thickness reached 1.5 nm. The mouse antibody protein of the present invention was diluted to 72.85 μg / mL (final concentration 500 nM) with PBST, diluted 7 times, and added to the above-mentioned sensor. Binding was performed for 120 seconds and dissociation was performed for 120 seconds. Figure 2 As shown in Table 2, the binding and dissociation curves of the murine antibody of the present invention binding to Omicron RBD were obtained and fitted, and its affinity was calculated to be <1.0E-12M.

[0136] Table 2

[0137]

[0138]

[0139] Example 8 Binding experiment of the mouse-derived antibody of the present invention with RBD of various mutant virus strains

[0140] 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. Pour off the coating solution, wash the plate with 300 μL per well of 1×PBST, wash 3 times with a plate washer, and pat dry on a flat sheet of paper. Block with 3% skimmed milk powder, 300 μL / well, incubate at 37°C for 1h20min, pour off the blocking solution, wash the plate with 300 μL per well of 1×PBST, wash 3 times with a plate washer, and pat dry on a dust-free paper. Dilute the mouse antibody of the present invention to 10 μg / mL with 3% skimmed milk powder, 100 μL / well, and incubate at 37°C for 1h. Discard the liquid in the well, wash 3 times with a plate washer, and pat dry on a flat sheet of paper. Dilute Goat pAb to Ms IgG (HRP) (Manufacturer: abcam, Lot: GR3393964-2) at 1:10000 with 3% skim milk powder, 100 μL / well, and incubate at 37°C for 45 minutes. Discard the liquid in the wells, wash the plate with 1×PBST (300 μL per well), wash 6 times with a plate washer, and pat dry on dust-free paper. Add a single-component TMB color development mixture, 100 μL / well, and wrap with aluminum foil, and color at 37°C in the dark for 3 minutes. Add 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water) to terminate the color development reaction, 100 μL / well. Read at 450 nm on an enzyme reader. Figure 3As shown, the mouse antibodies of the present invention can cross-bind to SARS-CoV-2 wild type, Delta, Omicron virus strain BA1, Omicron virus strain BA1.1, and Omicron virus strain BA2.

[0141] Example 9: Single-point blocking of the binding of Omicron RBD to hACE2 by the murine antibody of the present invention

[0142] SARS-CoV-2 Spike RBD, his tag (manufacturer: Acro, Cat: SPD-C522e) (BA.1 / Omicron) protein was diluted to 100 nM (final concentration 50 nM) with diluent (PBS + 2% FBS), and the mouse antibody of the present invention and unrelated antibody (negative control) were diluted to 2000 nM (final concentration 1000 nM). HEK293-human ACE2 cells (manufacturer: Acro) were washed twice, grouped, and 1×10 5 / well, wash twice, centrifuge and remove the supernatant. Add protein and antibody at 50μL / well, total volume 100μL, incubate at room temperature for 1h. After washing the cells, add secondary antibody Anti-6X-his tag antibody-FITC (manufacturer: Abcam, Cat: Ab1206) at 1:500, incubate at room temperature in the dark for 0.5h. Then use flow cytometry to detect FITC fluorescence readings. Figure 4 As shown, the mouse antibodies of the present invention can significantly inhibit the binding of ACE2 cell lines to SARS-CoV-2 Omicron RBD.

[0143] Example 10 The murine antibody of the present invention blocks the binding of Omicron RBD to hACE2 at multiple points

[0144] Dilute SARS-CoV-2Spike RBD, his tag (manufacturer: Acro, Cat: SPD-C522e) (BA.1 / Omikron) to 100nM (final concentration 50nM) with diluent (PBS + 2% FBS), and dilute the positive control antibody Anti-SARS-Cov2RBD antibody (manufacturer: Acro, Cat: SPD-M305), the mouse antibody of the present invention, and the unrelated antibody (negative control) to 2000nM (final concentration 1000nM). Add 100μL of antibody diluent to the first column, take 30μL to 60μL of diluent for 3-fold dilution, and so on, for a total of 5 gradients. Wash HEK293-Hu ACE2 cells twice, group them, and add 1×10 5 / well, wash twice, centrifuge and remove the supernatant. Add protein and antibody at 50μL / well, total volume 100μL, incubate at room temperature for 1h. After washing the cells, add secondary antibody Anti-6X-his tag antibody-FITC (manufacturer: Abcam, Cat: Ab1206) at 1:500, incubate at room temperature in the dark for 0.5h. Then use flow cytometry to detect FITC fluorescence readings. Figure 5 As shown, the mouse antibodies of the present invention can completely inhibit the binding of ACE2 cell line to SARS-CoV-2 Omicron RBD.

