WO2024022438A1 - 一种抗rbd抗体及其用途 - Google Patents

一种抗rbd抗体及其用途 Download PDF

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Publication number
WO2024022438A1
WO2024022438A1 PCT/CN2023/109571 CN2023109571W WO2024022438A1 WO 2024022438 A1 WO2024022438 A1 WO 2024022438A1 CN 2023109571 W CN2023109571 W CN 2023109571W WO 2024022438 A1 WO2024022438 A1 WO 2024022438A1
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antibody
amino acid
antigen
acid sequence
binding portion
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PCT/CN2023/109571
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English (en)
French (fr)
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杨化冰
殷刘松
姜晓玲
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盛禾(中国)生物制药有限公司
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Publication of WO2024022438A1 publication Critical patent/WO2024022438A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an anti-RBD antibody and its use.
  • the new coronavirus has four structural proteins, which mediate the assembly and infection of virus particles.
  • the N protein makes up the nucleocapsid outside the genome, followed by the viral envelope structure composed of membrane protein (M), spike protein (S), and envelope protein (E) on the outer layer.
  • M membrane protein
  • S spike protein
  • E envelope protein
  • the S protein of SARS-CoV-2 is a type of iconic transmembrane protein on the surface of the virus. It is a homotrimer formed by combining three identical subunits through non-covalent bonds. Its relative molecular mass is 141,178 , containing 1273 amino acids.
  • S protein mainly consists of signal peptide, N-terminal domain, receptor binding domain (RBD), fusion peptide, heptad repeat (HR)1, HR2, transmembrane domain, cytoplasmic domain, etc. , with both S1/S2 and S2 cleavage sites.
  • RBD receptor binding domain
  • HR heptad repeat
  • HR1 and HR2 will form a fusion core (6-HB) after the RBD of S1 combines with ACE2 of the target cell, improving the efficiency of virus fusion and infection.
  • the cellular receptor of SARS-CoV-2 is the main active peptide of the renin-angiotensin system. It is currently believed that in SARS-CoV-2 infection, ACE2 is the main receptor for the virus to enter cells.
  • ACE2 as a type I transmembrane protein containing zinc carboxypeptidase, is mostly expressed in the kidney, heart and male reproductive system. It is also expressed in tissues such as lung, small intestine and liver.
  • the spike protein plays a major role in the virus invasion process, and the S protein is the main immunogen. The protein It will stimulate the body to produce a large number of antibodies.
  • S protein trimer formation will be bind to the surface of the S protein, blocking the recognition of the virus and the host, inhibiting the shearing and allosteric transformation of the viral protein, thereby inhibiting virus invasion.
  • the S protein often aggregates in the form of trimers on the virus surface.
  • the S protein is a homotrimer with the C-terminus anchored to the viral membrane.
  • the S protein is the key for coronavirus to enter cells, so it is an attractive antiviral target.
  • Anti-RBD antibodies can prevent S protein trimer formation.
  • the present invention provides an anti-RBD antibody, including a monospecific antibody and a bispecific antibody, which can effectively inhibit virus invasion and interact with the virus. It has good binding activity, long half-life, and good broad-spectrum neutralizing activity against a broad spectrum of mutated viruses.
  • the invention provides isolated monoclonal human antibodies, or antigen-binding portions thereof, including monospecific antibodies and bispecific antibodies, which bind to a spike protein or a fragment thereof, such as an extracellular domain, or more specifically, a receptor of the spike protein.
  • a coronavirus such as SARS-CoV-2 at the body binding domain
  • a coronavirus such as SARS-CoV-2 at the body binding domain
  • prevents or blocks the entry of a coronavirus such as SARS-CoV-2 into a host cell and/or
  • induces complement-mediated inhibition of a coronavirus such as SARS-CoV-2 SARS-CoV-2 inactivation and/or phagocytosis.
  • 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 cellular 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.
  • the immune complexes can be recognized by antigen-presenting cells such as dendritic cells and induce body fluids. and cellular antiviral immune responses.
  • 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.
  • the present invention provides an isolated monoclonal antibody or an antigen-binding portion thereof that specifically binds to the spike protein of coronavirus or a fragment thereof, said antibody or an antigen-binding portion thereof comprising a heavy chain variable region and a light chain.
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1 or a variant of the shown amino acid sequence including up to three amino acid mutations;
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:2 or a variant of the amino acid sequence including at most three amino acid mutations;
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:3 The sequence or the amino acid sequence shown contains variants containing up to three amino acid mutations; and/or
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 4 or 10 or a variant of the amino acid sequence shown including at most three amino acid mutations; LCDR2 includes the amino acid sequence shown in SEQ ID NO:5 or the amino acid sequence shown includes variants of up to three amino acid mutations; the LCDR3 includes the amino acid sequence shown in SEQ ID NO:6 or the amino acid sequence shown includes up to three amino acid mutations. Amino acid mutation variants.
  • HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences set forth in SEQ ID NO: 1, 2, 3 or variants of the set forth amino acid sequences respectively comprising up to three amino acid mutations; and LCDR1, LCDR2 and LCDR3 Contains the amino acid sequence shown in SEQ ID NO: 4 or 10, 5, and 6 respectively or the amino acid sequence shown contains variants of up to three amino acid mutations.
  • the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 7, 13, 14, 42.
  • the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 8, 11, 47.
  • the heavy chain variable region and the light chain variable region comprise amino acid sequences corresponding to SEQ ID NO: 7, 13, 14, 42 and SEQ ID NO: 8, 11, 47, respectively. Amino acid sequences with at least 80% identity.
  • it is a full length antibody, Fab, Fab', (Fab')2, Fd, Fv, dAb, scFv or scFv-scFv.
  • it comprises an antibody or an antigen-binding portion thereof linked to a second antibody or an antigen-binding portion thereof, wherein the second antibody or an antigen-binding portion thereof has an antibody or an antigen-binding portion thereof linked to any of the foregoing.
  • Different binding specificities of the antigen-binding portion, the second antibody or its antigen-binding portion is capable of binding to another epitope of the coronavirus spike protein.
  • the second antibody, or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 17 or a variant of the amino acid sequence shown including at most three amino acid mutations; the HCDR2 includes The amino acid sequence shown in SEQ ID NO: 18 or a variant of the shown amino acid sequence comprising at most three amino acid mutations; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19 or The amino acid sequence shown contains variants containing up to three amino acid mutations; and/or
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 20 or 45 or a variant of the amino acid sequence shown including at most three amino acid mutations; LCDR2 includes the amino acid sequence shown in SEQ ID NO:21 or the amino acid sequence shown includes variants of up to three amino acid mutations; the LCDR3 includes the amino acid sequence shown in SEQ ID NO:22 or the amino acid sequence shown includes up to three amino acid mutations. Amino acid mutation variants.
  • the heavy chain variable region and the light chain variable region of the second antibody or antigen-binding portion thereof comprise SEQ ID NO: 23 or 45 and SEQ ID NO: 24 or 46, respectively.
  • Amino acid sequence An amino acid sequence having at least 80% identity.
  • the bispecific antibody comprises two single chain antibody fragments scFv, wherein the sequence of the heavy chain variable region VH of the scFv is set forth in SEQ ID NO: 27 or 14, and the scFv's The sequence of the light chain variable region VL is shown in SEQ ID NO: 28 or 47.
  • the scFv is linked to the N-terminus or C-terminus of two heavy chains or two light chains of the second antibody or its antigen-binding portion, respectively. In certain embodiments, the scFv is linked to the N-terminus of two light chains of the second antibody or antigen-binding portion thereof, respectively.
  • the heavy chain constant region is an IgG, IgA, IgD, IgE, and IgM constant region.
  • the heavy chain constant region is an IgGl, IgG2, IgG3 or IgG4 constant region.
  • the light chain constant region is a kappa or lambda constant region.
  • the heavy chain constant region optionally contains mutations.
  • the heavy chain constant region may contain mutations, such as the M252Y/S254T/T256E (YTE) mutation.
  • it binds to the spike protein of a coronavirus selected from the group consisting of SARS-CoV, SARS-CoV-2, and MERS-CoV, or a fragment thereof.
  • it binds to the receptor binding domain or extracellular domain of the coronavirus' spike protein. In certain embodiments, it (a) binds to the spike protein of SARS-CoV-2 or a fragment thereof, (b) inhibits binding of the spike protein of SARS-CoV-2 or a fragment thereof to human ACE2, (c) Prevent SARS-CoV-2 from entering host cells, (d) induce complement-mediated inactivation of SARS-CoV-2, (e) induce phagocytosis of SARS-CoV-2, (f) induce response to SARS-CoV-2 infection Complement-dependent cytotoxicity of cells, (g) induction of antibody-dependent cellular phagocytosis against SARS-CoV-2-infected cells, (h) induction of infection against SARS-CoV-2 antibody-dependent cell-mediated viral inhibition of infected cells, (i) inducing antibody-dependent cell-mediated cytotoxicity against SARS-CoV-2-infected cells, (j) inducing humoral and
  • it binds to the extracellular domain of the spike protein of SARS-CoV-2 and mediates antibody neutralization.
  • the invention also provides an immunoconjugate, such as an antibody-drug conjugate, which may comprise an antibody of the invention or an antigen-binding portion thereof linked to a therapeutic agent, such as a cytotoxin.
  • an immunoconjugate such as an antibody-drug conjugate, which may comprise an antibody of the invention or an antigen-binding portion thereof linked to a therapeutic agent, such as a cytotoxin.
  • the antibodies of the invention can be made part of a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the invention also provides immune cells, such as T cells, that may contain chimeric receptors for antigens.
  • the invention provides a nucleic acid molecule encoding an antibody of the invention or an antigen-binding portion thereof, as well as expression vectors that may contain such nucleic acid molecules and host cells that may contain the expression vectors.
  • the invention provides a method for preparing an antibody or an antigen-binding portion thereof of the invention using a host cell.
  • the method may comprise the following steps: (i) expressing the antibody or an antigen-binding portion thereof in the host cell and ( ii) Isolating the antibody or antigen-binding portion thereof from the host cell or cell culture thereof.
  • the invention provides a composition, which may comprise the antibody of the invention or an antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, an immune cell with a CAR or an expression vector, and a pharmaceutical acceptable carrier.
  • a composition may comprise more than one antibody of the invention, or antigen-binding portion thereof.
  • a composition may comprise a vector expressing more than one antibody of the invention, or antigen-binding portion thereof.
  • compositions may comprise immunoconjugates generated with more than one antibody of the invention, or antigen-binding portion thereof.
  • a composition may comprise a bispecific molecule having more than one antibody of the invention, or antigen-binding portion thereof.
  • compositions may comprise immune cells having more than one antibody of the invention, or antigen-binding portion thereof, in a CAR.
  • the invention provides a method of 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 invention or an antigen-binding portion thereof. In certain embodiments, more than one antibody or antigen-binding portion thereof is administered. In certain embodiments, the method may include administering a bispecific molecule, immunoconjugate, or CAR-containing molecule of the invention. Immune Cells.
  • the coronavirus can be SARS-CoV, MERS-CoV or SARS-CoV-2. In certain embodiments, the coronavirus is SARS-CoV-2.
  • the invention provides a method of preventing disease caused by coronavirus infection in a subject in need thereof, comprising administering to the subject an antibody of the invention or an antigen-binding portion thereof.
  • the coronavirus can be SARS-CoV, MERS-CoV, or SARS-CoV-2.
  • the coronavirus is SARS-CoV-2.
  • the subject is human.
  • a subject may be exposed to a disease caused by a coronavirus or to a coronavirus (eg, SARS-CoV-2) but not be protected by vaccination.
  • SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2, which belongs to the family Coronaviridae and the genus Betacoronavirus (including SARS-CoV and MERS-CoV).
  • spike protein is the transmembrane protein used by coronaviruses to invade host cells.
  • the spike protein of SARS-CoV-2 contains the first subunit (S1 subunit) that binds to host cell receptors and the second subunit (S2 subunit) that is used for fusion of the virus and the cell membrane.
  • S1 subunit first subunit
  • S2 subunit second subunit
  • RBD receptor binding domain
  • antibody includes immunoglobulin molecules including four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (eg, IgM).
  • Each L chain is connected to the H chain by a covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each heavy chain has a variable region at the N-terminus, followed by a constant region.
  • Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. This heavy chain constant region contains three regions (domains), CH1, CH2 and CH3.
  • Each light chain contains a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region contains a region (domain, CL1).
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conservative regions, called framework regions (FR, also known as skeleton regions, framework regions).
  • CDRs complementarity-determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Antibodies can be of different subclasses.
  • full-length antibody refers to an antibody in its substantially complete form as compared to antibody fragments. Specifically, full-length antibodies include 4-chain antibodies having a heavy chain and a light chain including an Fc region.
  • antigen-binding portion refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, SARS-CoV-2 spike protein). It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • binding fragments included within the term "antigen-binding portion" of an antibody include, but are not limited to: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) Fab' and F( ab')2 fragment, where F(ab')2 is a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; ( iv) Fv fragment consisting of the VL and VH domains of one arm of the antibody, (v) dAb fragment (Ward et al. (1989) Nature 341:544-546), which consists of the VH domain; (vi) scFv; (viii)scFv-scFv.
  • Fc region is 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 two identical fragments from the second and third constant domains (CH2 and CH3) of the antibody heavy chain, whereas the IgM and IgE Fc regions contain three heavy chain constant domains (CH domain 2- 4).
  • the Fc region can bind to complement component C1q to activate the classic complement cascade, and can bind to Fc receptors on phagocytes (i.e., macrophages, granulocytes, and dendritic cells) to induce phagocytosis of cells bound by the antibody.
  • immune effector cells mainly natural killer cells
  • antigen-presenting cells such as dendritic cells Binds to induce humoral and cellular antiviral immune responses.
  • chimeric antibody is an antibody molecule (or an antigen-binding portion thereof) in which (1) the constant region or portion thereof is altered, substituted, or replaced such that the antigen-binding site (variable region) is different from or Changing the type, effector function and/or type of constant region linkage, or linking to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confer new properties to the chimeric antibody; or (2) the The variable region or part thereof is altered, substituted or replaced with a variable region having different or altered antigen specificity.
  • a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. due to constant location
  • the chimeric antibody can retain its specificity for recognizing an antigen while having reduced antigenicity in humans compared to the original mouse antibody.
  • humanized antibody refers to a chimeric antibody containing amino acid residues derived from human antibody sequences.
  • Humanized antibodies 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. It can overcome the heterologous reaction induced by chimeric antibodies carrying a large number of heterologous protein components.
  • 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 activity caused by a decrease in immunogenicity, a minimum of reverse mutation or back mutation can be performed on the human antibody variable region framework sequence to maintain activity.
  • monoclonal antibody refers to a homogeneous antibody that targets only a specific antigenic epitope. In contrast to common polyclonal antibody preparations, which 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 uniform character of the antibody and is not to be construed as requiring that the antibody be produced by any particular method.
  • Monoclonal antibodies of the invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.
  • 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 other antibodies that specifically bind to the SARS-CoV-2 spike protein). Antibodies to antigens other than the SARS-CoV-2 spike protein). However, isolated antibodies that specifically bind the SARS-CoV-2 spike protein may have cross-reactivity with other antigens, for example, spike proteins from other viruses such as coronaviruses. Furthermore, isolated antibodies may be substantially free of other cellular material and/or chemicals.
  • bispecific antibody refers to a protein molecule capable of specifically binding to two target antigens or target antigen epitopes.
  • scFv refers to a molecule comprising an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) joined by a linker.
  • VH antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • 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 repeats of the GGGGS amino acid sequence or variants thereof, for example using 1 to 6 repeats of the GGGGS amino acid sequence or variants thereof.
  • affinity refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (eg, the antigen-binding module of a MIAC) and its binding partner (eg, an 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.
  • binding affinity refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • Kd dissociation constant
  • Affinity can be measured by common methods known in the art, such as by using surface plasmon resonance (SPR) techniques (eg, Instruments) or biolayer interferometry (eg, Instruments).
  • CAR chimeric antigen receptor
  • the term "chimeric antigen receptor” or “CAR” refers to an engineered receptor that grafts a defined specificity onto immune effector cells, usually T cells, and enhances T cell function.
  • the new generation of CAR includes an extracellular binding domain, including an scFv, a hinge region, a transmembrane domain and an intracellular signaling domain (mainly the cytoplasmic domain of CD3-zeta, which is responsible for the T cell activation signal primary transmitter), plus one or more co-stimulatory domains).
  • CARs may further possess factors that enhance T cell expansion, persistence, and antitumor activity, such as cytokines and costimulatory ligands.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules may 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.
  • vector refers to any molecule (eg, nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide-encoded information into a host cell.
  • vector covers all types of vectors, regardless of their function.
  • Vectors capable of directing the expression of an expressible nucleic acid to which they are operably linked are generally referred to as "expression vectors.”
  • expression vectors In this specification, “plasmid” and “vector” are used interchangeably, since plasmids are the most commonly used form of vectors.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest.
  • the term includes the offspring of a parent cell, regardless of whether the offspring is identical in morphology or genetic composition to the original parent cell, as long as the selected gene of interest is present in the offspring.
  • Commonly used host cells include bacteria, yeast, mammalian cells, etc.
  • treating means suppressing, eliminating, reducing and/or ameliorating symptoms, symptom severity and/or symptom frequency associated with the disease or condition being treated, such as a disease caused by coronavirus.
  • prevention refers to actions taken to prevent a specific disease, such as that caused by coronavirus. Prevention may include administration of antibodies to subjects exposed to the disease or disease-causing virus and not protected by vaccination.
  • administering means delivering or causing delivery of a therapeutic or pharmaceutical composition by the methods described herein to the behavior of the subject's body or as known in the art.
  • Administration of 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, jelly, powders or patches; oral dosage forms; inhalation powders, sprays, suspensions and rectal suppositories.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of onset of a condition, including biochemical, histological, and biological aspects of the condition, its complications, and intermediate pathological phenotypes present during the development of the condition. academic and/or behavioral symptoms.
  • beneficial or desirable results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various disorders associated with the target antigen of the invention, reducing the dosage of other agents required to treat the disorder. , enhance the efficacy of another agent, and/or delay the progression of a disorder associated with the target antigen of the invention in a patient.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic and sustained release agents, and the like that are compatible with drug administration. Including various excipients, diluents and buffers, etc., these substances are suitable for administration to humans and/or animals without excessive adverse side effects, and are suitable for maintaining the activity of the drugs or active agents located therein.
  • suitable vectors are described in the standard reference documents in Remington’s Pharmaceutical Sciences, latest edition, which is incorporated herein by reference in its entirety.
  • 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 can also be used.
  • the use of media and agents for pharmaceutically active substances is well known in the art. Except those conventional media or agents which are incompatible with the active ingredient, their use in the composition will achieve the desired effect.
  • identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a control polypeptide sequence after aligning the sequences and introducing gaps where necessary to obtain maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software, such as BLAST software or the FASTA package.
  • the term "at least 80% identity” means that the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the control polypeptide sequence is more than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
  • complement-dependent cytotoxicity refers to an antibody-mediated immune mechanism in which antibodies bind to the complement component C1q and activate the classical complement cascade, resulting in the formation of the membrane attack complex (MAC) on the cell surface and Subsequent cell lysis.
  • MAC membrane attack complex
  • antibody-dependent cellular phagocytosis 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 Induces phagocytosis of cells bound by the antibody.
  • phagocytes i.e., macrophages, granulocytes, and dendritic cells
  • antibody-dependent cell-mediated cytotoxicity refers to a cell-mediated immune mechanism in which the Fc portion of an antibody binds to the Fc receptor of immune effector cells (primarily natural killer cells), resulting in an immune effect The cells release cytotoxic granules, causing the death of the antibody-coated cells.
  • the antibody or antigen-binding portion thereof of the present invention specifically binds to the spike protein of a coronavirus such as SARS-CoV-2 or a fragment thereof, more specifically binds to the receptor binding domain of the spike protein, and inhibits binding of the spike protein to its Entry receptor ACE2.
  • Antibodies of the present disclosure, or antigen-binding portions thereof may also bind to spike proteins or fragments thereof, such as receptor binding domains, displayed on virus-infected host cells.
  • 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) targeting of coronavirus-infected host cells.
  • Complement-dependent cytotoxicity CDC
  • ADCP antibody-dependent cellular phagocytosis
  • ADCVI antibody-dependent cell-mediated viral inhibition against coronavirus-infected host cells
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • humoral and cellular antiviral immune responses e.g., CDC, iv) antibody-dependent cellular phagocytosis (ADCP) against infected host cells, (v) antibody-dependent cell-mediated viral inhibition against coronavirus-infected host cells ( ADCVI), (vi) antibody-dependent cell-mediated cytotoxicity (ADCC) against coronavirus-infected host cells, and/or (vii) humoral and cellular antivir
  • 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 heavy/light chain variable region sequences by other systems such as Chothia, AbM or Contact numbering systems/methods.
  • Antibodies of the invention may contain a heavy chain constant region, such as a human IgGl heavy chain constant region having the amino acid sequence set forth in, for example, SEQ ID NO: 9, or a variant thereof, and/or having, for example, SEQ ID NO: 12 The light chain constant region of the listed amino acid sequence or a variant thereof.
  • the antibodies of the invention may also contain other suitable heavy chain constant regions and/or light chain constant regions, or variants thereof.
  • Figure 1 shows the Omicron RBD binding activity of mouse-derived antibodies.
  • Figure 2 shows the viral RBD binding activities of multiple mutant strains of murine antibodies.
  • Figure 3 shows the Omicron pseudovirus neutralizing activity of mouse-derived antibodies.
  • Figure 4 shows the Omicron virus-binding activity of humanized antibody 1.
  • Figure 5 shows the Omicron virus-binding activity of humanized antibody 2.
  • Figure 6 shows the Omicron virus-binding activity of humanized antibody 3.
  • Figure 7 shows the pseudovirus neutralizing activities of mouse antibodies, chimeric antibodies, humanized antibody 1, humanized antibody 2, and humanized antibody 3.
  • Figure 8 is a schematic diagram of the structure of antibody a.
  • Figure 9 is a schematic diagram of the structure of antibody b.
  • Figure 10 is a schematic diagram of the structure of antibody c.
  • Figure 11 is a schematic diagram of the structure of antibody d.
  • Figure 12 shows the Omicron virus-binding activities of antibodies a1, b1, and c1.
  • Figure 13 shows the Omicron virus-binding activities of antibodies d1, a2, and b2.
  • Figure 14 shows the Omicron virus-binding activity of antibodies c2 and d2.
  • Figure 15 shows the Omicron pseudovirus neutralizing activity of antibodies a1, a2, b1, and b2.
  • Figure 16 shows the Omicron pseudovirus neutralizing activity of antibodies c1, c2, d1, and d2.
  • Figure 17 shows the pseudovirus broad-spectrum neutralizing activity of antibody d2.
  • Figure 18 shows the neutralizing activity of humanized bispecific antibodies against Delta and Omicron BA.2 pseudoviruses.
  • Figure 19 shows the neutralizing activity of batch 1 and batch 2 of humanized bispecific antibodies against Omicron BA.1 pseudovirus.
  • Figure 20 shows the neutralizing activity of batch 1 and batch 2 of humanized bispecific antibodies against Omicron BA.4 & BA.5 pseudoviruses.
  • Figure 21(a)-(f) show the responses of humanized bispecific antibodies to L452R, T478K/Delta, B.1.1.529/Omicron, BA.1.1/Omicron, and BA.2/Omicron respectively. Micron, BA.4&BA.5/Omicron, BF7&BA.4.6/ Omicron protein competitive activity fitting curve.
  • Figure 22 is the BA.2 lung viral load curve of each group after administration of humanized bispecific antibodies.
  • the serum titer was diluted, and the serum titer was detected by ELISA.
  • mice were sacrificed and the spleen and lymph nodes were removed, and the cells were ground through a cell mesh to obtain lymphocytes. Calculate the total number of cells, mix the obtained lymphocytes and myeloma SP2/0 cells at a ratio of 2:1, centrifuge the cell suspension at 1000 rpm for 8 minutes, and wash the cells twice with electrofusion solution. Resuspend the cells at a total cell density of 1 ⁇ 10 7 /mL, place the cell suspension in a fusion pool, and perform cell fusion according to conventional electroporation methods. The fusion was cultured in DMEM complete medium containing feeder cells and HAT, 5% CO 2 , and 37°C. The feeder cells used in fusion selection culture are obtained from macrophages in the peritoneal cavity of unimmunized animals to assist the growth of new hybrid B lymphocyte hybridomas.
  • OD450>1.0 negative control ⁇ 0.1, it is a positive clone. Blow up the positive clone wells to count the cells, and spread 1 cell/well into a 96-well culture plate in 5% CO 2 and 37°C for 7-10 days. After 7-10 days, the supernatant is taken for ELISA detection.
  • the single cell of the present invention Antibodies can bind to RBD of three virus strains and can be used as candidate molecules.
  • the monoclonal antibody is obtained by using an in vitro culture method. Expand the cell line in a T75 culture flask and culture until the cell coverage rate is 40-50%. Discard the cell supernatant, add 50 mL of hybridoma-SFM medium, and culture at 37°C and 5% CO2 . Cultivate for 6-7 days, until the cell survival rate is lower than 20%, collect the culture supernatant after low-speed centrifugation, and purify using a ProteinA affinity chromatography column to obtain purified mouse antibodies.
  • the DNA sequence of the variable region of the mouse antibody was determined using a degenerate primer-based PCR method.
  • the hybridoma cell lines were expanded and cultured separately, the cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol.
  • PCR amplifies the corresponding variable region DNA sequence using the first strand cDNA as a subsequent template.
  • the PCR product is recovered for TA cloning and sequencing to obtain the candidate hybridoma heavy chain variable region and
  • the light chain variable region sequence, the heavy chain variable region sequence includes the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region sequence includes the amino acid sequence shown in SEQ ID NO:8.
  • Put SARS-CoV-2 Spike RBD, His Tag (B.1.1.529/Omicron) (Manufacturer: Acro, Cat: SPD-C522e, Lot: 5716-21CCF2-11B) was diluted to 1 ⁇ g/mL, coated into a 96-well enzyme plate, 100 ⁇ L/well, and incubated at 4°C overnight. Pour off the coating solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 3 times with a plate washer, and pat dry on dust-free paper. Prepare 3% skimmed milk powder, 300 ⁇ L/well, and incubate at 37°C for 1 hour.
  • the mouse antibody and the positive control antibody were diluted to 20 ⁇ g/mL with 3% skimmed milk powder respectively. This was used as the initial concentration for 3-fold dilution. A total of 11 gradients were diluted. Another blank well was set and only the diluent was added. 100 ⁇ L/well, incubate at 37°C for 1 hour. Discard the liquid in the wells, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 3 times with a plate washer, and pat dry on dust-free paper.
  • Goat anti-mouse IgG Fc HRP
  • abcam Cat#ab97265
  • 3% skimmed milk powder 100 ⁇ L/well, and incubated at 37°C for 45 min.
  • Pour off the secondary antibody liquid wash the plate 6 times with 300 ⁇ L of 1 ⁇ PBST per well, and pat dry on dust-free paper.
  • Add one-component TMB color development mixture (manufacturer: Solebao, CAT: PR1200), 100 ⁇ L/well, and develop color for 7 minutes at 37°C in the dark.
  • stop solution 1M HCl 80mL 37% concentrated hydrochloric acid + 917mL pure water
  • mouse antibody Heavy chain variable region sequence is shown in SEQ ID NO:15, light chain variable region sequence is shown in SEQ ID NO:16) and Omicron RBD, as shown in the figure
  • the EC 50 values are 0.95 and 3.60 ⁇ g/mL respectively. It can be seen that the effect of mouse-derived antibodies is better than that of positive control antibodies.
  • the positive control antibody (heavy chain variable region sequence is shown in SEQ ID NO: 15, light chain variable region sequence is shown in SEQ ID NO: 16) and mouse antibody were diluted to 10 ⁇ g/mL with 3% skim milk powder. , 100 ⁇ L/well, incubate at 37°C for 1 hour. Discard the liquid in the wells, wash 3 times with a plate washer, and pat dry on flat paper.
  • both mouse-derived antibodies and positive control antibodies can cross-bind with SARS-CoV-2 wild type, Delta, Omicron virus strain BA.1, and Omicron virus strain BA.2.
  • the binding activity against Omicron virus strain BA.1.1 is slightly weak.
  • the diluted virus was added to a 96-well white plate at 25 ⁇ L/well, and incubated with the antibody diluent at room temperature for 1 hour.
  • HEK293-ACE2 hu cells were added to the culture plate at 5 ⁇ 10 4 cells/100 ⁇ L/well, and cultured in the incubator for 48 hours.
  • Table 2 The results of the single-point neutralization test between mouse antibodies and Omicron pseudovirus are shown in Table 2.
  • the mouse antibody has an inhibitory activity of more than 99.81% against Omicron pseudovirus, which is equivalent to the positive control antibody.
  • the mouse antibody, positive control antibody, and irrelevant antibody were melted at 4°C, and diluted with DMEM complete medium to 13.3 ⁇ g/ml (final concentration 10 ⁇ g/mL).
  • the first column of antibody diluent is 200 ⁇ L. Take 30 ⁇ L to 150 ⁇ L diluent and dilute it 6 times, and so on, for a total of 7 gradients.
  • the diluted virus was added to a 96-well white plate at 25 ⁇ L/well, and incubated with the antibody diluent at room temperature for 1 hour.
  • HEK293-ACE2hu cells were added to the culture plate at 5 ⁇ 10 4 cells/100 ⁇ L/well, and cultured in the incubator for 48 h.
  • mouse-derived antibodies and positive control antibodies have obvious neutralizing activity against Omicron pseudovirus, with IC 50 values of 0.0021 ⁇ g/mL and 0.0097 ⁇ g/mL respectively.
  • the activity of mouse-derived antibodies is higher than that of positive control antibodies.
  • Dilute SARS-CoV-2 Spike RBD, His tag. (Manufacturer: Acro, Cat: SPD-c522e Lot: 5716-21CCF2-Z4) to 0.8 ⁇ g/mL, and coat it into a 96-well enzyme plate, 100 ⁇ L/well , 4°C overnight. Pour off the coating solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 3 times with a plate washer, and pat dry on flat paper. Block with 3% skimmed milk powder, 300 ⁇ L/well, and incubate at 37°C for 1 hour and 20 minutes. Pour off the blocking solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 3 times with a plate washer, and pat dry on dust-free paper.
  • chimeric antibodies were used as positive control antibodies. Dilute the chimeric antibody, humanized antibody 1, humanized antibody 2, and humanized antibody 3 with 3% skim milk powder to 10 ⁇ g/mL, dilute 11 gradients of 3 times, 100 ⁇ L/well, and incubate at 37°C for 1 hour. Discard the liquid in the wells, wash 3 times with a plate washer, and pat dry on flat paper. Use 3% skimmed milk powder to dilute Goat anti-human IgG (HRP) (manufacturer: Jackson, Lot: 109-035-098) at 1:10000, 100 ⁇ L/well, and incubate at 37°C for 45 minutes.
  • HRP Goat anti-human IgG
  • Example 11 Pseudovirus neutralization experiments of mouse antibodies, chimeric antibodies, humanized antibodies 1, humanized antibodies 2, and humanized antibodies 3
  • the mouse antibody, chimeric antibody, humanized antibody 1, humanized antibody 2, humanized antibody 3, and irrelevant antibody at 4°C and dilute it with DMEM complete medium to 13.3 ⁇ g/mL (final concentration 10 nM) .
  • the first column of antibody diluent is 200 ⁇ L, and 30 ⁇ L is added to 150 ⁇ L diluent for 6-fold dilution, and so on, for a total of 7 gradients.
  • the diluted antibody was added to the 96-well white plate at 75 ⁇ L/well.
  • humanized antibody 1 humanized antibody 2, and humanized antibody 3 have obvious neutralizing activity against Omicron pseudovirus, with IC 50 values of 0.023nM, 0.022nM, and 0.021nM respectively.
  • bispecific antibodies were constructed based on mouse-derived antibodies and the mouse-derived antibodies in patent CN202210892796.0, which are antibodies a1, a2, b1, b2, c1, c2, d1, and d2, among which a1, b1, c1, and d1 are The Fc region does not contain mutations, and a2, b2, c2, and d2 are antibody structures that contain mutations in the Fc region.
  • Exogenous expression becomes a bispecific antibody suspension, in which antibodies a1 and a2 have the same structure (referred to as antibody a structure), b1 and b2 have the same structure (referred to as antibody b structure), and c1 and c2 have the same structure (referred to as antibody c structure). ), the structures of d1 and d2 are the same (referred to as antibody d structure), and the structures of antibodies a-d are shown in Figure 8-11 respectively.
  • Dilute SARS-CoV-2 Spike RBD, His tag. (Manufacturer: Acro, Cat: SPD-c522e Lot: 5716-21CCF2-Z4) to 0.8 ⁇ g/mL, and coat it into a 96-well enzyme plate, 100 ⁇ L/well , 4°C overnight. Pour off the coating solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 3 times with a plate washer, and pat dry on flat paper. Block with 3% skimmed milk powder, 300 ⁇ L/well, and incubate at 37°C for 1 hour and 20 minutes. Pour off the blocking solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 3 times with a plate washer, and pat dry on dust-free paper.
  • the humanized antibody in the patent CN202210892796.0 (the full-length heavy chain sequence is shown in SEQ ID NO: 40, the full-length light chain sequence is shown in SEQ ID NO: 41) is used as a positive control antibody, and the positive control antibody, Antibodies a1, a2, b1, b2, c1, c2, d1, and d2 were diluted with 3% skimmed milk powder to 5 ⁇ g/mL (final concentration 2.5 ⁇ g/mL), 3-fold dilution in 11 gradients, 100 ⁇ L/well, and incubated at 37°C. 1h. Discard the liquid in the wells, wash 3 times with a plate washer, and pat dry on flat paper.
  • HRP Goat anti-human IgG
  • 3% skimmed milk powder at 1:10000, 100 ⁇ L/well, and incubate at 37°C for 45 min. Discard the liquid in the wells and wash each well with 1 ⁇ PBST. 300 ⁇ L, washed 6 times with a plate washer, and patted dry on dust-free paper. Add one-component TMB color development mixture, 100 ⁇ L/well, wrap it in aluminum foil, and develop color for 3 minutes at 37°C in the dark. Add stop solution 1M HCl (83 mL 37% concentrated hydrochloric acid + 917 mL pure water) to stop the color development reaction, 100 ⁇ L/well.
  • HRP Goat anti-human IgG
  • Antibodies a1, b1, b2, c1, c2, d1, and d2 are all more active than the positive control antibody. .
  • the humanized antibody in the patent CN202210892796.0 (the full-length heavy chain sequence is shown in SEQ ID NO: 40, the full-length light chain sequence is shown in SEQ ID NO: 41) is used as a positive control antibody, and the positive control antibody, Antibodies a1, a2, b1, b2, c1, c2, d1, and d2 were melted at 4°C and diluted with DMEM complete medium to 13.3 ⁇ g/mL (final concentration 10 nM).
  • the first column of antibody diluent is 200 ⁇ L. Take 30 ⁇ l to 150 ⁇ L diluent and dilute it 6 times, and so on, for a total of 7 gradients.
  • the diluted virus was added to a 96-well white plate at 25 ⁇ L/well, and incubated with the antibody diluent at room temperature for 1 hour. Add HEK293-ACE2 hu cells to the culture plate at 5 ⁇ 10 4 cells/100 ⁇ L/well, and culture in the incubator for 48 h.
  • Antibody d2 was melted at 4°C and diluted with DMEM complete medium to 1.5 ⁇ g/mL (final concentration 10 nM).
  • the first column of antibody diluent is 200 ⁇ L. Take 30 ⁇ L to 150 ⁇ L diluent and dilute it 6 times, and so on, for a total of 7 gradients.
  • the diluted antibody was added to the 96-well white plate at 75 ⁇ L/well.
  • the pseudovirus SARS-Cov-2 spike (Amicron) Fluc-GFP Pseudovirus (manufacturer: Acro, Cat: PSSO-HLGB003), SARS-CoV-2 Spike (Delta) Fluc-GFP Pseudovirus (manufacturer: Acro, Cat : PSSD-HLGB002), SARS-CoV-2 Spike (Omicron BA.2) Fluc-GFP Pseudovirus (Manufacturer: Acro, Cat: PSSO-HLGB011) were reconstituted at room temperature, according to the pseudovirus: diluent 1: 125 dilution.