[0145] Example 11 Single-site neutralization of the mouse-derived antibody of the present invention with the Omicron pseudovirus

[0146] The mouse antibody of the present invention was 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 redissolved 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 4 100 μL / well of the sample wells were added to the culture plate and incubated in an incubator for 48 hours. The luciferase colorimetric solution and the 96-well white plate were removed and equilibrated to room temperature. 100 μL of culture medium in the sample wells 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 chemiluminescent signal was read using a microplate reader. The results of the single-point neutralization test of the murine antibodies of the present invention against the Omicron pseudovirus are shown in Table 3. The murine antibodies of the present invention exhibited over 99.8% inhibitory activity against the Omicron pseudovirus.

[0147] Table 3

[0148]

[0149] Example 12 Multi-point neutralization of the mouse-derived antibody of the present invention with the Omicron pseudovirus

[0150] The mouse antibody of the present invention and the irrelevant antibody (negative control) were melted at 4°C and diluted with DMEM complete medium to 13.3 μg / mL (final concentration 10 μg / mL). The first column of antibody diluent is 200 μL, and 30 μL is taken to 150 μL of diluent for 6-fold dilution, and so on, for a total of 7 gradients. The diluted antibody was added to a 96-well white plate at 75 μL / well. The pseudovirus SARS-Cov-2 spike (Acro) Fluc-GFP Pseudovirus (manufacturer: Acro, Cat: PSSO-HLGB003) was redissolved 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 / well of the sample 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 and equilibrate them to room temperature in advance. 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. Figure 6 As shown, the mouse-derived antibody of the present invention has significant neutralizing activity against Omicron pseudovirus, IC 50 The value is 0.0032μg / mL.

[0151] Example 13 Neutralization of the Mouse Antibody of the Present Invention with Live Omicron Virus

[0152] According to the operating procedures of the BSL-3 laboratory new coronavirus neutralization test (SOP-MEM / SZ / GF34-V00) of Jiangsu Provincial Center for Disease Control and Prevention and the test plan agreed upon with the test party, the monoclonal antibodies submitted for inspection were tested for anti-new coronavirus (SARS-CoV-2) neutralizing antibody activity. The detection method uses a microneutralization (MN) assay based on cytopathic effect to verify the activity of neutralizing antibodies. After mixing the fixed virus (SARS-CoV-2) with equal amounts of antibodies of different dilutions, Vero-E6 cells grown into a monolayer were infected, and duplicate wells were tested for each antibody dilution. The experiment also set up normal cell controls, virus controls, positive antibody controls and virus titer back titration. 3-5 days after inoculation, the cells were observed for changes in CPE (virus-induced cytopathic effect). The mouse-derived antibody of the present invention was submitted for inspection, with an original concentration of 0.5 mg / mL. After diluting to 50.00 μg / mL with DMEM, the antibody was serially diluted with 1:2 for 12 concentration gradients and mixed with 100 TCID50 SARS-CoV-2 virus (a virus strain isolated from a clinical case of novel coronavirus infection in Jiangsu Province, which is a novel coronavirus variant, Omicron strain, which has been sequenced and identified) to infect the cells. Each dilution concentration was tested in duplicate. The neutralizing activity concentration (IC50 ) was 6.25 μg / mL.

[0153] Example 14 Binding experiment of the humanized antibody of the present invention with Omicron RBD

[0154] 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 chimeric antibody and the humanized antibody of the present invention were diluted to 20 μg / mL with 3% skim milk powder, and 3-fold dilution was performed with this as the initial concentration, with a total of 11 dilution gradients. A blank well was set up and only the diluent was added. 100 μl / well, incubated at 37°C for 1 hour. Discard the liquid in the well, wash the plate with 1×PBST, 300 μL per well, wash 3 times with a plate washer, and pat dry on a dust-free paper. Dilute goat anti-mouse IgG Fc (HRP) (abcam, Cat#ab97265) at 1:10000 with 3% skim milk powder, 100 μL / well, and incubate at 37°C for 45 minutes. Pour out the secondary antibody liquid, wash the plate with 1×PBST, 300 μL per well with a plate washer, and pat dry on a dust-free paper. Add a single-component TMB color development mixture (manufacturer: Solebo, CAT:PR1200), 100 μL / well, and color at 37°C in the dark for 7 minutes. Add 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water) to stop the color reaction, 100 μL / well. Read at 450 nm on a microplate reader. Figure 7 As shown, the chimeric antibody of the present invention and the humanized antibody of the present invention bind to Omicron RBD, and its EC 50 The values were 0.019 and 0.013 μg / mL, respectively.