  • the diluted virus was added to a 96-well white plate at 25 ⁇ L/well, and incubated with the antibody diluent at room temperature for 1 hour.
  • Will HEK293-ACE2 hu cells were added to the culture plate at 5 ⁇ 10 4 cells/100 ⁇ L/well, and cultured in the incubator for 48 h.
  • antibody d2 has obvious broad-spectrum neutralizing activity against the new coronavirus pseudovirus, and the IC 50 values against Delta virus, BA.1, BA.2, and BA.4/BA.5 strains are 0.235nM respectively. , 0.028nM, 0.086nM, 0.089nM.
  • the antibody d2 was humanized to obtain a humanized bispecific antibody, in which the full-length amino acid sequence of the heavy chain is SEQ ID NO:43, and the full-length amino acid sequence of the scFv+ second antibody light chain is SEQ ID NO:44.
  • the neutralizing activity of humanized bispecific antibodies was studied using pseudovirus kits of different subtype mutant strains.
  • the pseudovirus can recognize the ACE2 receptor on the surface of HEK293-ACE2 cells through its surface RBD protein, leading to viral invasion and intracellular expression of luciferase.
  • Humanized bispecific antibodies can effectively inhibit the infection of host cells by wild-type Delta and different mutant strains Omicron BA.1, Omicron BA.2, and Omicron BA.4&5, and inhibit fluorescein. Expression of enzyme genes.
  • a comparative study was conducted on the neutralizing activities of batches 1 and 2 using pseudoviruses of BA.1/Omicron and BA.4&BA.5/Omicron strains. The research data showed that the neutralizing activities of batches 1 and 2 were The neutralizing activities of batch 1 and batch 2 against BA.1 and BA.4&5 mutant strains were completely consistent.
  • the specific test data are shown in Table 3 and Figures 18-20.
  • Example 18 In vitro neutralizing activity of humanized bispecific antibodies against live viruses
  • Table 4 shows the CPE statistical results of samples with various concentrations of neutralizing activity of humanized bispecific antibodies against the BA.2 strain.
  • the test results show that the humanized bispecific antibody has 100% live virus neutralizing ability against the BA.2 strain at a very low concentration of 2ng/mL.
  • Table 5 shows the CPE statistical results of samples with various concentrations of neutralizing activity of humanized bispecific antibodies against BA.5 strains.
  • Example 20 In vivo pharmacodynamic test of humanized bispecific antibody in animal infection model
  • the viral load in the lungs of the hamster control group and the treatment group 3 days after challenge and administration is shown in Figure 22.
  • Experimental data show that the humanized bispecific antibody has very obvious neutralizing activity against the BA.2 strain, can effectively inhibit the virus's lung infection in hamsters, and can effectively reduce the viral load in the lungs of hamsters, with statistical differences. .
  • the viral load in the lungs of 8 hamsters in the low-dose group was significantly reduced, and the viral load in the lungs of almost all hamsters in the high-dose group was completely reduced.
  • the decrease in lung viral load is dose-dependent.
  • the average lung viral load in the low-dose group decreased by 2 lg values, and the average lung viral load in the high-dose group decreased by 3.8 lg values.

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Abstract

本发明提供了一种抗RBD抗体及其用途。本发明提供的抗体包含单特异性抗体和双特异性抗体，能够特异性地与SARS-CoV-2的RBD蛋白结合，可有效抑制病毒入侵，与奥密克戎等变异病毒结合活性优异，针对广谱变异病毒均有良好的广谱中和活性。

Description

一种抗RBD抗体及其用途 技术领域
本发明属于生物技术领域，具体涉及一种抗RBD抗体及其用途。
背景技术
自SARS-CoV-2被发现以来，全世界范围内已有20多个变异株，截至2022年1月7日，WHO定义了5个关切变异株(variant of concern，VOC)，即阿尔法(α)、贝塔(β)、伽马(γ)、德尔塔(δ)和奥密克戎(ο)。2022年5月，世卫组织表示，已有128个国家和地区报告发现了奥密克戎变异株。在德尔塔变异毒株中突变的氨基酸残基数量为18个，而在新毒株奥密克戎中则有43个。这些变异多样化，且大部分位于与人体细胞相互作用的区域。由于病毒变异率高，传播范围广，大量的疫苗和药物也在大量研发，疫苗和抗体药物出现耐药以及脱靶风险提高，这也给新型抗体药物以及疫苗研发提出更高要求，也提供了更多机遇。
新冠病毒具有四个结构蛋白，结构蛋白介导病毒粒子的组装与感染。N蛋白组成了基因组外的核衣壳，随后在外层还有膜蛋白(M)，刺突蛋白(S)，包膜蛋白(E)所组成的病毒囊膜结构。
SARS-CoV-2的S蛋白为病毒表面的一类标志性跨膜蛋白，是由3个相同的亚基通过非共价键的方式结合形成的同源三聚体，其相对分子质量为141178，含有1273个氨基酸。S蛋白主要由信号肽、N端结构域、受体结合结构域(RBD)、融合肽段、七肽重复序列(heptad repeat，HR)1、HR2、跨膜结构域、胞质结构域等构成，同时具有S1/S2与S2两个切割位点。相关研究显示，HR1和HR2会在S1的RBD与靶细胞的ACE2结合后，形成融合核心(6-HB)，提高病毒融合和感染的效率。
SARS-CoV-2的细胞受体血管紧张素转换酶2(angiotensin-converting enzyme 2，ACE2)是肾素-血管紧张素***的主要活性肽。目前认为，在SARS-CoV-2感染中，ACE2为病毒进入细胞的主要受体。ACE2作为含锌羧肽酶的Ⅰ型跨膜蛋白，多表达于肾脏、心脏和男性生殖***，同时也在肺、小肠和肝脏等组织中表达。刺突蛋白在病毒入侵过程中起着主要的作用，并且S蛋白为主要免疫原。该蛋白 会刺激机体产生大量抗体，这些抗体会结合到S蛋白表面，阻断病毒与宿主识别，抑制病毒蛋白剪切变构，从而抑制病毒入侵。S蛋白在病毒表面常以三聚体形式聚集。S蛋白为同源三聚体，C端锚定于病毒膜上，S蛋白是冠状病毒进入细胞的关键，所以它是一个有吸引力的抗病毒靶点。抗RBD的抗体可以阻碍S蛋白三聚体形成。
发明内容
为了解决市面上没有针对奥密克戎病毒具有良好广谱中和活性的抗体，本发明提供了一种抗RBD抗体，包含单特异性抗体和双特异性抗体，可有效抑制病毒入侵，与病毒结合活性好，半衰期长，针对广谱变异病毒均有良好的广谱中和活性。
本发明提供了分离的单克隆人抗体或其抗原结合部分，包含单特异性抗体和双特异性抗体，其在刺突蛋白或其片段，例如胞外域，或更具体地，刺突蛋白的受体结合结构域处特异性结合冠状病毒如SARS-CoV-2，(i)防止或阻断冠状病毒如SARS-CoV-2进入宿主细胞，和/或(ii)诱导补体介导的冠状病毒如SARS-CoV-2失活和/或吞噬作用。本发明的抗体还可以结合在受感染宿主细胞上展示的冠状病毒如SARS-CoV-2的刺突蛋白，诱导补体依赖性细胞毒性(CDC)、抗体依赖性细胞吞噬作用(ADCP)、抗体依赖性细胞介导的病毒抑制作用(ADCVI)和/或抗体依赖性细胞介导的细胞毒性(ADCC)以消除受感染的细胞。本发明的抗体可包被在冠状病毒如SARS-CoV-2或展示冠状病毒刺突蛋白的感染细胞上形成免疫复合物，该免疫复合物可被抗原呈递细胞如树突状细胞识别，诱导体液和细胞抗病毒免疫反应。
该抗体或其抗原结合部分可用于体外检测冠状病毒如SARS-CoV-2，以及治疗或预防由冠状病毒引起的疾病。
因此，本发明提供了一种分离的单克隆抗体或其抗原结合部分，其特异性结合冠状病毒的刺突蛋白或其片段，所述抗体或其抗原结合部分包含重链可变区和轻链可变区，(a)所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR2包含SEQ ID NO:2所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR3包含SEQ ID NO:3所示的氨基酸 序列或所示氨基酸序列包含至多三个氨基酸突变的变体；和/或
(b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:4或10所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR2包含SEQ ID NO:5所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR3包含SEQ ID NO:6所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体。
在某些实施方案中，HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:1、2、3所示的氨基酸序列或所示氨基酸序列分别包含至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:4或10、5、6所示的氨基酸序列或所示氨基酸序列分别包含至多三个氨基酸突变的变体。
在某些实施方案中，所述重链可变区包含与SEQ ID NO:7、13、14、42所示氨基酸序列具有至少80％同一性的氨基酸序列。
在某些实施方案中，所述轻链可变区包含与SEQ ID NO:8、11、47所示氨基酸序列具有至少80％同一性的氨基酸序列。
在某些实施方案中，所述重链可变区和所述轻链可变区分别包含与SEQ ID NO:7、13、14、42和SEQ ID NO:8、11、47所示氨基酸序列具有至少80％同一性的氨基酸序列。
在某些实施方案中，其为全长抗体、Fab、Fab'、(Fab')2、Fd、Fv、dAb、scFv或scFv-scFv。
在某些实施方案中，其包含与第二抗体或其抗原结合部分连接的抗体或其抗原结合部分，其中所述第二抗体或其抗原结合部分具有与前述任一项所述的抗体或其抗原结合部分不同的结合特异性，所述第二抗体或其抗原结合部分能够结合冠状病毒刺突蛋白的另一个表位。
在某些实施方案中，所述第二抗体或其抗原结合部分包含重链可变区和轻链可变区，其中，
(a)所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:17所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR2包含SEQ ID NO:18所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR3包含SEQ ID NO:19所示的氨基酸序列或 所示氨基酸序列包含至多三个氨基酸突变的变体；和/或
(b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:20或45所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR2包含SEQ ID NO:21所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR3包含SEQ ID NO:22所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体。
在某些实施方案中，其中所述第二抗体或其抗原结合部分的重链可变区和轻链可变区分别包含与SEQ ID NO:23或45和SEQ ID NO:24或46所示氨基酸序列具有至少80％同一性的氨基酸序列。
在某些实施方案中，所述双特异性抗体包含两个单链抗体片段scFv，其中所述scFv的重链可变区VH的序列如SEQ ID NO:27或14所示，所述scFv的轻链可变区VL的序列如SEQ ID NO:28或47所示。
在某些实施方案中，所述scFv分别连接于所述第二抗体或其抗原结合部分的两条重链或两条轻链的N端或C端。在某些实施方案中，所述scFv分别连接于所述第二抗体或其抗原结合部分的两条轻链的N端。
在某些实施方案中，其包含重链恒定区和/或轻链恒定区。在某些实施方案中，所述重链恒定区是IgG、IgA、IgD、IgE和IgM恒定区。在某些实施方案中，所述重链恒定区是IgG1、IgG2、IgG3或IgG4恒定区。在某些实施方案中，所述轻链恒定区是κ或λ恒定区。
在某些实施方案中，其重链恒定区任选含有突变。在某些实施方案中，其重链恒定区可以含有突变，例如M252Y/S254T/T256E(YTE)突变。
在某些实施方案中，其与选自SARS-CoV、SARS-CoV-2和MERS-CoV的冠状病毒的刺突蛋白或其片段结合。
在某些实施方案中，其与冠状病毒的刺突蛋白的受体结合域或胞外域结合。在某些实施方案中，其(a)与SARS-CoV-2的刺突蛋白或其片段结合，(b)抑制SARS-CoV-2的刺突蛋白或其片段与人ACE2结合，(c)阻止SARS-CoV-2进入宿主细胞，(d)诱导补体介导的SARS-CoV-2失活，(e)诱导SARS-CoV-2的吞噬作用，(f)诱导针对SARS-CoV-2感染细胞的补体依赖性细胞毒性，(g)诱导针对SARS-CoV-2感染细胞的抗体依赖性细胞吞噬作用，(h)诱导针对SARS-CoV-2感 染细胞的抗体依赖性细胞介导的病毒抑制作用，(i)诱导针对SARS-CoV-2感染细胞的抗体依赖性细胞介导的细胞毒性，(j)诱导体液和细胞抗病毒免疫反应，和/或(k)阻碍刺突蛋白的三聚体形成。
在某些实施方案中，其与SARS-CoV-2的刺突蛋白的胞外域结合并介导抗体中和。
在另一个方面，本发明还提供了一种免疫偶联物，例如抗体-药物偶联物，其可以包含与治疗剂如细胞毒素连接的本发明的抗体或其抗原结合部分。
在另一个方面，本发明的抗体或其抗原结合部分可以制成嵌合抗原受体(CAR)的一部分。本发明还提供了可以包含抗原嵌合受体的免疫细胞，例如T细胞。
在另一个方面，本发明提供了一种编码本发明的抗体或其抗原结合部分的核酸分子，以及可以包含此类核酸分子的表达载体和可以包含所述表达载体的宿主细胞。
在另一个方面，本发明提供了一种使用宿主细胞制备本发明的抗体或其抗原结合部分的方法，该方法可以包括以下步骤：(i)在宿主细胞中表达抗体或其抗原结合部分和(ii)从宿主细胞或其细胞培养物中分离抗体或其抗原结合部分。
在另一个方面，本发明提供了一种组合物，该组合物可以包含本发明的抗体或其抗原结合部分、免疫偶联物、双特异性分子、具有CAR的免疫细胞或表达载体，以及药学上可接受的载体。在某些实施方案中，组合物可以包含多于一种本发明的抗体或其抗原结合部分。在某些实施方案中，组合物可以包含表达多于一种本发明的抗体或其抗原结合部分的载体。在某些实施方案中，组合物可以包含用多于一种本发明的抗体或其抗原结合部分产生的免疫偶联物。在某些实施方案中，组合物可以包含具有多于一种本发明的抗体或其抗原结合部分的双特异性分子。在某些实施方案中，组合物可以包含在CAR中具有多于一种本发明内容的抗体或其抗原结合部分的免疫细胞。
在另一个方面，本发明提供了一种在有需要的受试者中治疗由冠状病毒感染引起的疾病的方法，包括向受试者施用治疗有效量的本发明的抗体或其抗原结合部分。在某些实施方案中，施用多于一种抗体或其抗原结合部分。在某些实施方案中，该方法可以包括施用本发明的双特异性分子、免疫偶联物或具有CAR的 免疫细胞。冠状病毒可以是SARS-CoV、MERS-CoV或SARS-CoV-2。在某些实施方案中，冠状病毒是SARS-CoV-2。
在另一个方面，本发明提供了一种在有需要的受试者中预防由冠状病毒感染引起的疾病的方法，包括向受试者施用本发明的抗体或其抗原结合部分。在某些实施方案中，冠状病毒可以是SARS-CoV、MERS-CoV或SARS-CoV-2。在某些实施方案中，冠状病毒是SARS-CoV-2。在某些实施方案中，受试者是人。在某些实施方案中，受试者可能是暴露于冠状病毒引起的疾病或暴露于冠状病毒(例如SARS-CoV-2)的，但不能通过疫苗接种得到保护。
本发明的其他特征和优点将从以下详细描述和不应被解释为限制的示例中显而易见。在整个本申请中引用的所有参考文献、GenBank条目和专利的内容通过引用明确并入本文。
术语定义
除非另有定义，本文使用的所有领域术语、符号和其它科学术语旨在具有本发明所属领域技术人员通常理解的含义。在某些情况下，为了清楚起见和/或为了便于参考，本文定义了具有通常理解的含义的术语，并且在此包含此类定义不应被解释为表示与本领域中通常理解的事情的差异。
术语“SARS-CoV-2”是指严重急性呼吸综合征冠状病毒2号，属于冠状病毒科、β冠状病毒属(包括SARS-CoV和MERS-CoV)。
术语“刺突蛋白”是冠状病毒用来侵入宿主细胞的跨膜蛋白。SARS-CoV-2的刺突蛋白含有与宿主细胞受体结合的第一亚基(S1亚基)和用于病毒和细胞膜融合的第二亚基(S2亚基)。S1亚基中有一个受体结合域(RBD)，其与宿主细胞受体结合。
术语“抗体”包含包括通过双硫键相互连接的四条多肽链，二条重(H)链和二条轻(L)链的免疫球蛋白分子以及其多聚体(例如IgM)。每个L链通过一个共价二硫键连接于H链，而两个H链视H链同种型而定通过一个或多个二硫键彼此连接。每个重链在N末端具有可变区，继之以恒定区。各重链包含重链可变区(VH)和重链恒定区。这一重链恒定区包含三个区(结构域)，CH1、CH2和CH3。各轻链包含轻链可变区(VL)和轻链恒定区。轻链恒定区包含一个区 (结构域，CL1)。VH和VL区可进一步细分为高变区，称为互补决定区(CDR)，其间散布着较保守性区域，称为框架区(framework region，FR，也称骨架区、构架区)。各VH和VL是由三个CDR和四个FR所组成，以下列顺序由氨基端排列到羧基端：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。抗体可以是不同亚类(subclass)的抗体。
术语“全长抗体”是指与抗体片段相比呈其基本上完整形式的抗体。特定来说，全长抗体包括具有包括Fc区的重链和轻链的4链抗体。
术语“抗原结合部分”是指保留特异性结合抗原的能力的抗体的一个或多个片段(例如，SARS-CoV-2刺突蛋白)。已经表明，抗体的抗原结合功能可以通过全长抗体的片段来执行。包含在抗体的术语“抗原结合部分”内的结合片段的实例包括但不限于：(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)Fab'和F(ab')2片段，其中F(ab')2为一种二价片段，包含在铰链区通过二硫键连接的两个Fab片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段，(v)dAb片段(Ward等人，(1989)Nature 341:544-546)，其由VH结构域组成；(vi)scFv；(viii)scFv-scFv。
术语“Fc区”是抗体的尾部区域，它与Fc受体和补体***的一些蛋白质相互作用以激活免疫***。IgG、IgA和IgG Fc区由来自抗体重链的第二和第三恒定域(CH2和CH3)的两个相同片段组成，而IgM和IgE Fc区含有三个重链恒定域(CH域2-4)。Fc区可与补体成分C1q结合以激活经典的补体级联反应，可与吞噬细胞(即巨噬细胞、粒细胞和树突细胞)上的Fc受体结合以诱导被抗体结合的细胞的吞噬作用，可与免疫效应细胞(主要是自然杀伤细胞)的Fc受体结合以诱导免疫效应细胞释放细胞毒性颗粒，导致抗体包被细胞死亡，并可与抗原呈递细胞如树突状细胞的Fc受体结合以诱导体液和细胞抗病毒免疫反应。
术语“嵌合抗体”是抗体分子(或其抗原结合部分)，其中(1)所述恒定区或其部分被改变、置换或更换，使得所述抗原结合位点(可变区)与不同或改变的类型、效应子功能和/或种类的恒定区连接，或者与赋予嵌合抗体新特性的完全不同的分子(例如酶、毒素、激素、生长因素、药物等)连接；或(2)所述可变区或其部分被改变、置换或更换为具有不同或改变的抗原特异性的可变区。例如，可以通过用来自人免疫球蛋白的恒定区替代其恒定区来修饰小鼠抗体。由于被人恒定区置 换，所述嵌合抗体可以保留其识别抗原的特异性，同时与原始小鼠抗体相比在人体中具有降低的抗原性。
术语“人源化抗体”是指含有源于人抗体序列的氨基酸残基的嵌合抗体。人源化抗体可含有来自非人动物或合成抗体的CDR中的一些或全部，而抗体的框架区和恒定区含有源于人抗体序列的氨基酸残基。可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少的反向突变或回复突变，以保持活性。
术语“单克隆抗体”是指均一的、仅针对某一特定抗原表位的抗体。与典型地包括针对不同抗原决定簇(表位)的不同抗体的普通多克隆抗体制剂相比，每种单克隆抗体针对抗原上的单个抗原决定簇。修饰语“单克隆”表示抗体的均一特征，不解释为需要通过任何特定方法产生的抗体。本发明的单克隆抗体优选通过重组DNA方法产生，或通过本文其它地方描述的筛选方法获得。
术语“分离的单克隆抗体”是指基本上不含具有不同抗原特异性的其他抗体的抗体(例如，特异性结合SARS-CoV-2刺突蛋白的分离抗体基本上是不含特异性结合除SARS-CoV-2刺突蛋白以外的抗原的抗体)。然而，特异性结合SARS-CoV-2刺突蛋白的分离抗体可能与其他抗原具有交叉反应性，例如，来自其他病毒(如冠状病毒)的刺突蛋白。此外，分离的抗体可以基本上不含其他细胞材料和/或化学物质。
术语“双特异性抗体”是指能够与两个目标抗原或目标抗原表位特异性结合的蛋白分子。
术语“scFv”指包含通过接头连接的抗体重链可变结构域(VH)和抗体轻链可变结构域(VL)的分子。此类scFv分子可具有一般结构：NH2-VL-连接子-VH-COOH或NH2-VH-连接子-VL-COOH。合适的现有技术连接子由重复的GGGGS氨基酸序列或其变体组成，例如使用1-6个重复的GGGGS氨基酸序列或其变体。
术语“亲和力”是指分子的单个结合位点(例如，MIAC的抗原结合模块)与其结合配偶体(例如，抗原)之间的非共价相互作用的总和的强度。在各抗原位点内，抗体“臂”的可变区通过弱非共价力与抗原在多个氨基酸位点处相互作用； 相互作用愈大，亲和力愈强。除非另外指示，否则如本文所用的“结合亲和力”是指反映结合对的成员(例如，抗体与抗原)之间的1:1相互作用的固有结合亲和力。分子X对其搭配物Y的亲和力一般可由解离常数(Kd)表示。亲和力可通过本领域中已知的常用方法测量，例如通过使用表面等离子体共振(SPR)技术(例如仪器)或生物层干涉测量法(例如，仪器)来测量。