[0155] Example 15 Binding experiment of the humanized antibody of the present invention (containing Fc mutation and FR back mutation) and Omicron RBD

[0156] 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 in 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 a lint-free tissue. Prepare 3% skim milk powder at 300 μl / well, incubate at 37°C for 1 hour, 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 a lint-free tissue to block the protein. The chimeric antibody of the present invention and the humanized antibody of the present invention (containing Fc mutation and FR reverse mutation) were diluted to 20 μg / mL with 3% skim milk powder, and 3-fold dilution was performed with this as the initial concentration, with a total of 11 dilution gradients. A blank well was set up and only the diluent was added. 100 μL / well, incubated at 37°C for 1 hour. Discard the liquid in the well, wash the plate with 1×PBST 300 μL per well, wash 3 times with a plate washer, and pat dry on a dust-free paper. Dilute goat anti-mouse IgG Fc (HRP) (abcam, Cat#ab97265) at 1:10000 with 3% skim milk powder, 100 μL / well, and incubate at 37°C for 45 minutes. Pour out the secondary antibody liquid, wash the plate with 1×PBST 300 μL per well with a plate washer 6 times, and pat dry on a dust-free paper. Add 100 μL / well of TMB colorimetric mixture (manufacturer: Solebol, CAT: PR1200) and develop at 37°C in the dark for 7 min. Add 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water) to terminate the colorimetric reaction. Read the plate at 450 nm on a microplate reader. Figure 8 As shown, the chimeric antibody of the present invention and the humanized antibody of the present invention (containing Fc mutation and FR back mutation) bind to Omicron RBD, and its EC 50 The values were 0.029 and 0.063 μg / mL, respectively.

[0157] Example 16 Blocking experiments of humanized antibodies and humanized antibodies (containing Fc mutations and FR reversion mutations) of the present invention

[0158] Dilute SARS-CoV-2 Spike RBD, his tag (manufacturer: Acro, Cat: SPD-C522e) to 100 nM (final concentration 50 nM) with diluent (PBS + 2% FBS), and dilute the chimeric antibody of the present invention (positive control), the humanized antibody of the present invention, the humanized antibody of the present invention (containing Fc mutation and FR reverse mutation), and the unrelated antibody (negative control) to 2000 nM (final concentration 1000 nM). Add 100 μL of antibody diluent to the first column, take 30 μL to 60 μL of diluent for 3-fold dilution, and so on, for a total of 5 gradients. Wash HEK293-Hu ACE2 cells twice, group them, and add 1×10 5 / well, wash twice, centrifuge and remove the supernatant. Add protein and antibody at 50μL / well, total volume 100μL, incubate at room temperature for 1h. After washing the cells, add secondary antibody Anti-6X-his tag antibody-FITC (manufacturer: Abcam, Cat: Ab1206) at 1:500, incubate at room temperature in the dark for 0.5h. Then use flow cytometry to detect FITC fluorescence readings. Figure 9 As shown in Figure 2, the humanized antibody of the present invention and the humanized antibody of the present invention (containing Fc mutation and FR reversion mutation) can completely inhibit the binding of ACE2 cell line to SARS-CoV-2 Omicron RBD. Its blocking activity IC 50 The values were 4.449 μg / mL and 4.271 μg / mL respectively.

[0159] Example 17 Pseudovirus Neutralization Experiments with Humanized Antibodies of the Present Invention and Humanized Antibodies of the Present Invention (Containing Fc Mutations and FR Reversion Mutations)

[0160] The mouse antibody of the present invention (positive control), the humanized antibody of the present invention, the humanized antibody of the present invention (containing Fc mutation and FR reverse mutation), and the irrelevant antibody (negative control) were melted at 4°C and diluted with DMEM complete medium to 13.3 μg / mL (final concentration 10 μg / mL). 200 μL of the first column of antibody diluent was used, and 30 μL was taken to 150 μL of diluent for 6-fold dilution, and so on, for a total of 7 gradients. 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 4Add 100 μL / well of the sample 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 and equilibrate them to room temperature in advance. 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. Figure 10 As shown, the humanized antibody of the present invention and the humanized antibody of the present invention (containing Fc mutation and FR reversion mutation) have obvious neutralizing activity against Omicron pseudovirus, IC 50 The values were 0.010 μg / mL and 0.012 μg / mL, respectively.