术语“嵌合抗原受体”或“CAR”是指将确定的特异性移植到免疫效应细胞，通常是T细胞上并增强T细胞功能的工程化受体。新一代CAR包括一个胞外结合结构域，包括一个scFv、一个铰链区、一个跨膜结构域和一个胞内信号结构域(主要是CD3-zeta的胞质结构域，它是T细胞激活信号的主要传递器)，加上一个或多个共同刺激域)。CARs可能进一步具有增强T细胞扩增、持久性和抗肿瘤活性的因子，例如细胞因子和共刺激配体。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。
术语“载体(vector)”指用于将核苷酸编码信息转移到宿主细胞内的任一种分子(例如，核酸、质粒、或病毒等)。术语“载体”涵盖所有类型的载体，无论它们的功能如何。能够引导它们所操作性地连接的可表达核酸的表达的载体通常被称为“表达载体”。在本说明书中，“质粒”和“载体”可互换使用，因为质粒是最常用的载体形式。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
术语“治疗”是指抑制、消除、减少和/或改善与正在治疗的疾病或病症，例如冠状病毒引起的疾病相关的症状、症状严重程度和/或症状频率。
术语“预防”是指为预防特定疾病(例如冠状病毒引起的疾病)而采取的行动。预防可以包括向暴露于疾病或引起疾病的病毒且不能通过疫苗接种保护的对象施用抗体。
术语“施用”是指通过本文描述的方法将治疗或药物组合物递送或导致递送 至受试者身体的行为或本领域中已知的。施用治疗或药物组合物包括开具将被递送到患者体内的治疗或药物组合物的处方。示例性的给药形式包括口服剂型，例如片剂、胶囊剂、糖浆剂、混悬剂；可注射剂型，例如静脉内(IV)、肌内(IM)或腹膜内(IP)；透皮剂型，包括霜剂、果冻剂、粉剂或贴剂；口腔剂型；吸入粉剂、喷雾剂、混悬剂和直肠栓剂。
术语“有效量”是指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本发明靶抗原相关病症的发病率或改善所述病症的一个或多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本发明靶抗原相关病症的进展。
术语“药学上可接受的载体”包括与药物施用相容的任何或所有溶剂、分散剂、包被物、抗细菌和抗真菌药剂、等渗和缓释剂，及其类似物。包括各种赋形剂、稀释剂和缓冲剂等，这些物质适合于人和/或动物施用而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。合适的载体在Remington’s Pharmaceutical Sciences最新版中的标准参考文件中有所叙述，其通过在此引述而全部合并于本文。合适的载体或稀释剂例子包括但不局限于水、盐溶液、Ringer’s液、葡萄糖溶液和5％人血清白蛋白。也可以使用脂质体和疏水介质如不挥发油。药物活性物质的介质和药剂的使用在本领域中是熟知的。除了那些对于活性成分不相容的常规介质或试剂以外，其在成分中的使用都可以达到预期效果。
术语“同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，候选序列中与对照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST软件或FASTA程序包。术语“至少80％同一性”是指候选序列中与对照多肽序列中的氨基酸残基相同的氨基酸残基的百分率为80％以上，包括80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。
术语“补体依赖性细胞毒性”或“CDC”是指抗体介导的免疫机制，其中抗体与补体成分C1q结合并激活经典的补体级联反应，导致在细胞表面形成膜攻击复合物(MAC)和随后的细胞裂解。
术语“抗体依赖性细胞吞噬作用”或“ADCP”是指细胞介导的免疫机制，其中抗体的Fc部分与吞噬细胞(即巨噬细胞、粒细胞和树突细胞)上的Fc受体结合以诱导被抗体结合的细胞的吞噬作用。
术语“抗体依赖性细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫机制，其中抗体的Fc部分与免疫效应细胞(主要是自然杀伤细胞)的Fc受体结合，导致免疫效应细胞释放细胞毒性颗粒，导致抗体包被细胞死亡。
本发明的抗体或其抗原结合部分特异性结合冠状病毒例如SARS-CoV-2的刺突蛋白或其片段，更具体地结合刺突蛋白的受体结合结构域，并抑制结合刺突蛋白到其进入受体ACE2。本公开的抗体或其抗原结合部分还可以结合于病毒感染的宿主细胞上展示的刺突蛋白或其片段，例如受体结合结构域。
本发明的抗体或其抗原结合部分可以诱导(i)补体介导的冠状病毒例如SARS-CoV-2的失活，(ii)冠状病毒的吞噬作用，(iii)针对冠状病毒感染的宿主细胞的补体依赖性细胞毒性(CDC)，(iv)针对感染的宿主细胞的抗体依赖性细胞吞噬作用(ADCP)，(v)针对冠状病毒感染的宿主细胞的抗体依赖性细胞介导的病毒抑制作用(ADCVI)，(vi)针对冠状病毒感染的宿主细胞的抗体依赖性细胞介导的细胞毒性(ADCC)，和/或(vii)体液和细胞抗病毒免疫反应。
本发明的抗体或其抗原结合部分的重/轻链CDR和可变区的氨基酸序列和ID号列于下表1中。重链可变区CDR和轻链可变区CDR已由Kabat编号***定义。然而，如本领域公知的，CDR区也可以基于重链/轻链可变区序列通过其他***例如Chothia、AbM或Contact编号***/方法来确定。
本发明的抗体可以含有重链恒定区，例如具有在例如SEQ ID NO:9中列出的氨基酸序列的人IgGl重链恒定区或其变体，和/或具有在例如SEQ ID NO:12中列出的氨基酸序列的轻链恒定区或其变体。本发明的抗体还可以含有其他合适的重链恒定区和/或轻链恒定区，或其变体。
表1.抗体或抗原结合部分氨基酸序列




附图说明
图1为鼠源抗体的奥密克戎RBD结合活性。
图2为鼠源抗体的多种突变毒株病毒RBD结合活性。
图3为鼠源抗体的奥密克戎假病毒中和活性。
图4为人源化抗体1的奥密克戎病毒结合活性。
图5为人源化抗体2的奥密克戎病毒结合活性。
图6为人源化抗体3的奥密克戎病毒结合活性。
图7为鼠源抗体、嵌合抗体、人源化抗体1、人源化抗体2、人源化抗体3的假病毒中和活性。
图8为抗体a结构的示意图。
图9为抗体b结构的示意图。
图10为抗体c结构的示意图。
图11为抗体d结构的示意图。
图12为抗体a1、b1、c1的奥密克戎病毒结合活性。
图13为抗体d1、a2、b2的奥密克戎病毒结合活性。
图14为抗体c2、d2的奥密克戎病毒结合活性。
图15为抗体a1、a2、b1、b2的奥密克戎假病毒中和活性。
图16为抗体c1、c2、d1、d2的奥密克戎假病毒中和活性。
图17为抗体d2的假病毒广谱中和活性。
图18为人源化双特异性抗体对德尔塔、奥密克戎BA.2假病毒的中和活性。
图19为人源化双特异性抗体的批次1和批次2对奥密克戎BA.1假病毒的中和活性。
图20为人源化双特异性抗体的批次1和批次2对奥密克戎BA.4&BA.5假病毒的中和活性。
图21(a)-(f)分别为人源化双特异性抗体对L452R,T478K/德尔塔、B.1.1.529/奥密克戎、BA.1.1/奥密克戎、BA.2/奥密克戎、BA.4&BA.5/奥密克戎、BF7&BA.4.6/ 奥密克戎蛋白竞争活性拟合曲线。
图22为人源化双特异性抗体给药后各组BA.2肺部病毒载量曲线。
具体实施方式
以下结合附图对本发明技术方案进行详细说明。应当理解，此处所描述的具体实施方式仅用以解释本发明，并不用于限定本发明。本申请的范围并不受这些实施方式的限定，以申请专利的范围为准。
实施例1小鼠免疫
取3-5只6周龄雌性Balb/c小鼠，SARS-COV-2 Spike RBD,His Tag(B.1.1.529/奥密克戎)(Acro:SPD-C522E)作为免疫原进行腹腔注射。提前3天采取阴性血清，首次免疫，注射50μg经弗氏完全佐剂充分乳化的免疫原，第14天、第35天腹腔注射25μg经弗氏不完全佐剂充分乳化的免疫原进行第二次、三次免疫。7天后，尾部采血，滴度稀释血清，用ELISA法检测血清效价。用SARS-COV-2 Spike RBD,His Tag(B.1.1.529/奥密克戎)(Acro:SPD-C522E)蛋白包被ELISA板过夜，弃包被液，2％BSA进行封闭，封闭完成后，加入血清稀释液，进行反应，反应完毕后用PBST清洗，加入辣根过氧化物酶标记的羊抗鼠二抗，PBST洗板。加入TMB显色液显色，1M盐酸终止反应，于450nm下测光密度。当效价结果满足要求时，可收获小鼠脾脏和***，进行细胞融合。
实施例2杂交瘤细胞融合
将小鼠处死取出脾脏和***，将细胞研磨过细胞筛网，得到淋巴细胞。计算总细胞数，将获得的淋巴细胞与骨髓瘤细胞SP2/0细胞量按2:1混合，细胞悬液1000rpm，8min，离心，并将细胞用电融合液清洗两次后，用电融合液按总细胞密度为1×107/mL重悬细胞，将细胞悬液置于融合池中，按常规电转方法进行细胞融合。将融合体在含有饲养细胞及HAT的DMEM完全培养基中，5％CO2，37℃培养。融合体选择培养中所用的饲养细胞取自未经免疫的动物腹腔中的巨噬细胞，以辅助新的杂种B淋巴细胞杂交瘤生长。
实施例3本发明的阳性克隆筛选
采用ELISA方法筛选出与SARS-COV-2 Spike RBD,His Tag(B.1.1.529/奥密克戎)(Acro:SPD-C522E)，SARS-COV-2 Spike RBD(L4S2R T478K),His Tag(Acro:SPD-C52Hh)，SARS-COV-2 S1 Protein RBD,His Tag(MALS verifiled)(Acro:SPD-C52H3)均结合的克隆。上述蛋白均来自于市售。
将目的蛋白包被到酶标板上，4℃，过夜，2％BSA进行封闭，37℃，1h；将杂交瘤细胞培养上清加入ELISA板中，以SP2/0细胞上清作为阴性对照，融合前小鼠血清稀释100倍作为阳性对照，100μL/孔，37℃，1h；PBST洗涤3次，加入HRP标记的羊抗鼠IgG-Fc抗体，100μL/孔，37℃，45min；洗涤后，TMB显色10min，1M盐酸终止反应；酶标仪测定OD450读数。OD450>1.0，阴性对照<0.1，为阳性克隆。将阳性克隆孔吹起对细胞进行计数，将细胞按1个/孔，铺进96孔培养板中，5％CO2，37℃7-10天后，取上清进行ELISA检测，本发明的单抗体可结合三种病毒毒株RBD，可作为候选分子。
实施例4鼠源抗体的生产
获得稳定的本发明筛选出的杂交瘤细胞系后，采用体外培养法获取单克隆抗体。将细胞株扩增至在T75培养瓶内，培养至细胞覆盖率为40-50％，将细胞上清弃去，加入50mL杂交瘤-SFM培养基，37℃，5％CO2培养。培养6-7天，待细胞存活率低于20％，低速离心后收集培养上清，用ProteinA亲和层析柱进行纯化，获得纯化的鼠源抗体。
实施例5鼠源抗体可变区序列的获得
用基于简并引物PCR的方法，测定由鼠源抗体可变区的DNA序列。将杂交瘤细胞株分别扩大培养，1000rpm离心收集细胞，并以Trizol提取总RNA。以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板PCR扩增对应的可变区DNA序列，回收PCR产物进行TA克隆及测序，得到候选杂交瘤重链可变区和轻链可变区序列，重链可变区序列包含SEQ ID NO:7所示的氨基酸序列，轻链可变区序列包含SEQ ID NO:8所示的氨基酸序列。
实施例6鼠源抗体与奥密克戎RBD结合活性
将SARS-CoV-2 Spike RBD,His Tag(B.1.1.529/奥密克戎)(厂家：Acro，Cat： SPD-C522e，Lot：5716-21CCF2-11B)稀释为1μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤3次，并在无尘纸上拍干。配置3％脱脂奶粉，300μL/孔，37℃孵育1h，倒掉封闭液，1×PBST洗板每孔300μL，洗板机洗涤3次，无尘纸上拍干，将蛋白封闭。将鼠源抗体和阳性对照抗体分别用3％脱脂奶粉稀释为20μg/mL，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，1×PBST洗板每孔300μL，洗板机洗涤3次，无尘纸上拍干。用3％脱脂奶粉将山羊抗小鼠IgG Fc(HRP)(abcam，Cat#ab97265)按1：10000稀释，100μL/孔，37℃孵育45min。倒掉二抗液体，1×PBST洗板每孔300μL洗板机洗涤6次，无尘纸上拍干。加入单组分TMB显色混合液(厂家：索莱宝，CAT:PR1200)，100μL/孔，37℃避光显色7min。加入终止液1M HCl(83mL 37％浓盐酸+917mL纯水)终止显色反应，100μL/孔。在酶标仪上450nm处读数。获得鼠源抗体、阳性对照抗体(重链可变区序列如SEQ ID NO:15所示，轻链可变区序列如SEQ ID NO:16所示)与奥密克戎RBD结合曲线，如图1所示，EC50值分别为0.95和3.60μg/mL，可见鼠源抗体效果优于阳性对照抗体。
实施例7鼠源抗体与多种突变毒株病毒RBD结合实验