[0161] Example 18 Blocking experiment of humanized antibodies (containing Fc mutations) of the present invention

[0162] Dilute SARS-CoV-2 Spike RBD, his tag (manufacturer: Acro, Cat: SPD-C522e) to 100 nM (final concentration 50 nM) with diluent (PBS + 2% FBS), and dilute the chimeric antibody of the present invention (positive control) and the humanized antibody of the present invention (containing Fc mutation) to 2000 nM (final concentration 1000 nM). Add 100 μL of antibody diluent to the first column, take 30 μL to 60 μL of diluent and perform 3-fold dilution, and so on, for a total of 5 gradients. Wash HEK293-Hu ACE2 cells twice, group them, and add 1×10 5 / well, wash twice, centrifuge and remove the supernatant. Add protein and antibody at 50μL / well, total volume 100μL, incubate at room temperature for 1h. After washing the cells, add secondary antibody Anti-6X-his tag antibody-FITC (manufacturer: Abcam, Cat: Ab1206) at 1:500, incubate at room temperature in the dark for 0.5h. Then use flow cytometry to detect FITC fluorescence readings. Figure 11 As shown, it can completely inhibit the binding of ACE2 cell line to SARS-CoV-2 Omicron RBD, and its blocking activity IC 50 The value is 0.34nM.

[0163] Example 19 Pseudovirus Neutralization Experiment with Humanized Antibodies (Containing Fc Mutations) of the Present Invention

[0164] The chimeric antibody of the present invention (positive control), the humanized antibody of the present invention, the humanized antibody of the present invention (containing Fc mutation and FR reversion mutation), the humanized antibody of the present invention (containing Fc mutation), and the unrelated antibody (negative control) were melted at 4°C and diluted with DMEM complete medium to 1.33 μg / mL (final concentration 10 nM). 200 μL of the first column of antibody diluent was used, and 30 μL was taken to 150 μL of diluent for 6-fold dilution, and so on, for a total of 7 gradients. 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 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 / well of the sample 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 and equilibrate them to room temperature in advance. 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. Figure 12 As shown, the humanized antibody of the present invention (containing Fc mutation) has obvious neutralizing activity against Omicron pseudovirus, IC 50 The neutralizing activity of the humanized antibody of the present invention, the humanized antibody of the present invention (containing Fc mutation and FR back mutation), and the humanized antibody of the present invention (containing Fc mutation) showed little difference.

[0165] Example 20 Pseudovirus broad-spectrum neutralization experiment of the humanized antibody of the present invention (containing Fc mutation and FR reversion mutation)

[0166] Thaw the humanized antibody of the present invention (containing an Fc mutation and a FR reversion mutation) and an unrelated antibody (negative control) at 4°C and dilute to 1.5 μg / ml (final concentration 10 nM) in complete DMEM medium. For the first column, add 200 μL of the antibody dilution to 150 μL of the dilution, then perform a 6-fold dilution. Repeat this process for a total of 7 dilutions. Add 75 μL / well of the diluted antibody to a 96-well plate. The pseudoviruses SARS-CoV-2 spike (Omicron) 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 (Omicron BA.2) Fluc-GFP Pseudovirus (manufacturer: Acro, Cat: PSSO-HLGB011) were reconstituted 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 / well of the sample 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 and equilibrate them to room temperature in advance. 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. Figure 13 As shown, the humanized antibody of the present invention (containing Fc mutation and FR reversion mutation) has obvious broad-spectrum neutralizing activity against the new coronavirus pseudovirus, and has no significant effect on the IC of delta, BA1, and BA2 strains. 50 The values were 0.17 nM, 0.12 nM and 0.14 nM, respectively.

[0167] Example 21 Affinity Detection of the Humanized Antibody of the Present Invention, the Humanized Antibody of the Present Invention (Containing Fc Mutation), and the Humanized Antibody of the Present Invention (Containing Fc Mutation and FR Reversion Mutation) with Omicron RBD

[0168] HISIK Biosensors (manufacturer: ForteBio. Inc., Cat: 18-5120) were pre-wetted with PBST buffer for 20 minutes and added to the Omicron RBD (manufacturer: Acro, Cat: SPD-C522e) protein diluted to 20 μg / mL with PBST, so that the target protein loading thickness reached 1.5 nm. The humanized antibody of the present invention, the humanized antibody of the present invention (containing Fc mutation), and the humanized antibody of the present invention (containing Fc mutation and FR reversion mutation) protein were diluted to 30 μg / mL (final concentration 200 nM) with PBST, diluted 7 times in a gradient, added to the above-mentioned sensor, bound for 120 seconds, and dissociated for 120 seconds. The results obtained were fitted to the binding and dissociation curves of the humanized antibody of the present invention, the humanized antibody of the present invention (containing Fc mutation), and the humanized antibody of the present invention (containing Fc mutation and FR reversion mutation) binding to Omicron RBD, as shown respectively. Figure 14-16 As shown in Table 4, the calculated affinities of the three antibodies were all <1.0E-12M.

[0169] Table 4

[0170] Test sample <![CDATA[K D (M)]]> <![CDATA[R Max ]]> R^2 Humanized antibodies of the present invention <1.0E-12 1.5335 0.9969 Humanized antibodies of the present invention (containing Fc mutations) <1.0E-12 1.6527 0.9969 Humanized antibodies of the present invention (containing Fc mutations and FR back mutations) <1.0E-12 1.4031 0.9958

[0171] 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 the spike protein of the coronavirus SARS-COV-2, 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 has the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 has the amino acid sequence shown in SEQ ID NO: 2; and the HCDR3 has the amino acid sequence shown in SEQ ID NO: 3; and (b) The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 has the amino acid sequence shown in SEQ ID NO:4; the LCDR2 has the amino acid sequence shown in SEQ ID NO:5; and the LCDR3 has the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody or antigen-binding portion thereof according to claim 1, 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, or 16.

3. The antibody or antigen-binding portion thereof according to claim 1, 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 or 14.

4. The antibody or antigen-binding portion thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOs: 7, 13, 16 and 8, 14, respectively. The antibody or antigen-binding portion thereof according to claim 1 , comprising a heavy chain constant region and / or a light chain constant region.

6. The antibody, or antigen binding portion thereof, of claim 5, wherein the heavy chain constant region is an IgG, IgA, IgD, IgE, or IgM constant region.

7. The antibody, or antigen binding portion thereof, of claim 5 or 6, wherein the heavy chain constant region is an IgG1, IgG2, IgG3 or IgG4 constant region.

8. The antibody or antigen-binding portion thereof according to claim 5, wherein the heavy chain constant region has a sequence containing amino acid mutations as shown in SEQ ID NO:

15.

9. The antibody, or antigen binding portion thereof, of claim 5, wherein the light chain constant region is a kappa or lambda constant region.

10. The antibody, or antigen binding portion thereof, of claim 9, wherein the light chain constant region is a human kappa constant region having the amino acid sequence of SEQ ID NO:

12. The antibody or antigen-binding portion thereof according to claim 10 , which is a human antibody or antigen-binding portion thereof.

12. The antibody or antigen-binding portion thereof according to claim 11, which is a full-length IgG antibody or a Fab fragment.

13. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of claim 1. An expression vector comprising the nucleic acid molecule according to claim 13 . A host cell comprising the expression vector according to claim 14 .

16. A composition comprising the antibody or antigen-binding portion thereof of claim 1, the nucleic acid molecule of claim 13, the expression vector of claim 14, or the host cell of claim 15.

17. Use of the composition according to claim 16 for preparing a medicament for treating a disease caused by infection with the coronavirus SARS-CoV-2.

18. The use according to claim 17, wherein the subject is a human.

19. Use of the antibody or antigen-binding portion thereof according to any one of claims 1 to 12 for the preparation of a medicament for treating a disease caused by infection with the coronavirus SARS-CoV-2.

20. The use according to claim 19, wherein the subject is a human.

21. The use according to claim 20, wherein the subject is not protected by vaccination.

22. Use of the antibody or antigen-binding portion thereof according to any one of claims 1 to 12 for the preparation of a medicament for diagnosing coronavirus SARS-CoV-2 infection in a subject, characterized in that It is contacted with a tissue sample from a subject.

Images