将SARS-CoV-2 Spike RBD，His tag.(BA.1/奥密克戎)(厂家：Acro，Cat：SPD-c522e，Lot：5716-21CCF2-Z4)、SARS-CoV-2 Spike RBD，His tag.(BA.1.1/奥密克戎)(厂家：Acro，Cat：SPD-c522j，Lot：6159-2248F1-12M)、SARS-CoV-2 Spike RBD，His tag.(BA.2/奥密克戎)(厂家：Acro，Cat：SPD-c522g，Lot：5911-223BF1-11Z)、SARS-CoV-2 Spike RBD，His tag.(德尔塔)(厂家：Acro)、SARS-CoV-2 Spike RBD，His tag.(野生型)(厂家：Acro)稀释为0.8μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤3次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h20min，倒掉封闭液，1×PBST洗板每孔300μL，洗板机洗涤3次，无尘纸上拍干。将阳性对照抗体(重链可变区序列如SEQ ID NO:15所示，轻链可变区序列如SEQ ID NO:16所示)和鼠源抗体分别用3％脱脂奶粉稀释为10μg/mL，100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤3次，平板纸上拍干。用3％脱脂奶粉将Goat pAb to Ms IgG(HRP)(厂家：abcam，Lot:GR3393964-2)按1：10000稀 释，100μL/孔，37℃孵育45min。弃去孔中液体，1×PBST洗板每孔300μL，洗板机洗涤6次，无尘纸上拍干。加入单组分TMB显色混合液，100μL/孔，并用铝箔纸包好，37℃避光显色3min。加入终止液1M HCl(83mL 37％浓盐酸+917mL纯水)终止显色反应，100μL/孔。在酶标仪上450nm处读数。如图2所示，鼠源抗体及阳性对照抗体，均可以与SARS-CoV-2野生型、德尔塔、奥密克戎病毒株BA.1、奥密克戎病毒株BA.2交叉结合，对奥密克戎病毒株BA.1.1结合活性稍弱。
实施例8鼠源抗体与奥密克戎假病毒单点中和
将鼠源抗体和阳性对照抗体(重链可变区序列如SEQ ID NO:15所示，轻链可变区序列如SEQ ID NO:16所示)分别于4℃融化，用DMEM完全培养基稀释成13.3μg/mL(终浓度10μg/mL)。稀释好的抗体以75μL/孔加入96孔白板中。将假病毒SARS-Cov-2 spike(奥密克戎)Fluc-GFP Pseudovirus(厂家：Acro，Cat：PSSO-HLGB003)于室温复溶，按照假病毒：稀释液＝1：125稀释。稀释好的病毒以25μL/孔加入96孔白板中，和抗体稀释液一起室温孵育1h。将HEK293-ACE2 hu细胞以5×104个/100μL/孔加入培养板中，培养箱中培养48h后。提前取出荧光酶显色液和96孔白板平衡至室温，弃去样品孔100μL培养基，立即加入100μL荧光酶显色液，室温孵育3-5min，酶标仪读取化学荧光信号。鼠源抗体与奥密克戎假病毒单点中和试验结果见表2，鼠源抗体对于奥密克戎假病毒具有99.81％以上的抑制活性，和阳性对照抗体效果相当。
表2.鼠源抗体与奥密克戎假病毒单点中和试验结果
实施例9鼠源抗体与奥密克戎假病毒中和实验
将鼠源抗体、阳性对照抗体、无关抗体于4℃融化，用DMEM完全培养基稀释成13.3μg/ml(终浓度10μg/mL)。首列抗体稀释液200μL，取30μL至150μL稀释液中进行6倍稀释，以此类推，共7梯度。稀释好的抗体以75μg/孔加入96 孔白板中。将假病毒SARS-Cov-2 spike(奥密克戎)Fluc-GFP Pseudovirus(厂家：Acro，Cat：PSSO-HLGB003)于室温复溶，按照假病毒：稀释液＝1：125稀释。稀释好的病毒以25μL/孔加入96孔白板中，和抗体稀释液一起室温孵育1h。将HEK293-ACE2hu细胞以5×104个/100μL/孔加入培养板中，培养箱中培养48h。提前取出荧光酶显色液和96孔白板平衡至室温，弃去样品孔100μL培养基，立即加入100μL荧光酶显色液，室温孵育3-5min，酶标仪读取化学荧光信号。如图3所示，鼠源抗体、阳性对照抗体对奥密克戎假病毒有明显中和活性，IC50值分别为0.0021μg/mL和0.0097μg/mL。鼠源抗体活性要高于阳性对照抗体。
实施例10人源化抗体与奥密克戎病毒结合实验
将SARS-CoV-2 Spike RBD，His tag.(厂家：Acro,Cat：SPD-c522e Lot：5716-21CCF2-Z4)稀释为0.8μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤3次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h 20min，倒掉封闭液，1×PBST洗板每孔300μL，洗板机洗涤3次，无尘纸上拍干。实验中，以嵌合抗体作为阳性对照抗体。将嵌合抗体、人源化抗体1、人源化抗体2、人源化抗体3用3％脱脂奶粉稀释为10μg/mL，3倍比稀释11个梯度，100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤3次，平板纸上拍干。用3％脱脂奶粉将Goat anti-人IgG(HRP)(厂家：Jackson，Lot:109-035-098)，按1：10000稀释，100μL/孔，37℃孵育45min。弃去孔中液体，1×PBST洗板每孔300μL，洗板机洗涤6次，无尘纸上拍干。加入单组分TMB显色混合液，100μL/孔，并用铝箔纸包好，37℃避光显色3min。加入终止液1M HCl(83mL 37％浓盐酸+917mL纯水)终止显色反应，100μL/孔。在酶标仪上450nm处读数。如图4-6所示，人源化抗体1、人源化抗体2、人源化抗体3活性差异不大，人源化抗体3相对活性更好。
实施例11鼠源抗体、嵌合抗体、人源化抗体1、人源化抗体2、人源化抗体3的假病毒中和实验
将鼠源抗体、嵌合抗体、人源化抗体1、人源化抗体2、人源化抗体3、无关抗体于4℃融化，用DMEM完全培养基稀释成13.3μg/mL(终浓度10nM)。首列抗体稀释液200μL，取30μL至150μL稀释液中进行6倍稀释，以此类推，共 7梯度。稀释好的抗体以75μL/孔加入96孔白板中。将假病毒SARS-Cov-2 spike(奥密克戎)Fluc-GFP Pseudovirus(厂家：Acro，Cat：PSSO-HLGB003)于室温复溶，按照假病毒：稀释液＝1：125稀释。稀释好的病毒以25μL/孔加入96孔白板中，和抗体稀释液一起室温孵育1h。将HEK293-ACE2 hu细胞以5×104个/100μL/孔加入培养板中，培养箱中培养48h。提前取出荧光酶显色液和96孔白板平衡至室温，弃去样品孔100μL培养基，立即加入100μL荧光酶显色液，室温孵育3-5min，酶标仪读取化学荧光信号。如图7所示，人源化抗体1、人源化抗体2、人源化抗体3对于奥密克戎假病毒有明显中和活性，IC50值分别为0.023nM、0.022nM和0.021nM。
实施例12本发明的双特异性抗体构建
基于鼠源抗体以及专利CN202210892796.0中的鼠源抗体构建八种双特异性抗体，分别为抗体a1、a2、b1、b2、c1、c2、d1、d2，其中a1、b1、c1、d1为Fc区不含突变的抗体结构，a2、b2、c2、d2为Fc区含突变的抗体结构。外源表达成为双特异性抗体悬液，其中抗体a1、a2的结构相同(简称抗体a结构)，b1、b2的结构相同(简称抗体b结构)，c1、c2的结构相同(简称抗体c结构)，d1、d2的结构相同(简称抗体d结构)，抗体a-d结构分别如图8-11所示。
实施例13本发明的双特异性抗体与奥密克戎病毒的结合实验
将SARS-CoV-2 Spike RBD，His tag.(厂家：Acro,Cat：SPD-c522e Lot：5716-21CCF2-Z4)稀释为0.8μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤3次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h 20min，倒掉封闭液，1×PBST洗板每孔300μL，洗板机洗涤3次，无尘纸上拍干。将专利CN202210892796.0中的人源化抗体(重链全长序列如SEQ ID NO:40所示，轻链全长序列如SEQ ID NO:41所示)作为阳性对照抗体，将阳性对照抗体、抗体a1、a2、b1、b2、c1、c2、d1、d2用3％脱脂奶粉稀释为5μg/mL(终浓度2.5μg/mL)，3倍比稀释11个梯度，100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤3次，平板纸上拍干。用3％脱脂奶粉将Goat anti-human IgG(HRP)(厂家：Jackson，Lot:109-035-098)，按1：10000稀释，100μL/孔，37℃孵育45min。弃去孔中液体，1×PBST洗板每孔 300μL，洗板机洗涤6次，无尘纸上拍干。加入单组分TMB显色混合液，100μL/孔，并用铝箔纸包好，37℃避光显色3min。加入终止液1M HCl(83mL37％浓盐酸+917mL纯水)终止显色反应，100μL/孔。在酶标仪上450nm处读数。如图12-14所示，抗体a1、a2、b1、b2、c1、c2、d1、d2活性差异不大，抗体a1、b1、b2、c1、c2、d1、d2均比阳性对照抗体活性强。
实施例14本发明的双特异性抗体的奥密克戎假病毒中和实验
将专利CN202210892796.0中的人源化抗体(重链全长序列如SEQ ID NO:40所示，轻链全长序列如SEQ ID NO:41所示)作为阳性对照抗体，将阳性对照抗体、抗体a1、a2、b1、b2、c1、c2、d1、d2于4℃融化，用DMEM完全培养基稀释成13.3μg/mL(终浓度10nM)。首列抗体稀释液200μL，取30μl至150μL稀释液中进行6倍稀释，以此类推，共7梯度。稀释好的抗体以75μL/孔加入96孔白板中。将假病毒SARS-Cov-2 spike(奥密克戎)Fluc-GFP Pseudovirus(厂家：Acro，Cat：PSSO-HLGB003)于室温复溶，按照假病毒：稀释液＝1：125稀释。稀释好的病毒以25μL/孔加入96孔白板中，与抗体稀释液一起室温孵育1h。将HEK293-ACE2 hu细胞以5×104个/100μL/孔加入培养板中，培养箱中培养48h。提前取出荧光酶显色液和96孔白板平衡至室温，弃去样品孔100μL培养基，立即加入100μL荧光酶显色液，室温孵育3-5min，酶标仪读取化学荧光信号。如图15、16所示，抗体a1、a2、b1、b2、c1、c2、d1、d2对奥密克戎假病毒都有明显中和活性，并且比阳性对照抗体活性强，其中抗体d2活性最佳。
实施例15抗体d2的假病毒广谱中和实验
将抗体d2于4℃融化，用DMEM完全培养基稀释成1.5μg/mL(终浓度10nM)。首列抗体稀释液200μL，取30μL至150μL稀释液中进行6倍稀释，以此类推，共7梯度。稀释好的抗体以75μL/孔加入96孔白板中。将假病毒SARS-Cov-2 spike(奥密克戎)Fluc-GFP Pseudovirus(厂家：Acro，Cat：PSSO-HLGB003)、SARS-CoV-2 Spike(Delta)Fluc-GFP Pseudovirus(厂家：Acro，Cat：PSSD-HLGB002)、SARS-CoV-2 Spike(奥密克戎BA.2)Fluc-GFP Pseudovirus(厂家：Acro，Cat：PSSO-HLGB011)于室温复溶，按照假病毒：稀释液＝1：125稀释。稀释好的病毒以25μL/孔加入96孔白板中，和抗体稀释液一起室温孵育1h。将 HEK293-ACE2 hu细胞以5×104个/100μL/孔加入培养板中，培养箱中培养48h。提前取出荧光酶显色液和96孔白板平衡至室温，弃去样品孔100μL培养基，立即加入100μL荧光酶显色液，室温孵育3-5min，酶标仪读取化学荧光信号。如图17所示，抗体d2对于新冠假病毒有明显广谱中和活性，对德尔塔病毒、BA.1、BA.2、BA.4/BA.5毒株的IC50值分别为0.235nM、0.028nM、0.086nM、0.089nM。
实施例16抗体d2的人源化
将抗体d2人源化，得到人源化双特异性抗体，其中重链全长氨基酸序列为SEQ ID NO:43，scFv+第二抗体轻链全长的氨基酸序列为SEQ ID NO:44。
实施例17人源化双特异性抗体对野生型和不同突变毒株的中和活性
人源化双特异性抗体的中和活性采用不同亚型突变株的假病毒试剂盒进行研究。假病毒可通过其表面RBD蛋白识别HEK293-ACE2细胞表面的ACE2受体，从而导致病毒入侵，在胞内表达荧光素酶。人源化双特异性抗体可以有效抑制野生型Delta和不同突变毒株奥密克戎BA.1、奥密克戎BA.2、奥密克戎BA.4&5对宿主细胞的感染，抑制荧光素酶基因的表达。采用BA.1/奥密克戎和BA.4&BA.5/奥密克戎2个毒株的假病毒对批次1和批次2的中和活性进行了比对研究，研究数据显示，批次1和批次2对BA.1和BA.4&5突变株的中和活性完全一致。具体试验数据见表3和图18-20所示。
表3.人源化双特异性抗体对野生型和不同突变毒株的中和活性(EC50值，ng/mL)
实施例18人源化双特异性抗体对活病毒的体外中和活性
人源化双特异性抗体对与各种亚型毒株RBD蛋白的结合活性和竞争活性，我们委托昆明动物所采用活病毒毒株，进一步验证人源化双特异性抗体的细胞中和活性。人源化双特异性抗体体外活性评价采用的新冠奥密克戎变异株(B A.2)和最新流行毒株BA.5，评价方法为人源化双特异性抗体抑制病毒毒株对Vero E6细胞的感染，观察记录人源化双特异性抗体完全抑制细胞病变(CPE)的浓度。首先在孔板中采用培养基依照浓度倍比稀释的抗体药物，然后向每个稀释后的样品中加入等体积100TCID50病毒，混匀，置于37℃，5％CO2培养箱中孵育1h。然后将上述样品与病毒的混合液及阳性对照、阴性对照分别加入到铺有Vero-E6的细胞板中，置于37℃，5％CO2培养箱中培养4～6天。显微镜下观察并记录各孔中细胞病变情况(CPE)，依据细胞病变情况，计算具有50％以上中和效应的样品稀释倍数，以此作为样品的中和抗体滴度。表4为人源化双特异性抗体针对BA.2毒株中和活性各浓度样品CPE统计结果。试验结果显示人源化双特异性抗体针对BA.2毒株在极低2ng/mL浓度即具有100％的活病毒中和能力。表5为人源化双特异性抗体针对BA.5毒株中和活性各浓度样品CPE统计结果。
表4.人源化双特异性抗体针对BA.2毒株中和活性各浓度样品CPE统计结果

注：分母代表复孔数，分子代表未观察到CPE的孔数，百分比代表该浓度样品的中和抗体
保护率。
表5.人源化双特异性抗体针对BA.5毒株中和活性各浓度样品CPE统计结果
实施例19人源化双特异性抗体对不同突变毒株蛋白竞争活性试验
采用流式细胞术的方法，分别检测人源化双特异性抗体对不同突变毒株蛋白L452R,T478K德尔塔、B.1.1.529/奥密克戎、BA.1.1/奥密克戎、BA.2/奥密克戎、BA.4&BA.5/奥密克戎、BF7&BA.4.6/奥密克戎的竞争活性。流式细胞术检测结果显示，不同突变毒株的RBD蛋白与表达ACE2的HEK293细胞具有较强的结合活性，人源化双特异性抗体具有浓度依赖的竞争不同突变毒株RBD蛋白与表达ACE2的HEK293细胞的结合，批次1和批次2对不同突变毒株蛋白竞争活性结果一致。具体试验数据见表6和图21(a)-(f)。
表6.人源化双特异性抗体对不同突变毒株蛋白竞争活性(EC50值，μg/mL)
实施例20人源化双特异性抗体动物感染模型体内药效学试验
人源化双特异性抗体动物感染模型体内有效性委托中国科学院昆明动物所进行，已有试验数据显示奥密克戎突变毒株BA.2感染仓鼠后，单次给与人源化双特异性抗体腹腔注射后，可有效抑制奥密克戎突变毒株BA.2对于仓鼠肺部的感染，高剂量组给药后，几乎抑制了所有仓鼠的肺部感染。具体试验方案和结果如下。
将5-7周龄叙利亚黄金仓鼠饲养后，利用滴鼻方式，采用1*104TICD50新冠奥密克戎突变毒株BA.2(由中国科学院昆明动物所分离培养)感染仓鼠。仓鼠感染后2h，利用腹腔注射方式，单次给与仓鼠不同剂量的人源化双特异性抗体，其中低剂量给药组为25mg/kg，高剂量给药组为50mg/kg。给药后三天，解剖仓鼠，检测其肺组织病毒载量，并进行肺组织切片进行病理观察。具体给药实验方案见表7。
表7.人源化双特异性抗体BA.2感染模型体内有效性试验方案
攻毒和给药后3天仓鼠对照组和治疗组仓鼠肺部的病毒载量见图22。试验数据显示，人源化双特异性抗体具有非常明显BA.2毒株中和活性，可以有效抑制病毒对于仓鼠的肺部感染，可以有效降低仓鼠肺部感染的病毒载量，具有统计学差异。低剂量给药组有8只仓鼠的肺部病毒载量明显下降，高剂量组几乎所有仓鼠的肺部病毒载量完全下降。肺部病毒载量的下降具有剂量依赖性，低剂量组平均肺部病毒载量下降2个lg值,高剂量组平均肺部病毒载量下降3.8个lg值。
表8.人源化双特异性抗体给药后各组BA.2肺部病毒载量(病毒载量copies/1μg RNA)
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (34)

  1. 一种分离的单克隆抗体或其抗原结合部分，其特异性结合冠状病毒的刺突蛋白或其片段，所述抗体或其抗原结合部分包含重链可变区和轻链可变区，其特征在于，
    (a)所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR2包含SEQ ID NO:2所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR3包含SEQ ID NO:3所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；和/或
    (b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:4或10所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR2包含SEQ ID NO:5所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR3包含SEQ ID NO:6所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体。
  2. 根据权利要求1所述的抗体或其抗原结合部分，其特征在于，HCDR1、HCDR2和HCDR3分别包含SEQ ID NO:1、2、3所示的氨基酸序列或所示氨基酸序列分别包含至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO:4或10、5、6所示的氨基酸序列或所示氨基酸序列分别包含至多三个氨基酸突变的变体。
  3. 根据权利要求1或2所述的抗体或其抗原结合部分，其特征在于，所述重链可变区包含与SEQ ID NO:7、13、14、42所示氨基酸序列具有至少80％同一性的氨基酸序列。
  4. 根据权利要求1或2所述的抗体或其抗原结合部分，其特征在于，所述轻链可变区包含与SEQ ID NO:8、11、47所示氨基酸序列具有至少80％同一性的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合部分，其特征在于，所述重链可变区和所述轻链可变区分别包含与SEQ ID NO:7、13、14、42和SEQ ID NO:8、11、47所示氨基酸序列具有至少80％同一性的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的抗体或其抗原结合部分，其特征在于，其为 全长抗体、Fab、Fab'、(Fab')2、Fd、Fv、dAb、scFv或scFv-scFv。
  7. 一种双特异性抗体或其抗原结合部分，其包含与第二抗体或其抗原结合部分连接的权利要求1-6中任一项所述的抗体或其抗原结合部分，其中所述第二抗体或其抗原结合部分具有与权利要求1-6中任一项所述的抗体或其抗原结合部分不同的结合特异性，所述第二抗体或其抗原结合部分能够结合冠状病毒刺突蛋白的另一个表位。
  8. 根据权利要求7所述的双特异性抗体或其抗原结合部分，其特征在于，所述第二抗体或其抗原结合部分包含重链可变区和轻链可变区，其中，
    (a)所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:17所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR2包含SEQ ID NO:18所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述HCDR3包含SEQ ID NO:19所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；和/或
    (b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:20所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR2包含SEQ ID NO:21所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体；所述LCDR3包含SEQ ID NO:22所示的氨基酸序列或所示氨基酸序列包含至多三个氨基酸突变的变体。
  9. 根据权利要求8所述的双特异性抗体或其抗原结合部分，其特征在于，其中所述第二抗体或其抗原结合部分的重链可变区和轻链可变区分别包含与SEQ ID NO:23或45和SEQ ID NO:24或46所示氨基酸序列具有至少80％同一性的氨基酸序列。
  10. 根据权利要求9所述的双特异性抗体或其抗原结合部分，其特征在于，所述双特异性抗体包含两个单链抗体片段scFv，其中所述scFv的重链可变区VH的序列如SEQ ID NO.27或14所示，所述scFv的轻链可变区VL的序列如SEQ ID NO.28或47所示。
  11. 根据权利要求10所述的双特异性抗体或其抗原结合部分，其特征在于，所述scFv分别连接于所述第二抗体或其抗原结合部分的两条重链或两条轻链的N端或C端；优选地，所述scFv分别连接于所述第二抗体或其抗原结合部分的两 条轻链的N端。
  12. 根据权利要求1-11中任一项所述的抗体或其抗原结合部分，其特征在于，其包含重链恒定区和/或轻链恒定区。
  13. 根据权利要求12所述的抗体或其抗原结合部分，其特征在于，其中所述重链恒定区是IgG、IgA、IgD、IgE和IgM恒定区。
  14. 根据权利要求12所述的抗体或其抗原结合部分，其特征在于，其中所述重链恒定区是IgG1、IgG2、IgG3或IgG4恒定区。
  15. 根据权利要求12所述的抗体或其抗原结合部分，其特征在于，其中所述轻链恒定区是κ或λ恒定区。
  16. 根据权利要求1-15中任一项所述的抗体或其抗原结合部分，其特征在于，其中所述重链恒定区任选含有突变。
  17. 根据权利要求1-15中任一项所述的抗体或其抗原结合部分，其特征在于，其与选自SARS-CoV、SARS-CoV-2和MERS-CoV的冠状病毒的刺突蛋白或其片段结合。
  18. 根据权利要求1-15中任一项所述的抗体或其抗原结合部分，其特征在于，其与冠状病毒的刺突蛋白的受体结合域或胞外域结合。
  19. 根据权利要求1-18中任一项所述的抗体或其抗原结合部分，其特征在于，其(a)与SARS-CoV-2的刺突蛋白或其片段结合，(b)抑制SARS-CoV-2的刺突蛋白或其片段与人ACE2结合，(c)阻止SARS-CoV-2进入宿主细胞，(d)诱导补体介导的SARS-CoV-2失活，(e)诱导SARS-CoV-2的吞噬作用，(f)诱导针对SARS-CoV-2感染细胞的补体依赖性细胞毒性，(g)诱导针对SARS-CoV-2感染细胞的抗体依赖性细胞吞噬作用，(h)诱导针对SARS-CoV-2感染细胞的抗体依赖性细胞介导的病毒抑制作用，(i)诱导针对SARS-CoV-2感染细胞的抗体依赖性细胞介导的细胞毒性，(j)诱导体液和细胞抗病毒免疫反应，和/或(k)阻碍刺突蛋白的三聚体形成。
  20. 根据权利要求1-19中任一项所述的抗体或其抗原结合部分，其特征在于，其与SARS-CoV-2的刺突蛋白的胞外域结合并介导抗体中和。
  21. 一种核酸分子，其编码权利要求1-20中任一项所述的抗体或其抗原结合部分。
  22. 一种表达载体，其含有权利要求21所述的核酸分子。
  23. 一种宿主细胞，其含有权利要求22所述的表达载体。
  24. 一种组合物，其包含权利要求1-20中任一项所述的抗体或其抗原结合部分，权利要求21所述的核酸分子，权利要求22所述的表达载体，和/或权利要求23所述的宿主细胞。
  25. 一种在有需要的受试者中治疗由冠状病毒感染引起的疾病的方法，包括向受试者施用权利要求24所述的组合物。
  26. 根据权利要求25所述的方法，其中所述疾病是由SARS-CoV、MERS-CoV或SARS-CoV-2引起的。
  27. 根据权利要求26所述的方法，其中所述疾病是由SARS-CoV-2引起的。
  28. 根据权利要求25-27中任一项所述的方法，其中所述受试者是人。
  29. 一种在有需要的受试者中预防由冠状病毒感染引起的疾病的方法，包括向受试者施用权利要求1-20中任一项所述的抗体或其抗原结合部分。
  30. 根据权利要求29所述的方法，用于预防由SARS-CoV、MERS-CoV或SARS-CoV-2引起的疾病。
  31. 根据权利要求30所述的方法，用于预防由SARS-CoV-2引起的疾病。
  32. 根据权利要求29-31中任一项所述的方法，其中所述受试者是人。
  33. 根据权利要求29-32中任一项所述的方法，其中所述受试者不受疫苗接种的保护。
  34. 一种诊断受试者中冠状病毒感染的方法，包括使权利要求1-20中任一项所述的抗体或其抗原结合部分与来自受试者的组织样品接触。
PCT/CN2023/109571 2022-07-29 2023-07-27 一种抗rbd抗体及其用途 WO2024022438A1 (zh)

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